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.     

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     

Fungal Parasitism: Life Cycle,Dynamics and Impact on Cyanobacterial Blooms

Many species of phytoplankton are susceptible to parasitism by fungi from the phylum Chytridiomycota (i.e. chytrids). However, few studies have reported the effects of fungal parasites on filamentous cyanobacterial blooms. To investigate the missing components of bloom ecosystems, we examined an entire field bloom of the cyanobacterium Anabaena macrospora for evidence of chytrid infection in a productive freshwater lake, using a high resolution sampling strategy. A. macrospora was infected by two species of the genus Rhizosiphon which have similar life cycles but differed in their infective regimes depending on the cellular niches offered by their host. R. crassum infected both vegetative cells and akinetes while R. akinetum infected only akinetes. A tentative reconstruction of the developmental stages suggested that the life cycle of R. crassum was completed in about 3 days. The infection affected 6% of total cells (and 4% of akinètes), spread over a maximum of 17% of the filaments of cyanobacteria, in which 60% of the cells could be parasitized. Furthermore, chytrids may reduce the length of filaments of Anabaena macrospora significantly by “mechanistic fragmentation” following infection. All these results suggest that chytrid parasitism is one of the driving factors involved in the decline of a cyanobacteria blooms, by direct mortality of parasitized cells and indirectly by the mechanistic fragmentation, which could weaken the resistance of A. macrospora to grazing.     

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.     

Evidence from beta-tubulin phylogeny that microsporidia evolved from within the fungi

Microsporidia are obligate intracellular parasites that were thought to be an ancient eukaryotic lineage based on molecular phylogenies using ribosomal RNA and translation elongation factors. However, this ancient origin of microsporidia has been contested recently, as several other molecular phylogenies suggest that microsporidia are closely related to fungi. Most of the protein trees that place microsporidia with fungi are not well sampled, however, and it is impossible to resolve whether microsporidia evolved from a fungus or from a protistan relative of fungi. We have sequenced beta-tubulins from 3 microsporidia, 4 chytrid fungi, and 12 zygomycete fungi, expanding the representation of beta-tubulin to include all four fungal divisions and a wide diversity of microsporidia. In phylogenetic trees including these new sequences, the overall topology of the fungal beta-tubulins generally matched the expected relationships among the four fungal divisions, although the zygomycetes were polyphyletic in some analyses. The microsporidia consistently fell within this fungal diversification, and not as a sister group to fungi. Overall, beta-tubulin phylogeny suggests that microsporidia evolved from a fungus sometime after the divergence of chytrids. We also found that chytrid alpha- and beta-tubulins are much less divergent than are tubulins from other fungi or microsporidia. In trees in which the only fungal representatives were the chytrids, microsporidia still branched with fungi (i.e., with chytrids), suggesting that the affiliation between microsporidian and fungal tubulins is not an artifact of long-branch attraction.     

Chytrid infecting the bloom-forming marine diatom Skeletonema sp.: Morphology,phylogeny and distribution of a novel species within the Rhizophydiales

Chytrids have long been recognised as important parasites of microalgae in freshwater systems, able to shape the dynamics of blooms, the gene pool of their host and phytoplankton succession. In the sea however, where the presence of these organisms is erratic and ephemeral, studies concerning chytrids are sparse and confined to metabarcoding surveys or microscopy observations. Despite the scarcity of data, chytrid epidemics are supposed to play an important role in marine biogeochemical cycles, being one of the drivers of phytoplankton dynamics. Here we combine microscopy observations and in silico mining of a single-cell whole genome to molecularly and morphologically characterise a novel chytrid parasite of the dominant diatom genus Skeletonema. Morphological observations highlight features of the thallus and ascertain the parasitic nature of the interaction whilst the genetic markers obtained allows for a phylogenetic reconstruction, placing the new species in the order Rhizophydiales. Thanks to the molecular data obtained we are also able to provide a first investigation of the global distribution of this organism by screening the Ocean Sampling Day (OSD) dataset, highlighting a northern transatlantic dissemination.     

