The Effect of Light and Darkness Upon Infection of Asterionella formosa Hassall by the Chytrid Rhizophydium planktonicum Canter emend

Observations, both experimental and microscopic, indicate thatzoospores of Rhizophydium planktonicum Canter emend., can remainalive, but rarely become adherent upon cells of Asterionellaformosa Hassall under conditions of very low light or in completedarkness. The behaviour patterns of zoospores towards host cells underdarkened conditions were compared with those which took placeunder well illuminated conditions leading to the settlementof zoospores on host cells. The differences noted may help toexplain the lack of zoospores found upon inadequately illuminateddiatom cells. Some evidence suggests that young zoospores lack the abilityto adhere to host cells. After the encystment of zoospores uponAsterionella cells in the light, their further growth and developmentcan continue in darkness. Rhizophydium planktonicum Canter emend., Asterionella formosa Hassall, chytrid, diatom, infra-red illumination, zoospore     

Effects of phosphorus limitation on the epidemiology of a chytrid phytoplankton parasite

SUMMARY 1. The effect of phosphorus limitation of the diatom Asterionella formosa Hass. on growth, survival and epidemic development of its fungal parasite Rhizophydium planktonicum Canter emend. was estimated, using measurements of production and infectivity of the zoospores of the chytrid grown on host cultures with different phosphorus-limited growth rates.
2. Phosphorus-limited host cells were less susceptible to infection with zoospores of the parasite than non-limited host cells.
3. The sporangia on phosphorus-limited algae produced substantially less zoospores, but the development time of these sporangia was only slightly reduced.
4. As a result of these effects, Rhizophydium will reach lower growth rates at a given host density, and survival of the parasite will require higher host densities when Asterionella is phosphorus-limited.
5. The zoospore production remained high enough to enable the parasite to grow faster than the alga at sufficiently high host densities, both at limiting and non-limiting phosphorus levels.
6. In spite of the reduced growth rate of the parasite, phosphorus limitation of Asterionella was found to facilitate the development of a Rhizophydium epidemic. This was a consequence of the reduced algal growth rate at phosphorus limitation, which makes the host population more easily outgrown by the parasite.
7. Phosphorus limitation of the host could reduce the threshold host density required for the development of an epidemic by a factor of 2.5.     

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.     

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 Isolation, Maintenance and Host Range Studies of a Chytrid Rhizophydium planktonicum Canter emend., Parasitic on Asterionella formosa Hassall

The methods of isolation and maintenance in dual clone cultureof the chytrid Rhizophydium planktonicum Canter emend, parasiticon Asterionella formosa Hass. from the plankton are described.The ability of a single fungal isolate to infect other clonesof Asterionella, Fragilaria, Tabellaria, Synedra and Cyclotella,as well as dead material was also tested. All the clones of Asterionella proved to be highly susceptibleto infection whereas only in rare instances could a sporangiumbe found which had grown and dehisced on Fragilaria and Synedra.Very few zoospores encysted on Tabellaria and Cyclotella andthose which did died without further growth. There was no evidenceto suggest that the chytrid could complete its life historyon dead material. freshwater phytoplankton, culture, parasitism, host–parasite relationships, chytridiomycetes, Rhizophydium, Zygorhizidium, diatoms, Asterionella, Fragilaria     

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.     

Parasitic chytrids could promote copepod survival by mediating material transfer from inedible diatoms

Diatoms form large spring blooms in lakes and oceans, providing fuel for higher trophic levels at the start of the growing season. Some of the diatom blooms, however, are not grazed by filter-feeding zooplankton like Daphnia due to their large size. Several of these large diatoms are susceptible to chytrid infections. Zoospores of chytrids appeared to be excellent food for Daphnia, both in terms of size, shape, and quality (PUFAs and cholesterol). Thus, zoospores of chytrids can bridge the gap between inedible diatoms and Daphnia. In order to examine the effects of diatoms and chytrids on the survival of copepods, we performed one grazing and one survival experiment. The grazing experiment revealed that the diatom, Asterionella formosa, was not grazed by the copepod, Eudiaptomus gracilis, even after being infected by the chytrid Zygorhizidium planktonicum. However, carbon and nitrogen concentrations were significantly reduced by E. gracilis only when A. formosa was infected by Z. planktonicum, indicating that the chytrids might facilitate material transfer from inedible diatoms to the copepods. The survival experiment revealed that E. gracilis lived shorter with A. formosa than with the cryptophyta Cryptomonas pyrenoidifera. However, the survival of E. gracilis increased significantly in the treatment where A. formosa cells were infected by Z. planktonicum. Since E. gracilis could not graze A. formosa cells due to their large colonial forms, E. gracilis may acquire nutrients by grazing on the zoospores, and were so able to survive in the presence of the A. formosa. This provides new insights into the role of parasitic fungi in aquatic food webs, where chytrids may improve copepod survival during diatom blooms.     

