Seasonality of parasitic and saprotrophic zoosporic fungi: linking sequence data to ecological traits

Zoosporic fungi of the phylum Chytridiomycota (chytrids) regularly dominate pelagic fungal communities in freshwater and marine environments. Their lifestyles range from obligate parasites to saprophytes. Yet, linking the scarce available sequence data to specific ecological traits or their host ranges constitutes currently a major challenge. We combined 28 S rRNA gene amplicon sequencing with targeted isolation and sequencing approaches, along with cross-infection assays and analysis of chytrid infection prevalence to obtain new insights into chytrid diversity, ecology, and seasonal dynamics in a temperate lake. Parasitic phytoplankton-chytrid and saprotrophic pollen-chytrid interactions made up the majority of zoosporic fungal reads. We explicitly demonstrate the recurrent dominance of parasitic chytrids during frequent diatom blooms and saprotrophic chytrids during pollen rains. Distinct temporal dynamics of diatom-specific parasitic clades suggest mechanisms of coexistence based on niche differentiation and competitive strategies. The molecular and ecological information on chytrids generated in this study will aid further exploration of their spatial and temporal distribution patterns worldwide. To fully exploit the power of environmental sequencing for studies on chytrid ecology and evolution, we emphasize the need to intensify current isolation efforts of chytrids and integrate taxonomic and autecological data into long-term studies and experiments.Subject terms: Fungal ecology, Limnology     

Phylogenetic Position of Parasitic Chytrids on Diatoms: Characterization of a Novel Clade in Chytridiomycota

Chytrids are true fungi that reproduce with posteriorly uniflagellate zoospores. In the last decade, environmental DNA surveys revealed a large number of uncultured chytrids as well as undescribed order‐level novel clades in Chytridiomycota. Although many species have been morphologically described, only some DNA sequence data of parasitic chytrids are available from the database. We herein discuss five cultures of parasitic chytrids on diatoms Aulacoseira spp. and Asterionella formosa. In order to identify the chytrids examined, thallus morphologies were observed using light microscopy. We also conducted a phylogenetic analysis using 18S, 5.8S, and 28S rDNA sequences to obtain their phylogenetic positions. Based on their morphological characteristics, two cultures parasitic on As. formosa were identified as Rhizophydium planktonicum and Zygorhizidium planktonicum. The other three cultures infecting Aulacoseira spp. (two on Aulacoseira ambigua and the other on Aulacoseira granulata) were regarded as Zygorhizidium aff. melosirae. The results of the molecular phylogenetic analysis revealed that R. planktonicum belonged to the known order Chytridiales, while the two species of Zygorhizidium were placed in a novel clade that was previously reported as an undescribed clade composed of only the environmental sequences of uncultured chytrids.     

PCR primers for assessing community structure of aquatic fungi including Chytridiomycota and Cryptomycota

Fungi are diverse and have the potential for material cycling in freshwater ecosystems. Species composition of aquatic fungi and their seasonal dynamics are often key to their role in the functioning of the ecosystems. However, community structure of aquatic fungi, especially of Chytridiomycota (Chytrids) and Cryptomycota, is still limited because few primer sets are available to examine species composition. In this study, we validated six primer sets for the detection of aquatic fungi by denaturing gradient gel electrophoresis (DGGE) analysis and found that FF390W/EF3r showed the highest specificity among the primer sets tested. We detected both Chytrids and Cryptomycota from Lake Inba by DGGE analysis using the FF390W/EF3r-GC primer set. Further study with our established analysis revealed community structures of aquatic fungi and their seasonal succession patterns in the lake. Results of our study are useful for selection of the primer set for studying community structures of aquatic fungi and their seasonal succession.     

Ontogenetic allometry of the bluemouth, <Emphasis Type="Italic">Helicolenus dactylopterus dactylopterus</Emphasis> (Teleostei: Scorpaenidae), in the Northeast Atlantic and Mediterranean based on geometric morphometrics

