A Double Staining Method Using SYTOX Green and Calcofluor White for Studying Fungal Parasites of Phytoplankton

We propose a double staining method based on the combination of two fluorochromes, calcofluor white (CFW; specific chitinous fluorochrome) and SYTOX green (nucleic acid stain), coupled to epifluorescence microscopy for counting, identifying, and investigating the fecundity of parasitic fungi of phytoplankton and the putative relationships established between hosts and their chytrid parasites. The method was applied to freshwater samples collected over two successive years during the terminal period of autumnal cyanobacterial blooms in a eutrophic lake. The study focused on the uncultured host-parasite couple Anabaena macrospora (cyanobacterium) and Rhizosiphon akinetum (Chytridiomycota). Our results showed that up to 36.6% of cyanobacterial akinetes could be parasitized by fungi. Simultaneously, we directly investigated the zoosporic content inside the sporangia and found that both the host size and intensity of infection conditioned the final size and hence fecundity of the chytrids. We found that relationships linking host size, final parasite size, and chytrid fecundity were conserved from year to year and seemed to be host-chytrid couple specific. We concluded that our double staining method was a valid procedure for improving our knowledge of uncultured freshwater phytoplankton-chytrid couples and so of the quantitative ecology of chytrids in freshwater ecosystems.     

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     

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.     

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.     

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.     

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.     

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.     

Parasites and phytoplankton, with special emphasis on dinoflagellate infections

Planktonic members of most algal groups are known to harbor intracellular symbionts, including viruses, bacteria, fungi, and protozoa. Among the dinoflagellates, viral and bacterial associations were recognized a quarter century ago, yet their impact on host populations remains largely unresolved. By contrast, fungal and protozoan infections of dinoflagellates are well documented and generally viewed as playing major roles in host population dynamics. Our understanding of fungal parasites is largely based on studies for freshwater diatoms and dinoflagellates, although fungal infections are known for some marine phytoplankton. In freshwater systems, fungal chytrids have been linked to mass mortalities of host organisms, suppression or retardation of phytoplankton blooms, and selective effects on species composition leading to successional changes in plankton communities. Parasitic dinoflagellates of the genus Amoebophrya and the newly described Perkinsozoa, Parvilucifera infectans, are widely distributed in coastal waters of the world where they commonly infect photosynthetic and heterotrophic dinoflagellates. Recent work indicates that these parasites can have significant impacts on host physiology, behavior, and bloom dynamics. Thus, parasitism needs to be carefully considered in developing concepts about plankton dynamics and the flow of material in marine food webs.     

HOST PARASITE INTERACTIONS BETWEEN FRESHWATER PHYTOPLANKTON AND CHYTRID FUNGI (CHYTRIDIOMYCOTA)1

Some chytrids are host‐specific parasiticfungithat may have a considerable impact on phytoplankton dynamics. The phylum Chytridiomycota contains one class, the Chytridiomycetes, and is composed of five different orders. Molecular studies now firmly place the Chytridiomycota within the fungal kingdom. Chytrids are characterized by having zoospores, a motile stage in their life cycle. Zoospores are attracted to the host cell by specific signals. No single physical–chemical factor has been found that fully explains the dynamics of chytrid epidemics in the field. Fungal periodicity was primarily related to host cell density. The absence of aggregated distributions of chytrids on their hosts suggested that their hosts did not vary in their susceptibility to infection. A parasite can only become epidemic when it grows faster than the host. Therefore, it has been suggested that epidemics in phytoplankton populations arise when growth conditions for the host are unfavorable. No support for such a generalization was found, however. Growth of the parasitic fungus Rhizophydium planktonicum Canter emend, parasitic on the diatom Asterionella formosa Hassal, was reduced under stringent nutrient limitation,because production and infectivity of zoospores were affected negatively. A moderate phosphorous or light limitation favored epidemic development, however. Chytrid infections have been shown to affect competition between their algal hosts and in this way altered phytoplankton succession. There is potential for coevolution between Asterionella and the chytrid Zygorhizidium planktonicum Canter based on clear reciprocal fitness costs, absence of overall infective parasite strains, and possibly a genetic basis for host susceptibility and parasite infectivity.     