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.     

GENETIC VARIATION IN ASTERIONELLA FORMOSA (BACILLARIOPHYCEAE): IS IT LINKED TO FREQUENT EPIDEMICS OF HOST‐SPECIFIC PARASITIC FUNGI?1

Understanding of the genetic basis for susceptibility and resistance is still lacking for most aquatic host–parasite systems, for instance, for phytoplankton and their fungal parasites. Fungal parasites can have significant effects on phytoplankton populations, mainly through their ability to decimate algal host populations during epidemics. We used random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analysis to study levels of genetic variation within a population of the freshwater diatom Asterionella formosa Hassall in relation to parasitism by the obligate, host‐specific, fungal parasite Zygorhizidium planktonicum Canter. The level of genetic variation within the A. formosa population in Lake Maarsseveen, The Netherlands was found to be high despite the presumed absence or very low frequency of sexual reproduction in this species, the limited gene flow, and the severity of parasite attack that would purge the population from susceptible genotypes. RAPD analysis revealed four distinct banding patterns, with 3 of 21 markers (14%) being polymorphic. In AFLP analysis, every single isolate of A. formosa showed a unique banding pattern, and 120 of the 210 AFLP markers (57%) were found to be polymorphic. Furthermore, character compatibility analysis revealed that sexual reproduction may be one of the mechanisms that generates and maintains genetic variation in the A. formosa population in Lake Maarsseveen. The presence of genetic variation in A. formosa was reflected in infection experiments, which showed that genetically different A. formosa strains differed in their susceptibility to various Z. planktonicum strains and that parasite strains differed in their ability to infect particular host strains.     

The influence of phosphorus limitation of the diatomAsterionella Formosa on the zoospore production of its fungal parasiteRhizophydium Planktonicum

The influence of phosphorus limitedAsterionella on the zoospore production of its fungal parasiteRhizophydium planktonicum was measured, using laboratory cultures of host and parasite. At saturated phosphorus concentrations the host reached a specific growth rate of 0.95.d^–1. Growing on these host cells, the mean parasite zoospore production was 26 spores per sporangium, and the mean development time of a sporangium was 45 hours. Growing on phosphorus limited hosts, the zoospore production decreased to less then 9 spores per sporangium, and the development time decreased to 40 hours. On phosphorus limited hosts, zoospores were produced at a slower rate. The algal growth rate was reduced to a greater extent than the fungal growth rate. Therefore, it could be concluded that phosphorus limitation ofAsterionella will facilitate the development of an epidemic of its parasiteRhizophidium, at least at high diatom densities, when possible differences in infectability of the algae play a minor role.     

Infection of the diatom Asterionella by a chytrid. I. Effects of light on reproduction and infectivity of the parasite

The influence of hight limitation of the diatom Asterionellaformosa Hass, on the growth-determining parameters of its fungalparasiteRhizophydium planktonicum Canter emend, was measured,using laboratory cultures of both organisms. The experimentswere earned out at 6°C under a 15:9 h light-dark cycle.At saturating light conditions, the mean zoospore productionof the parasite was 23.4 zoospores sporangium^–1, and themean development time of the sporangia was 7 9 days. Light limitationof the host caused a substantial decrease of the zoospore production,while the development time was only slightly reduced. The improvedzoospore production at high light intensities was mainly theresult of incorporation of photosynthetic products generatedby the host after infection. Under limiting light conditions,Asterionella cells were less susceptible to infection withfungal zoospores. No infection at all occurred below 2 µEm^–2 s^–1, a light intensity that still supportedsome algal growth The maximum infection rate indicated thatchemotactic attraction of the parasite's zoospores by extracellularproducts of the host is involved. The infective lifetime ofthe zoospores of the parasite did not depend on light conditions,and was estimated at 8 days. The measured zoospore productionrates, both under limiting and saturating light conditions,enable the parasite to exceed the specific growth rate of thehost, and thus become epidemic, at sufficiently high host densities.     