Since the emergence of the ‘microbial loop’ concept, heterotrophic flagellates have received particular attention as grazers in aquatic ecosystems. These microbes have historically been regarded incorrectly as a homogeneous group of bacterivorous protists in aquatic systems. More recently, environmental rDNA surveys of small heterotrophic flagellates in the pelagic zone of freshwater ecosystems have provided new insights. (i) The dominant phyla found by molecular studies differed significantly from those known from morphological studies with the light microscope, (ii) the retrieved phylotypes generally belong to well-established eukaryotic clades, but there is a very large diversity within these clades and (iii) a substantial part of the retrieved sequences cannot be assigned to bacterivorous but can be assigned instead to parasitic and saprophytic organisms, such as zoosporic true fungi (chytrids), fungus-like organisms (stramenopiles), or virulent alveolate parasites (Perkinsozoa and Amoebophrya sp.). All these microorganisms are able to produce small zoospores to assure dispersal in water during their life-cycles. Based on the existing literature on true fungi and fungus-like organisms, and on the more recently published eukaryotic rDNA environmental studies and morphological observations, we conclude that previously overlooked microbial diversity and related ecological potentials require intensive investigation (i) for an improved understanding of the roles of heterotrophic flagellates in pelagic ecosystems and (ii) to properly integrate the concept of ‘the microbial loop’ into modern pelagic microbial ecology.     

Unveiling fungal zooflagellates as members of freshwater picoeukaryotes: evidence from a molecular diversity study in a deep meromictic lake

This study presents an original 18S rRNA PCR survey of the freshwater picoeukaryote community, and was designed to detect unidentified heterotrophic picoflagellates (size range 0.6-5 microm) which are prevalent throughout the year within the heterotrophic flagellate assemblage in Lake Pavin. Four clone libraries were constructed from samples collected in two contrasting zones in the lake. Computerized statistic tools have suggested that sequence retrieval was representative of the in situ picoplankton diversity. The two sampling zones exhibited similar diversity patterns but shared only about 5% of the operational taxonomic units (OTUs). Phylogenetic analysis clustered our sequences into three taxonomic groups: Alveolates (30% of OTUs), Fungi (23%) and Cercozoa (19%). Fungi thus substantially contributed to the detected diversity, as was additionally supported by direct microscopic observations of fungal zoospores and sporangia. A large fraction of the sequences belonged to parasites, including Alveolate sequences affiliated to the genus Perkinsus known as zooparasites, and chytrids that include host-specific parasitic fungi of various freshwater phytoplankton species, primarily diatoms. Phylogenetic analysis revealed five novel clades that probably include typical freshwater environmental sequences. Overall, from the unsuspected fungal diversity unveiled, we think that fungal zooflagellates have been misidentified as phagotrophic nanoflagellates in previous studies. This is in agreement with a recent experimental demonstration that zoospore-producing fungi and parasitic activity may play an important role in aquatic food webs.     

Comparison of sterol and fatty acid profiles of chytrids and their hosts reveals trophic upgrading of nutritionally inadequate phytoplankton by fungal parasites

Chytrids are ubiquitous fungal parasites in aquatic ecosystems, infecting representatives of all major phytoplankton groups. They repack carbon from inedible phytoplankton hosts into easily ingested chytrid propagules (zoospores), rendering this carbon accessible to zooplankton. Grazing on zoospores may circumvent bottlenecks in carbon transfer imposed by the dominance of inedible or poorly nutritious phytoplankton (mycoloop). We explored qualitative aspects of the mycoloop by analysing lipid profiles (fatty acids, sterols) of two chytrids infecting two major bloom-forming phytoplankton taxa of contrasting nutritional value: the diatom Asterionella formosa and the filamentous cyanobacterium Planktothrix agardhii. The polyunsaturated fatty acid composition of chytrids largely reflected that of their hosts, highlighting their role as conveyors of otherwise inaccessible essential lipids to higher trophic levels. We also showed that chytrids are capable of synthesizing sterols, thus providing a source of these essential nutrients for grazers even when sterols are absent in their phytoplankton hosts. Our findings reveal novel qualitative facets of the mycoloop, showing that parasitic chytrids, in addition to making carbon and essential lipids available from inedible sources, also upgrade their host's biochemical composition by producing sterols de novo, thereby enhancing carbon and energy fluxes in aquatic food webs.     

