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.

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].

Fitness and eco-physiological response of a chytrid fungal parasite infecting planktonic cyanobacteria to thermal and host genotype variation.

Understanding how individual parasite traits contribute to overall fitness, and how they are modulated by both external and host environment, is crucial for predicting disease outcome. Fungal (chytrid) parasites of phytoplankton are important yet poorly studied pathogens with the potential to modulate the abundance and composition of phytoplankton communities and to drive their evolution. Here, we studied life-history traits of a chytrid parasite infecting the planktonic, bloom-forming cyanobacterium Planktothrix spp. under host genotype and thermal variation. When expressing parasite fitness in terms of transmission success, disease outcome was largely modulated by temperature alone. Yet, a closer examination of individual parasite traits linked to different infection phases, such as (i) the establishment of the infection (i.e. intensity of infection) and (ii) the exploitation of host resources (i.e. size of reproductive structures and propagules), revealed differential host genotype and temperature × host genotype modulation, respectively. This illustrates how parasite fitness results from the interplay of individual parasite traits that are differentially controlled by host and external environment, and stresses the importance of combining multiple traits to gain insights into underlying infection mechanisms.

Differences in planktonic microbial communities associated with three types of macrophyte stands in a shallow lake.

Little is known about how various substances from living and decomposing aquatic macrophytes affect the horizontal patterns of planktonic bacterial communities. Study sites were located within Lake Kolon, which is a freshwater marsh and can be characterised by open-water sites and small ponds with different macrovegetation (Phragmites australis, Nymphea alba and Utricularia vulgaris). Our aim was to reveal the impact of these macrophytes on the composition of the planktonic microbial communities using comparative analysis of environmental parameters, microscopy and pyrosequencing data. Bacterial 16S rRNA gene sequences were dominated by members of phyla Proteobacteria (36%-72%), Bacteroidetes (12%-33%) and Actinobacteria (5%-26%), but in the anoxic sample the ratio of Chlorobi (54%) was also remarkable. In the phytoplankton community, Cryptomonas sp., Dinobryon divergens, Euglena acus and chrysoflagellates had the highest proportion. Despite the similarities in most of the measured environmental parameters, the inner ponds had different bacterial and algal communities, suggesting that the presence and quality of macrophytes directly and indirectly controlled the composition of microbial plankton.

Growth, Grazing, and Starvation Survival in Three Heterotrophic Dinoflagellate Species.

To assess the effects of fluctuating prey availability on predator population dynamics and grazing impact on phytoplankton, we measured growth and grazing rates of three heterotrophic dinoflagellate species-Oxyrrhis marina, Gyrodinium dominans and Gyrodinium spirale-before and after depriving them of phytoplankton prey. All three dinoflagellate species survived long periods (> 10 d) without algal prey, coincident with decreases in predator abundance and cell size. After 1-3 wks, starvation led to a 17-57% decrease in predator cell volume and some cells became deformed and transparent. When re-exposed to phytoplankton prey, heterotrophs ingested prey within minutes and increased cell volumes by 4-17%. At an equivalent prey concentration, continuously fed predators had ~2-fold higher specific growth rates (0.18 to 0.55 d-1 ) than after starvation (-0.16 to 0.25 d-1 ). Maximum specific predator growth rates would be achievable only after a time lag of at least 3 d. A delay in predator growth poststarvation delays predator-induced phytoplankton mortality when prey re-emerges at the onset of a bloom event or in patchy prey distributions. These altered predator-prey population dynamics have implications for the formation of phytoplankton blooms, trophic transfer rates, and potential export of carbon.

In situ associations between marine photosynthetic picoeukaryotes and potential parasites - a role for fungi?

Photosynthetic picoeukaryotes (PPEs) are important components of the marine picophytoplankton community playing a critical role in CO2 fixation but also as bacterivores, particularly in the oligotrophic gyres. Despite an increased interest in these organisms and an improved understanding of the genetic diversity of this group, we still know little of the environmental factors controlling the abundance of these organisms. Here, we investigated the quantitative importance of eukaryotic parasites in the free-living fraction as well as in associations with PPEs along a transect in the South Atlantic. Using tyramide signal amplification-fluorescence in situ hybridization (TSA-FISH), we provide quantitative evidence of the occurrence of free-living fungi in open ocean marine systems, while the Perkinsozoa and Syndiniales parasites were not abundant in these waters. Using flow cytometric cell sorting of different PPE populations followed by a dual-labelled TSA-FISH approach, we also demonstrate fungal associations, potentially parasitic, occurring with both pico-Prymnesiophyceae and pico-Chrysophyceae. These data highlight the necessity for further work investigating the specific role of marine fungi as parasites of phytoplankton to improve understanding of carbon flow in marine ecosystems.

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.

Seasonal variations of marine protist community structure based on taxon-specific traits using the eastern English Channel as a model coastal system.

Previous microscopy-based studies in the eastern English Channel have revealed it to be a productive meso-eutrophic coastal ecosystem, characterized by strong repeating patterns in microplankton succession. The present study examines the seasonal structure of the entire protistan community from March 2011 to July 2013, using tag pyrosequencing of the V2-V3 hypervariable region of the 18S rRNA gene. A total of 1242 OTUs and 28 high-level taxonomic groups, which included previously undetected taxa in the area, were identified. The detected OTUs were considered according to taxon-specific traits, which included their trophic role, abundance and specialization level. Taxa differentiation based on specialization level rather than abundance was more informative in describing community organization. While generalists were always abundant, numerous specialists that were either rare or absent in most samples, increased in abundance for short periods, appearing to be overall abundant. Statistical and network analyses showed that the protistan seasonal organization was influenced by environmental parameters. It also highlighted that in addition to grazers, fungi and parasites played potentially significant roles during phytoplankton blooms. Overall, while the protistan succession was mainly shaped by environmental variations, biotic interactions among co-occurring taxa were the main structural drivers of the temporal assemblages.

Unexpected importance of potential parasites in the composition of the freshwater small-eukaryote community.

The diversity of small eukaryotes (0.2 to 5 mum) in a mesotrophic lake (Lake Bourget) was investigated using 18S rRNA gene library construction and fluorescent in situ hybridization coupled with tyramide signal amplification (TSA-FISH). Samples collected from the epilimnion on two dates were used to extend a data set previously obtained using similar approaches for lakes with a range of trophic types. A high level of diversity was recorded for this system with intermediate trophic status, and the main sequences from Lake Bourget were affiliated with ciliates (maximum, 19% of the operational taxonomic units [OTUs]), cryptophytes (33%), stramenopiles (13.2%), and cercozoa (9%). Although the comparison of TSA-FISH results and clone libraries suggested that the level of Chlorophyceae may have been underestimated using PCR with 18S rRNA primers, heterotrophic organisms dominated the small-eukaryote assemblage. We found that a large fraction of the sequences belonged to potential parasites of freshwater phytoplankton, including sequences affiliated with fungi and Perkinsozoa. On average, these sequences represented 30% of the OTUs (40% of the clones) obtained for each of two dates for Lake Bourget. Our results provide information on lacustrine small-eukaryote diversity and structure, adding to the phylogenetic data available for lakes with various trophic types.

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.