Infection of the diatom Asterionella by a chytrid. II. Effects of light on survival and epidemic development of the parasite

A model is formulated to investigate the ability of chytridparasites to survive or become epidemic within populations oftheir algal hosts The model is used for an analysis of the effectsof light on the occurrence of Rhizophydium planktonicum Canteremend., a chytrid parasite of the freshwater diatom Asterionellaformosa Hass., using the information on the growth parametersof host and parasite presented in the first part of this article(J. Plankton Res ., 13, 103–117). According to the model,conditions for survival of the parasite are optimal when thehost grows at saturating light conditions. Under limiting lightconditions, Rhizophydium needs higher host densities in orderto maintain itself. The parasite is not able to survive prolongedperiods of severe light limitation of the host Epidemic development,however, turned out to be facilitated by a moderate light limitationof the host. Both light saturation and severe light limitationhamper epidemic development, but in the first case, epidemicdevelopment is still possible at sufficiently high host densities.     

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.     

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.     

Temperature Alters Host Genotype-Specific Susceptibility to Chytrid Infection

The cost of parasitism often depends on environmental conditions and host identity. Therefore, variation in the biotic and abiotic environment can have repercussions on both, species-level host-parasite interaction patterns but also on host genotype-specific susceptibility to disease. We exposed seven genetically different but concurrent strains of the diatom Asterionella formosa to one genotype of its naturally co-occurring chytrid parasite Zygorhizidium planktonicum across five environmentally relevant temperatures. We found that the thermal tolerance range of the tested parasite genotype was narrower than that of its host, providing the host with a “cold” and “hot” thermal refuge of very low or no infection. Susceptibility to disease was host genotype-specific and varied with temperature level so that no genotype was most or least resistant across all temperatures. This suggests a role of thermal variation in the maintenance of diversity in disease related traits in this phytoplankton host. The duration and intensity of chytrid parasite pressure on host populations is likely to be affected by the projected changes in temperature patterns due to climate warming both through altering temperature dependent disease susceptibility of the host and, potentially, through en- or disabling thermal host refugia. This, in turn may affect the selective strength of the parasite on the genetic architecture of the host population.     

Isolation and characterization of a novel chytrid species (phylum Blastocladiomycota), parasitic on the green alga Haematococcus

A parasite was found in cultures of the green microalga Haematococcus pluvialis that grew epibiotically on algal cells and caused epidemics resulting in damage to the host cultures. The parasite was isolated into axenic culture on solid and liquid media. It was demonstrated to be the sole causative agent of the epidemics. According to its life cycle and phylogenetic analysis based on 18S ribosomal DNA sequences, the pathogen appears to represent a novel chytrid fungus closely related to the vascular plant pathogen Physoderma (Blastocladiomycota), although it differs from all other known chytrids by its infective stage, a wall-less propagule endowed with amoeboid motion and lacking the group's typical flagellum.     

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.     

Parasite Fitness Traits Under Environmental Variation: Disentangling the Roles of a Chytrid’s Immediate Host and External Environment

Parasite environments are heterogeneous at different levels. The first level of variability is the host itself. The second level represents the external environment for the hosts, to which parasites may be exposed during part of their life cycle. Both levels are expected to affect parasite fitness traits. We disentangle the main and interaction effects of variation in the immediate host environment, here the diatom Asterionella formosa (variables host cell volume and host condition through herbicide pre-exposure) and variation in the external environment (variables host density and acute herbicide exposure) on three fitness traits (infection success, development time and reproductive output) of a chytrid parasite. Herbicide exposure only decreased infection success in a low host density environment. This result reinforces the hypothesis that chytrid zoospores use photosynthesis-dependent chemical cues to locate its host. At high host densities, chemotaxis becomes less relevant due to increasing chance contact rates between host and parasite, thereby following the mass-action principle in epidemiology. Theoretical support for this finding is provided by an agent-based simulation model. The immediate host environment (cell volume) substantially affected parasite reproductive output and also interacted with the external herbicide exposed environment. On the contrary, changes in the immediate host environment through herbicide pre-exposure did not increase infection success, though it had subtle effects on zoospore development time and reproductive output. This study shows that both immediate host and external environment as well as their interaction have significant effects on parasite fitness. Disentangling these effects improves our understanding of the processes underlying parasite spread and disease dynamics.