Importance of Saprotrophic Freshwater Fungi for Pollen Degradation

Fungi and bacteria are the major organic matter (OM) decomposers in aquatic ecosystems. While bacteria are regarded as primary mineralizers in the pelagic zone of lakes and oceans, fungi dominate OM decomposition in streams and wetlands. Recent findings indicate that fungal communities are also active in lakes, but little is known about their diversity and interactions with bacteria. Therefore, the decomposer niche overlap of saprotrophic fungi and bacteria was studied on pollen (as a seasonally recurring source of fine particulate OM) by performing microcosm experiments with three different lake types. Special emphasis was placed on analysis of fungal community composition and diversity. We hypothesized that (I) pollen select for small saprotrophic fungi and at the same time for typical particle-associated bacteria; (II) fungal communities form specific free-living and attached sub-communities in each lake type; (III) the ratio between fungi or bacteria on pollen is controlled by the lake''s chemistry. Bacteria-to-fungi ratios were determined by quantitative PCR (qPCR), and bacterial and fungal diversity were studied by clone libraries and denaturing gradient gel electrophoresis (DGGE) fingerprints. A protease assay was used to identify functional differences between treatments. For generalization, systematic differences in bacteria-to-fungi ratios were analyzed with a dataset from the nearby Baltic Sea rivers. High abundances of Chytridiomycota as well as occurrences of Cryptomycota and yeast-like fungi confirm the decomposer niche overlap of saprotrophic fungi and bacteria on pollen. As hypothesized, microbial communities consistently differed between the lake types and exhibited functional differences. Bacteria-to-fungi ratios correlated well with parameters such as organic carbon and pH. The importance of dissolved organic carbon and nitrogen for bacteria-to-fungi ratios was supported by the Baltic Sea river dataset. Our findings highlight the fact that carbon-to-nitrogen ratios may also control fungal contributions to OM decomposition in aquatic ecosystems.     

Pendulichytrium sphaericum gen. et sp. nov. (Chytridiales,Chytriomycetaceae), a new chytrid parasitic on the diatom,Aulacoseira granulata

During the last decade, our concept of chytrid systematics has dramatically changed based on molecular phylogeny and zoospore ultrastructure. In contrast with well-studied saprotrophic chytrids, only a few obligate parasitic chytrids have been investigated with modern methods. Here, we investigate the novel chytrid culture KS93 that is parasitic on the diatom Aulacoseira granulata. Thallus morphology of KS93 was characterized by a spherical, stalked zoosporangium with a single, apical inoperculate discharge pore and zoospore discharge as a mass in a vesicle. A cross-inoculation experiment revealed that the infection of KS93 was specific to A. granulata. Zoospores of KS93 possessed the characters of the Group I type zoospore of the Chytriomycetaceae in the Chytridiales, but does not appear to have a paracrystalline inclusion. Additionally, KS93 also possessed a globule-type KAS (kinetosome-associated structure), first reported here for members of Chytriomycetaceae. In our molecular phylogeny, KS93 was placed in the basal position of the Chytriomycetaceae and was distinguished from any known species in the family. Morphological features of KS93 were distinct from those of any other taxa in the Chytriomycetaceae and from any described chytrids. Based on these results, we describe this chytrid as Pendulichytrium sphaericum gen. et sp. nov. in the family Chytriomycetaceae.     

Phytoplankton chytridiomycosis: community structure and infectivity of fungal parasites in aquatic ecosystems

Fungal parasitism is recurrent in plankton communities, especially in the form of parasitic chytrids. However, few attempts have been made to study the community structure and activity of parasites at the natural community level. To analyse the dynamics of zoosporic fungal parasites (i.e. chytrids) of phytoplankton, samples were collected from February to December 2007 in two freshwater lakes. Infective chytrids were omnipresent in lakes, with higher diversity of parasites and infected phytoplankton than in previous studies. The abundance and biomass of parasites were significantly higher in the productive Lake Aydat than in the oligomesotrophic Lake Pavin, while the infection prevalence in both lakes were similar and averaged about 20%. The host species composition and their size appeared as critical for chytrid infectivity, the larger hosts being more vulnerable, including pennate diatoms and desmids in both lakes. The highest prevalence (98%) was noted for the autumn bloom of the cyanobacterium Anabaena flosaquae facing the parasite Rhizosiphon crassum in Lake Aydat. Because parasites killed their hosts, this implies that cyanobacterial blooms, and other large size inedible phytoplankton blooms as well, may not totally represent trophic bottlenecks because their zoosporic parasites can release dissolved substrates for microbial processes through host destruction, and provide energetic particles as zoospores for grazers. Overall, we conclude that the parasitism by zoosporic fungi represents an important ecological driving force in the food web dynamics of aquatic ecosystems, and infer general empirical models on chytrid seasonality and trophodynamics in lakes.     

