Characterizing the "fungal shunt": Parasitic fungi on diatoms affect carbon flow and bacterial communities in aquatic microbial food webs.

Microbial interactions in aquatic environments profoundly affect global biogeochemical cycles, but the role of microparasites has been largely overlooked. Using a model pathosystem, we studied hitherto cryptic interactions between microparasitic fungi (chytrid Rhizophydiales), their diatom host Asterionella, and cell-associated and free-living bacteria. We analyzed the effect of fungal infections on microbial abundances, bacterial taxonomy, cell-to-cell carbon transfer, and cell-specific nitrate-based growth using microscopy (e.g., fluorescence in situ hybridization), 16S rRNA gene amplicon sequencing, and secondary ion mass spectrometry. Bacterial abundances were 2 to 4 times higher on individual fungal-infected diatoms compared to healthy diatoms, particularly involving Burkholderiales. Furthermore, taxonomic compositions of both diatom-associated and free-living bacteria were significantly different between noninfected and fungal-infected cocultures. The fungal microparasite, including diatom-associated sporangia and free-swimming zoospores, derived ∼100% of their carbon content from the diatom. By comparison, transfer efficiencies of photosynthetic carbon were lower to diatom-associated bacteria (67 to 98%), with a high cell-to-cell variability, and even lower to free-living bacteria (32%). Likewise, nitrate-based growth for the diatom and fungi was synchronized and faster than for diatom-associated and free-living bacteria. In a natural lacustrine system, where infection prevalence reached 54%, we calculated that 20% of the total diatom-derived photosynthetic carbon was shunted to the parasitic fungi, which can be grazed by zooplankton, thereby accelerating carbon transfer to higher trophic levels and bypassing the microbial loop. The herein termed "fungal shunt" can thus significantly modify the fate of photosynthetic carbon and the nature of phytoplankton-bacteria interactions, with implications for diverse pelagic food webs and global biogeochemical cycles.

A Widely Distributed Thraustochytrid Parasite of Diatoms Isolated from the Arctic Represents a gen. and sp. nov.

A unicellular, heterotrophic, eukaryotic parasite was isolated from nearshore Arctic marine sediment in association with the diatom Pleurosigma sp. The parasite possessed ectoplasmic threads that could penetrate diatom frustules. Healthy and reproducing Pleurosigma cultures would begin to collapse within a week following the introduction of this parasite. The parasite (2-10 µm diameter) could reproduce epibiotically with biflagellate zoospores, as well as binary division inside and outside the diatom host. While the parasite grew, diatom intracellular content disappeared. Evaluation of electron micrographs from co-cultures revealed the presence of hollow tubular processes and amorphic cells that could transcend the diatom frustule, generally at the girdle band, as well as typical thraustochytrid ultrastructure, such as the presence of bothrosomes. After nucleotide extraction, amplification, and cloning, database queries of DNA revealed closest molecular affinity to environmental thraustochytrid clone sequences. Testing of phylogenetic hypotheses consistently grouped this unknown parasite within the Thraustochytriidae on a distinct branch within the environmental sequence clade Lab19. Reclassification of Arctic high-throughput sequencing data, with appended reference datasets that included this diatom parasite, indicated that the majority of thraustochytrid sequences, previously binned as unclassifiable stramenopiles, are allied to this new isolate. Based on the combined information acquired from electron microscopy, life history, and phylogenetic testing, this unknown isolate is described as a novel species and genus.

The oomycete Lagenisma coscinodisci hijacks host alkaloid synthesis during infection of a marine diatom.

Flagellated oomycetes frequently infect unicellular algae, thus limiting their proliferation. Here we show that the marine oomycete Lagenisma coscinodisci rewires the metabolome of the bloom-forming diatom Coscinodiscus granii, thereby promoting infection success. The algal alkaloids ß-carboline and 4-carboxy-2,3,4,9-tetrahydro-1H-ß-carboline are induced during infection. Single-cell profiling with AP-MALDI-MS and confocal laser scanning microscopy reveals that algal carbolines accumulate in the reproductive form of the parasite. The compounds arrest the algal cell division, increase the infection rate and induce plasmolysis in the host. Our results indicate that the oomycete manipulates the host metabolome to support its own multiplication.

Intracellular Infection of Diverse Diatoms by an Evolutionary Distinct Relative of the Fungi.