Alogomyces tanneri gen. et sp. nov., a chytrid in Lobulomycetales from horse manure

The order Lobulomycetales contains chytrids from soil, freshwater and marine habitats; environmental DNA sampling has indicated that representatives of this order might be found in deep ocean localities. We describe Alogomyces tanneri as the first lobulomycetalean chytrid isolated from horse manure; A. tanneri is also the first species in the order to possess a rumposome in its zoospore. This species widens the range of habitats, ultrastructural variation and thallus morphology for Lobulomycetales.     

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.     

Access to organic and insoluble sources of phosphorus varies among soil Chytridiomycota

The sources of minerals accessed by fungi in the Chytridiomycota (chytrid) in soil are largely unknown. The ability of ten species of soil chytrids to use various sources of phosphorus was examined in vitro. While all grew on orthophosphate, fifty per cent of isolates grew on phytic acid, and one isolate grew on DNA as the sole source of phosphorus. All isolates solubilised and utilised CaHPO₄. Most isolates utilised hydroxyapatite when NH₄⁺ was the nitrogen source. When ammonium was omitted, 50% of isolates solubilised hydroxyapatite. Many soil chytrids may utilise phosphomonoesters as the sole source of phosphorus, and access to DNA appears limited. We suggest that the capacity to use different sources of phosphorus may influence the diversity of chytrids found in Australian soils.     

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

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Community Structure of Planktonic Fungi and the Impact of Parasitic Chytrids on Phytoplankton in Lake Inba,Japan

Freshwater fungi have received little attention by scientific research in recent years, especially fungi of the pelagic zone. Recently, parasitic fungi, termed chytrids, have been found to play important roles in aquatic food webs. Yet, the diversity and community structure of planktonic fungi including chytrids are not well studied. In this study, we examined the temporal fluctuations of freshwater fungi, including chytrids, in Lake Inba by using molecular techniques of denaturing gradient gel electrophoresis (DGGE). DGGE profiles, and associated sequence analysis, indicated that chytrids were present on all sampling dates from May to October (n = 12). In addition, analysis showed that a large proportion of the sequences belonged to chytrids of both parasitic and saprotrophic species. This finding was supported by microscopic observations using Calcofluor white to stain chytrids infecting various phytoplankton species. The percentages of infection by chytrids on two dominant diatom species, Aulacoseira granulata and Aulacoseira ambigua, showed a similar seasonal pattern in the DGGE band profiles. From the phylogenetic analysis and microscopic identification, the chytrids infecting the two diatoms are likely to be affiliated to Chytriomyces sp. and Zygorhizidium sp.. This is the first study to show that DGGE is a useful preliminary approach for examining the diversity of planktonic fungi including chytrids. Our results indicate both parasitic and saprotrophic chytrids are a significant component of freshwater fungi inhabiting the pelagic zone of Lake Inba, Japan. Further modification of DGGE, together with new molecular techniques and microscopic observation, would reveal the hidden diversity and ecological significance of planktonic fungi in aquatic ecosystems.     

Potential roles for recently discovered chytrid parasites in the dynamics of harmful algal blooms

Zoosporic true fungi belonging to the phylum Chytridiomycota, commonly referred to as chytrids, are ubiquitous in aquatic environments, however their role in phytoplankton population and eco-physiological dynamics is not fully understood. With the rising occurrence of harmful algal blooms (HABs) of phytoplankton worldwide, there is a growing need to investigate the factors affecting toxicity in algae, with a view to determining the significance of these factors in light of the current trends in global climate change. In this review we present current knowledge regarding the parasitism of phytoplankton by chytrids, including incidence of chytrid epidemics, methodologies used in their isolation and classification, their life cycles and infection strategies, and their potential role in toxin production in algae. We outline key areas in phytoplankton host–parasite dynamics that are poorly understood, discuss the potential roles of chytrids in these areas, and highlight future research directions for the furthering of our knowledge regarding algal ecophysiology. The synthesis of current knowledge in these fields will help researchers develop new hypotheses to further our understanding of primary production in aquatic ecology, and thus enhance our understanding of aquatic ecology, for more effective management of 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.     