The ecology of chytrids in aquatic ecosystems: roles in food web dynamics

Chytrids are very important components of freshwater ecosystems, but the ecological significance of this group of fungi is not well understood. This review considers some of the significant environmental factors affecting growth and population composition of chytrids in aquatic habitats. The physical factors include primarily salinity, dissolved oxygen concentration and temperature. The biological factors include the role of chytrids as saprobes and parasites and methods of dispersal of propagules throughout the ecosystem. Dispersal depends upon both zoospores for short range and whole thalli for long range dispersal. Five roles for chytrids in food-web dynamics are proposed: (1) chytrid zoospores are a good food source for zooplankton, (2) chytrids decompose particulate organic matter, (3) chytrids are parasites of aquatic plants, (4) chytrids are parasites of aquatic animals and (5) chytrids convert inorganic compounds into organic compounds. New molecular methods for analysis of chytrid diversity in aquatic environments have the potential to provide accurate quantitative data necessary for better understanding of ecological processes in aquatic ecosystems.     

Functional effects of parasites on food web properties during the spring diatom bloom in Lake Pavin: a linear inverse modeling analysis

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.     

Molecular and microscopic diversity of planktonic eukaryotes in the oligotrophic Lake Stechlin (Germany)

The aim of this study was to compare a molecular and a microscopic approach to study the planktonic eukaryotic diversity of an oligotrophic lake. Plankton samples from the temperate Lake Stechlin were assessed in winter and summer 2008 by comparison of 18S rRNA gene clone libraries to light microscopic evaluations. For both approaches identical samples were used. There were remarkable differences between the main groups recovered by the contrasting methods. The microscopic analyses showed predominance of autotrophic planktonic organisms, whereas most of them could not be discovered by the molecular method which resulted in a higher diversity of heterotrophic flagellates. The microscopic survey revealed high diversity of Chlorophyta and Cryptophyta as well as the Stramenopiles groups of Bacillariophyceae and Chrysophyceae. The clone libraries, based on full-length 18S rRNA gene sequences, displayed highest diversity of Alveolata belonging to seven different subclades. Notably, Antarctic Dinophyta-related clones were detected. The occurrence of the marine phagotrophic flagellate Telonema was also documented. Comparing the two sampling seasons, rich diversity suggests that flagellates played an important role in late winter (February), however, there is relatively low diversity in summer (August). The newly discovered molecular diversity of planktonic eukaryotes in Stechlin will help to understand the biodiversity patterns in freshwater lakes.     

Community Structure of Bacteria Associated with Sheaths of Freshwater and Brackish Thioploca Species

Bacterial communities associated with sheaths of Thioploca spp. from two freshwater lakes (Lake Biwa, Japan, and Lake Constance, Germany) and one brackish lake (Lake Ogawara, Japan) were analyzed with denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. The comparison between the DGGE band patterns of bulk sediment and Thioploca filaments of Lake Biwa suggested the presence of specific bacterial communities associated with Thioploca sheaths. As members of sheath-associated communities, bacteria belonging to Bacteroidetes were detected from the samples of both freshwater lakes. A DGGE band from Thioploca of Lake Biwa, belonging to candidate division OP8, was quite closely related to another DGGE band detected from that of Lake Constance. In contrast to the case of freshwater lakes, no bacterium of Bacteroidetes or OP8 was detected from Thioploca of Lake Ogawara. However, two DGGE bands from Lake Ogawara, belonging to Chloroflexi, were quite closely related to a DGGE band from Lake Constance. Two DGGE bands obtained from Lake Biwa were closely related to phylogenetically distant dissimilatory Fe(III)-reducing bacteria. Cloning analyses for a dissimilatory sulfite reductase gene were performed on the same samples used for DGGE analysis. The results of the analyses suggest that sheaths of freshwater/brackish Thioploca have little ecological significance for the majority of sulfate reducers.     