The Fungi are a diverse kingdom, dominating terrestrial environments and driving important ecologies. Although fungi, and the related Opisthosporidia, interact with photosynthetic organisms on land and in freshwater as parasites, symbionts, and/or saprotrophic degraders [1, 2], such interactions in the marine environment are poorly understood [3-8]. One newly identified uncultured marine lineage has been named novel chytrid-like-clade-1 (NCLC1) [4] or basal-clone-group-I [5, 6]. We use ribosomal RNA (rRNA) encoding gene phylogenies to demonstrate that NCLC1 is a distinct branch within the Opisthosporidia (Holomycota) [7]. Opisthosporidia are a diverse and largely uncultured group that form a sister branch to the Fungi or, alternatively, the deepest branch within the Fungi, depending on how the boundary to this kingdom is inferred [9]. Using culture-free lineage-specific rRNA-targeted fluorescent in situ hybridization (FISH) microscopy, we demonstrate that NCLC1 cells form intracellular infection of key diatom species, establishing that intracellular colonization of a eukaryotic host is a consistent lifestyle across the Opisthosporidia [8-11]. NCLC1 infection-associated loss and/or envelopment of the diatom nuclei infers a necrotrophic-pathogenic interaction. Diatoms are one of the most diverse and ecologically important phytoplankton groups, acting as dominant primary producers and driving carbon fixation and storage in many aquatic environments [12-14]. Our results provide insight into the diversity of microbial eukaryotes that interact with diatoms. We suggest that such interactions can play a key role in diatom associated ecosystem functions, such as the marine carbon pump through necrotrophic-parasitism, facilitating the export of diatoms to the sediment [15, 16].

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.

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.

Mapping glycoconjugate-mediated interactions of marine Bacteroidetes with diatoms.

The degradation of diatoms is mainly catalyzed by Bacteroidetes and this process is of global relevance for the carbon cycle. In this study, a combination of catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) and fluorescent lectin binding analysis (FLBA) was used to identify and map glycoconjugates involved in the specific interactions of Bacteroidetes and diatoms, as well as detritus, at the coastal marine site Helgoland Roads (German Bight, North Sea). The study probed both the presence of lectin-specific extracellular polymeric substances (EPS) of Bacteroidetes for cell attachment and that of glycoconjugates on diatoms with respect to binding sites for Bacteroidetes. Members of the clades Polaribacter and Ulvibacter were shown to form microcolonies within aggregates for which FLBA indicated the presence of galactose containing slime. Polaribacter spp. was shown to bind specifically to the setae of the abundant diatom Chaetoceros spp., and the setae were stained with fucose-specific lectins. In contrast, Ulvibacter spp. attached to diatoms of the genus Asterionella which bound, among others, the mannose-specific lectin PSA. The newly developed CARD-FISH/FLBA protocol was limited to the glycoconjugates that persisted after the initial CARD-FISH procedure. The differential attachment of bacteroidetal clades to diatoms and their discrete staining by FLBA provided evidence for the essential role that formation and recognition of glycoconjugates play in the interaction of bacteria with phytoplankton.

Chytrid epidemics may increase genetic diversity of a diatom spring-bloom.

Contrary to expectation, populations of clonal organisms are often genetically highly diverse. In phytoplankton, this diversity is maintained throughout periods of high population growth (that is, blooms), even though competitive exclusion among genotypes should hypothetically lead to the dominance of a few superior genotypes. Genotype-specific parasitism may be one mechanism that helps maintain such high-genotypic diversity of clonal organisms. Here, we present a comparison of population genetic similarity by estimating the beta-dispersion among genotypes of early and peak bloom populations of the diatom Asterionella formosa for three spring-blooms under high or low parasite pressure. The Asterionella population showed greater beta-dispersion at peak bloom than early bloom in the 2 years with high parasite pressure, whereas the within group dispersion did not change under low parasite pressure. Our findings support that high prevalence parasitism can promote genetic diversification of natural populations of clonal hosts.

Herbicides in the environment alter infection dynamics in a microbial host-parasite system.

Parasites play an important role in the regulation of host population growth. How these ubiquitous stressors interact with anthropogenic stressors is less often studied. In a full factorial experiment we explored the independent and combined effects of the widely used herbicide diuron and a chytrid parasite on the fitness of genetically different monoclonal diatom populations. Furthermore, we evaluated how herbicide exposure influenced infection dynamics, parasite fitness and the impact of infectious disease on host populations. We found no evidence of host genetic variation for diuron sensitivity and parasite resistance. Instead, host population phenotype was a decisive factor in controlling parasite growth. Although herbicide exposure initially posed a constraint on disease transmission, it enhanced the spread of disease over time. Consequently, the nature of the parasite-toxicant stressor interaction shifted from antagonistic (on exponential host growth) towards additive (on final uninfected host density). We conclude that herbicide exposure can modify infection dynamics and impact of disease on host populations through the complex interplay between host and parasite growth dynamics and host population phenotype.

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.