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.     

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.     

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.     

Ecological impacts of parasitic chytrids,syndiniales and perkinsids on populations of marine photosynthetic dinoflagellates

Parasitism is a widespread interaction that plays significant roles in ecosystem balance and evolution. Historically the biology of zoosporic parasites is often a neglected field when studying aquatic ecological dynamics, especially in marine ecosystems. In the marine environment, dinoflagellates represent a significantly large component of primary production, and may be infected by a variety of fungal and fungus-like parasites including chytrids, syndiniales, and perkinsids as well as other microorganisms. The relationship between these organisms and their dinoflagellate hosts constitutes a highly significant pathosystem given the increasing importance of aquaculture. Because of their small size and lack of morphological characteristics these organisms are difficult to identify. This review compares the taxonomy, life cycle, host range, infection strategies, and ecological roles of species of Parvilucifera, Amoebophrya and Dinomyces which are parasites of dinoflagellates. Most of these species have only been described recently. Implications for environmental management are discussed.     

Effects of eukaryotic pathogens (Chytridiomycota and Oomycota) on marine benthic diatom communities in the Solthörn tidal flat (southern North Sea,Germany)

The presence of eukaryotic parasites (i.e. chytridiomycetes and oomycetes) infecting benthic marine diatoms was revealed by a reconnaissance survey in the Solthörn tidal flat (southern North Sea, Germany) followed by 5 months of regular monitoring in order to assess the impact of these zoosporic fungal pathogens on microphytobenthic diatom communities. Additionally, variation of environmental factors such as sediment composition and nutrient concentrations were monitored. Pre-treatment of sediment samples using short ultrasound pulses and gradient centrifugation, in combination with CalcoFluor White, were used for the visualization of both pathogen groups. The highest prevalence of infected benthic diatoms was observed in late September (6.3% of the total benthic diatom community), correlating with the highest abundance of benthic diatoms recorded during the survey (6.5 ± 1.3 × 10⁴ cells cm^–2). Most infections were caused by chytrids (up to 99.8%) and, only in a few cases, by oomycetes. The analysis of individual host abundances/infection prevalence showed in most cases a decline in cell numbers of the host species with increasing numbers of the eukaryotic parasite. Several shifts in the diatom community composition were observed. Statistical analysis revealed that the abundance of the benthic diatom hosts and their parasites was related to seasonal variation in temperature, irradiance and nutrient availability, particularly of dissolved inorganic nitrogen.     

Warming accelerates termination of a phytoplankton spring bloom by fungal parasites

Climate change is expected to favour infectious diseases across ecosystems worldwide. In freshwater and marine environments, parasites play a crucial role in controlling plankton population dynamics. Infection of phytoplankton populations will cause a transfer of carbon and nutrients into parasites, which may change the type of food available for higher trophic levels. Some phytoplankton species are inedible to zooplankton, and the termination of their population by parasites may liberate otherwise unavailable carbon and nutrients. Phytoplankton spring blooms often consist of large diatoms inedible for zooplankton, but the zoospores of their fungal parasites may serve as a food source for this higher trophic level. Here, we investigated the impact of warming on the fungal infection of a natural phytoplankton spring bloom and followed the response of a zooplankton community. Experiments were performed in ca. 1000 L indoor mesocosms exposed to a controlled seasonal temperature cycle and a warm (+4 °C) treatment in the period from March to June 2014. The spring bloom was dominated by the diatom Synedra. At the peak of infection over 40% of the Synedra population was infected by a fungal parasite (i.e. a chytrid) in both treatments. Warming did not affect the onset of the Synedra bloom, but accelerated its termination. Peak population density of Synedra tended to be lower in the warm treatments. Furthermore, Synedra carbon: phosphorus stoichiometry increased during the bloom, particularly in the control treatments. This indicates enhanced phosphorus limitation in the control treatments, which may have constrained chytrid development. Timing of the rotifer Keratella advanced in the warm treatments and closely followed chytrid infections. The chytrids' zoospores may thus have served as an alternative food source to Keratella. Our study thus emphasizes the importance of incorporating not only nutrient limitation and grazing, but also parasitism in understanding the response of plankton communities towards global warming.     