Carbon and nutrients of indigestible pollen are transferred to zooplankton by chytrid fungi

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The parasitic chytrid, Zygorhizidium, facilitates the growth of the cladoceran zooplankter, Daphnia, in cultures of the inedible alga, Asterionella

In food-web studies, parasites are often ignored owing to their insignificant biomass. We provide evidence that parasites may affect trophic transfer in aquatic food webs. Many phytoplankton species are susceptible to parasitic fungi (chytrids). Chytrid infections of diatoms in lakes may reach epidemic proportions during diatom spring blooms, so that numerous free-swimming fungal zoospores (2-3 microm in diameter) are produced. Analysis shows that these zoospores are rich in polyunsaturated fatty acids and sterols (particularly cholesterol), which indicates that they provide excellent food for zooplankters such as Daphnia. In life-table experiments using the large diatom Asterionella formosa as food, Daphnia growth increased significantly in treatments where a parasite was present. By grazing on the zoospores, Daphnia acquired important supplementary nutrients and were able to grow. When large inedible algae are infected by parasites, nutrients within the algal cells are consumed by these chytrids, some of which, in turn, are grazed by Daphnia. Thus, chytrids transfer energy and nutrients from their hosts to zooplankton. This study suggests that parasitic fungi alter trophic relationships in freshwater ecosystems and may be the important components in shaping the community and the food-web dynamics of lakes.     

Phylogeny and biogeography of an uncultured clade of snow chytrids

Numerous studies have shown that snow can contain a diverse array of algae known as ‘snow algae’. Some reports also indicate that parasites of algae (e.g. chytrids) are also found in snow, but efforts to phylogenetically identify ‘snow chytrids’ have not been successful. We used culture‐independent molecular approaches to phylogenetically identify chytrids that are common in long‐lived snowpacks of Colorado and Europe. The most remarkable finding of the present study was the discovery of a new clade of chytrids that has representatives in snowpacks of Colorado and Switzerland and cold sites in Nepal and France, but no representatives from warmer ecosystems. This new clade (‘Snow Clade 1’ or SC1) is as deeply divergent as its sister clade, the Lobulomycetales, and phylotypes of SC1 show significant (P < 0.003) genetic‐isolation by geographic distance patterns, perhaps indicating a long evolutionary history in the cryosphere. In addition to SC1, other snow chytrids were phylogenetically shown to be in the order Rhizophydiales, a group with known algal parasites and saprotrophs. We suggest that these newly discovered snow chytrids are important components of snow ecosystems where they contribute to snow food‐web dynamics and the release of nutrients due to their parasitic and saprotrophic activities.     

Diatom evidence for the timing and causes of eutrophication in Lake Victoria,East Africa

The determination of the history and causes of recent eutrophication and intensified thermal stratification in Lake Victoria is still hampered by the sparsity of paleolimnologic coverage of the enormous lake. Five new diatom records from Ugandan waters now show that a transition from Aulacoseira-dominated planktonic assemblages to those dominated by long Nitzschia spp., occurred in northern coastal sites from the mid-1970s to mid-1980s. Similar transitions developed from the late 1960s to early 1970s offshore and from the 1940s to early 1950s along the Kenyan coast, suggesting a time-transgressive process. These changes are not readily attributable to the trophic effects of Nile perch population growth during the early 1980s, but more likely reflect responses to long-term nutrient enrichment and climatic instability in the region. The diversity of planktonic diatom communities has declined dramatically, and a namesake variety of Aulacoseira nyassensis may now be nearly extirpated. Although local phytoplankton communities varied considerably in the past, the current domination of diatom assemblages by Nitzschia is apparently unprecedented in the 15,000-year history of Lake Victoria.     

Anaerobic Oxalate Degradation: Widespread Natural Occurrence in Aquatic Sediments

Significant concentrations of oxalate (dissolved plus particulate) were present in sediments taken from a diversity of aquatic environments, ranging from 0.1 to 0.7 mmol/liter of sediment. These included pelagic and littoral sediments from two freshwater lakes (Searsville Lake, Calif., and Lake Tahoe, Calif.), a hypersaline, meromictic, alkaline lake (Big Soda Lake, Nev.), and a South San Francisco Bay mud flat and salt marsh. The oxalate concentration of several plant species which are potential detrital inputs to these aquatic sediments ranged from 0.1 to 5.0% (wt/wt). In experiments with litter bags, the oxalate content of Myriophyllum sp. samples buried in freshwater littoral sediments decreased to 7% of the original value in 175 days. This suggests that plant detritus is a potential source of the oxalate within these sediments. [¹⁴C]oxalic acid was anaerobically degraded to ¹⁴CO₂ in all sediment types tested, with higher rates evident in littoral sediments than in the pelagic sediments of the lakes studied. The turnover time of the added [¹⁴C]oxalate was less than 1 day in Searsville Lake littoral sediments. The total sediment oxalate concentration did not vary significantly between littoral and pelagic sediments and therefore did not appear to be controlling the rate of oxalate degradation. However, depth profiles of [¹⁴C]oxalate mineralization and dissolved oxalate concentration were closely correlated in freshwater littoral sediments; both were greatest in the surface sediments (0 to 5 cm) and decreased with depth. The dissolved oxalate concentration (9.1 μmol/liter of sediment) was only 3% of the total extractable oxalate (277 μmol/liter of sediment) at the sediment surface. These results suggest that anaerobic oxalate degradation is a widespread phenomenon in aquatic sediments and may be limited by the dissolved oxalate concentration within these sediments.     