Environmental Conditions Determine the Course and Outcome of Phytoplankton Chytridiomycosis

Chytrid fungi are highly potent parasites of phytoplankton. They are thought to force phytoplankton organisms into an evolutionary arms race with high population diversity as the outcome. The underlying selection regime is known as Red Queen dynamics. However, our study suggests a more complex picture for chytrid parasitism in the cyanobacterium Planktothrix. Laboratory experiments identified a “cold thermal refuge”, inside which Planktothrix can grow without chytrid infection. A field study in two Norwegian lakes underlined the ecological significance of this finding. The study utilized sediment DNA as a biological archive in combination with existing monitoring data. In one lake, temperature and light conditions forced Planktothrix outside the thermal refuge for most of the growing season. This probably resulted in Red Queen dynamics as suggested by a high parasitic pressure exerted by chytrids, an increase in Planktothrix genotype diversity over time, and a correlation between Planktothrix genotype diversity and duration of bloom events. In the second lake, a colder climate allowed Planktothrix to largely stay inside the thermal refuge. The parasitic pressure exerted by chytrids and Planktothrix genotype diversity remained low, indicating that Planktothrix successfully evaded the Red Queen dynamics. Episodic Planktothrix blooms were observed during spring and autumn circulation, in the metalimnion or under the ice. Interestingly, both lakes were dominated by the same or related Planktothrix genotypes. Taken together, our data suggest that, depending on environmental conditions, chytrid parasitism can impose distinct selection regimes on conspecific phytoplankton populations with similar genotype composition, causing these populations to behave and perhaps to evolve differently.     

Potentials and limitations of quantification of fungi in freshwater environments based on PLFA profiles

Aquatic fungi are increasingly recognized for their contribution to carbon cycling in aquatic ecosystems, both as saprotrophs and parasites. Their quantification in mixed communities is crucial to assess their ecological significance but remains challenging. We characterized the phospholipid-derived fatty acid (PLFA) composition of fifteen aquatic fungal isolates from Chytridiomycota (chytrids) and Dikarya. Additionally, we identified PLFA patterns of chytrids infecting phytoplankton and their zoospores. PLFA composition of zoospores was highly similar among different taxa, but were distinct from their respective sporangial life-stage. Finally, we applied a fatty acid-based Bayesian mixed model (FASTAR) and tested its potential to quantify fungi in complex mixtures with bacteria and phytoplankton using PLFA profiles. While the quantification of chytrid biomass in low quantities was rather imprecise, the model predicted the contribution of filamentous fungi and other components with fair accuracy, supporting the suitability of this approach to quantify fungal biomass in aquatic environments.     

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.     

Parasitic chytrids: their effects on phytoplankton communities and food-web dynamics

Many phytoplankton species are susceptible to fungal parasitism. Parasitic fungi of phytoplankton mainly belong to the Chytridiomycetes (chytrids). Here, we discuss the progression made in the study of chytrids that parasitize phytoplankton species. Specific fluorescent stains aid in the identification of chytrids in the field. The established culturing methods and the advances in molecular science offer good potential to gain a better insight into the mechanisms of epidemic development of chytrids and coevolution between chytrids and their algal hosts. Chytrids are often considered to be highly host-specific parasites, but the extent of host specificity has not been fully investigated. Chytrids may prefer larger host cells, since they would gain more resources, but whether hosts are really selected on the basis of size is not clear. The dynamics of chytrids epidemics in a number of studies were partly explained by environmental factors such as light, temperature, nutrients, pH, turbulence and zooplankton grazing. No generalization was made about the epidemic conditions; some state unfavorable conditions for the host growth support epidemic development, while others report epidemics even under optimal growth conditions for the host. Phytoplankton is not defenseless, and several mechanisms have been suggested, such as a hypersensitivity response, chemical defense, maintaining a high genetic diversity and multitrophic indirect defenses. Chytrids may also play an important role in food webs, because zoospores of chytrids have been found to be a good food source for zooplankton.