Communities of metazoan parasites in open water fishes of Cold Lake, Alberta

Communities of metazoan parasites in ten species of fishes from Cold Lake, Alberta are described and compared. Relative abundances in the overall community of parasites in the lake were estimated using data on abundance in each host species, plus estimates of the relative abundances of the species of hosts. Parasites of the numerically dominant salmonid fishes dominated the overall community, with over half of the individual parasites being Metechinorhynchus salmonis . Exchange of parasites between host species was greatest between related and/or abundant host species. Parasite communities in cisco and whitefish, Coregonus spp., were relatively rich in species and diverse (Simpson's index) compared with communities in those species in other lakes in North America; communities in lake trout, Salvelinus spp., and the non-salmonid fishes were poor in species and low in diversity compared with communities in other lakes. Parasite species overlaps (Jaccard index) between related host species in Cold Lake were greater than overlaps within host species between lakes. The same pattern is seen in data from some other lakes. These features support the hypothesis of Wisiewski (1958); that the parasite community within an ecosystem is characterized by parasites of the numerically dominant hosts.     

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.     

Seasonal dietary shifts enhance parasite transmission to lake salmonids during ice cover

Changes in abiotic and biotic factors between seasons in subarctic lake systems are often profound, potentially affecting the community structure and population dynamics of parasites over the annual cycle. However, few winter studies exist and interactions between fish hosts and their parasites are typically confined to snapshot studies restricted to the summer season whereas host‐parasite dynamics during the ice‐covered period rarely have been explored. The present study addresses seasonal patterns in the infections of intestinal parasites and their association with the diet of sympatric living Arctic charr (Salvelinus alpinus) and brown trout (Salmo trutta) in Lake Takvatn, a subarctic lake in northern Norway. In total, 354 Arctic charr and 203 brown trout were sampled from the littoral habitat between June 2017 and May 2018. Six trophically transmitted intestinal parasite taxa were identified and quantified, and their seasonal variations were contrasted with dietary information from both stomachs and intestines of the fish. The winter period proved to be an important transmission window for parasites, with increased prevalence and intensity of amphipod‐transmitted parasites in Arctic charr and parasites transmitted through fish prey in brown trout. In Arctic charr, seasonal patterns in parasite infections resulted mainly from temporal changes in diet toward amphipods, whereas host body size and the utilization of fish prey were the main drivers in brown trout. The overall dynamics in the community structure of parasites chiefly mirrored the seasonal dietary shifts of their fish hosts.     

Spatiotemporal patterns of avian host–parasite interactions in the face of biogeographical range expansions

Exploration of interactions between hosts and parasitic symbionts is important for our understanding of the temporal and spatial distribution of organisms. For example, host colonization of new geographical regions may alter levels of infections and parasite specificity, and even allow hosts to escape from co‐evolved parasites, consequently shaping spatial distributions and community structure of both host and parasite. Here we investigate the effect of host colonization of new regions and the elevational distribution of host–parasite associations between birds and their vector‐transmitted haemosporidian blood parasites in two geological and geographical settings: mountains of New Guinea and the Canary Islands. Our results demonstrate that bird communities in younger regions have significantly lower levels of parasitism compared to those of older regions. Furthermore, host–parasite network analyses demonstrate that blood parasites may respond differently after arriving to a new region, through adaptations that allow for either expanding (Canary Islands) or retaining (New Guinea) their host niches. The spatial prevalence patterns along elevational gradients differed in the two regions, suggesting that region‐specific biotic (e.g., host community) and abiotic factors (e.g., temperature) govern prevalence patterns. Our findings suggest that the spatiotemporal range dynamics in host–parasite systems are driven by multiple factors, but that host and parasite community compositions and colonization histories are of particular importance.