Changes in Pelagic Bacteria Communities Due to Leaf Litter Addition

In many limnetic systems, the input of allochthonous organic matter, e.g., leaf litter, is a substantial source of dissolved organic carbon (DOC) for pelagic bacteria, especially in fall and winter when autochthonous DOC production is low. However, relatively little is known about community changes of pelagic lake bacteria due to leaf litter input which includes both the release of leaf leachates and microorganisms from the leaf litter into the surrounding water. Therefore, we have experimentally studied the effects of different types of leaf litter (Betula pendula, Fagus silvatica, and Pinus silvestris) on the pelagic bacterial community composition by adding leaves to different treatments of epilimnic water samples (unfiltered, 0.2 μm and 5.0 μm-pre-filtered) from humic Lake Grosse Fuchskuhle (Northeastern Germany). The addition of leaf litter led to a significant increase in DOC concentration in lake water, and each leaf litter type produced significantly different amounts of DOC (p = <0.001) as well as of specific DOC fractions (p = <0.001), except of polysaccharides. DGGE banding patterns varied over time, between types of leaf litter, and among treatments. Bacteria belonging to known bacterial phylotypes in the southwest basin of Lake Grosse Fuchskuhle were frequently found and even persisted after leaf litter additions. Upon leaf litter addition, α-proteobacteria (Azospirillum, Novosphingobium, and Sphingopyxis) as well as β-proteobacteria (Curvibacter and Polynucleobacter) were enriched. Our results indicate that supply of leaf litter DOM shifted the bacterial community in the surrounding water towards specific phylotypes including species capable of assimilating the more recalcitrant DOC pools. Statistical analyses, however, show that DGGE banding patterns are not only affected by DOC pools but also by treatment. This indicates that biological factors such as source community and grazing may be also important for shifts in bacterial community structure following leaf litter input into different lakes.     

Fluorescence in situ hybridization of uncultured zoosporic fungi: Testing with clone-FISH and application to freshwater samples using CARD-FISH

Recently, molecular environmental surveys of the eukaryotic microbial community in lakes have revealed a high diversity of sequences belonging to uncultured zoosporic fungi commonly known as chytrids. These microorganisms have two different stages in their life cycle and are known as algal parasites (i.e. host-attached infective sporangia) and as food sources for zooplankton (i.e. free-living zooflagellate propagules) in aquatic systems. However, because of their small size and their lack of distinctive morphological features, traditional microscopy does not allow the detection of chytrids, particularly of zoospores which have probably been misidentified as phagotrophic flagellates in previous studies. Hence, quantitative data on chytrids in natural environments is missing. We have adapted a clone-FISH approach known from prokaryotes to optimize the hybridization conditions of a designed oligonucleotidic probe specific to Chytridiales (i.e. the largest group of the true-fungal division of Chytridiomycota), before application to natural samples using the CARD-FISH approach. When these conditions were applied, the CARD-FISH assay demonstrated high specificity and sensitivity, and offers a promising tool for quantitative assessment of natural zoosporic fungi, primarily of zoospores which contributed up to 60% of the total abundance of heterotrophic flagellates. Although the field results from the CARD-FISH approach were considered preliminary and mainly as ‘proof of concept’, findings were consistent with ecological considerations known from pelagic habitats and host versus parasite populations, with recurrent ecological patterns in two contrasting lake ecosystems. We conclude that this approach will contribute to a better understanding of the ecological significance of zoosporic organisms in microbial food webs of pelagic ecosystems.