Marlène Jobard

Exploring and quantifying fungal diversity in freshwater lake ecosystems using rDNA cloning/sequencing and SSU tag pyrosequencing.

Water samples were collected along transects from the shore to the centre of two French lakes: the deep, volcanic, oligomesotrophic and low allochthonic-impacted Lake Pavin, and the productive and higher allochthonic-impacted Lake Aydat. The biodiversity was analysed using two approaches: the classical approach consisting of cloning/sequencing of the 18S, ITS1, 5.8S, ITS2 and partial 28S region using primers designed for fungus sequences, and the pyrosequencing of 18S rRNA hypervariable V2, V3 and V5 regions using two primer sets (one universal for eukaryotes and one for fungi). The classical approach yielded 146 (Lake Pavin) and 143 (Lake Aydat) sequences, corresponding to 46 and 63 operational taxonomic units (OTUs) respectively. Fungi represented half of the OTUs identified in Lake Pavin and 30% in Lake Aydat, and were dominated by sequences from Chytridiomycota found throughout Lake Pavin but mostly in the central pelagic zone of Lake Aydat. The pyrosequencing approach yielded 42,064 (Pavin) and 61,371 (Aydat) reads, of which 12-15% and 9-19% reads were assigned to fungi in Lakes Pavin and Aydat respectively. Chytridiomycota members were also dominant among these reads, with OTUs displaying up to > 33-fold overrepresentation in the centre compared with the riparian areas of Lake Aydat. Besides fungi, both approaches revealed other major eukaryote groups, with the highest diversity in the central areas of lakes. One of the major findings of our study was that the two lakes displayed contrasting spatial distributions, homogenous for Lake Pavin and heterogeneous for Lake Aydat, which may be related to their peculiarities. This study represents the first unveiling of microbial eukaryote and fungus diversity assessed with two complementary molecular methods, and is considered a major milestone towards understanding the dynamics and ecology of fungi in freshwater lake ecosystems, which are directly link to the abundance and distribution of taxa.

Use of Calcofluor White for Detection, Identification, and Quantification of Phytoplanktonic Fungal Parasites

ABSTRACT We propose a routine protocol based on size fractionation of pelagic samples and the use of the fluorochrome calcofluor white (which binds to β-1,3 and β-1,4 polysaccharides) for diagnosing, identifying, and counting chitinaceous fungal parasites (i.e., the sporangia of chytrids) of phytoplankton. The protocol was applied to freshwater samples collected during different seasons (spring and summer/autumn) in two lakes whose trophic statuses varied. Because few samples were collected (i.e., two dates per site), the findings are considered preliminary and mainly a “proof of concept” rather than a valid comparison of sites versus seasons. The results from the proposed protocol indicate higher diversity of infected host and parasite communities than in previous studies. Chytrid epidemics were omnipresent, infecting diverse phytoplankton host communities, primarily diatoms, chlorophytes, and colonial and filamentous cyanobacteria. The diversity and numerical abundance of sporangia and of hosts, and the prevalence of infection (range, <1 to 24% of total host cells) as well, increased from the oligotrophic Lake Pavin to the eutrophic Lake Aydat, while the temporal changes in parasites were apparently more influenced by the host community composition. We conclude that the proposed protocol offers a valid method for the quantitative ecology of chytrid epidemics in aquatic ecosystems and food web dynamics.

Diversity and functions of microscopic fungi: a missing component in pelagic food webs

Fungi are a highly complex group of organisms of the kingdom Eumycota (i.e. the true-fungi) and other fungus-like organisms traditionally studied by mycologists, such as slime molds (Myxomycota) and oomycota (Straminopiles or Heterokonts). They constitute a significant proportion of the as yet undiscovered biota that is crucial in ecological processes and human well-being, through at least three main trophic modes: saprophytism, parasitism, or symbiosis. In addition to direct benefit (sources of antibiotics) or adverse effects (agents of disease), fungi can impact many environmental processes, particularly those associated with the decomposition of organic matter. They are present in almost all regions and climates, even under extreme conditions. However, studies have focussed mostly on economically interesting species, and knowledge of their diversity and functions is mainly restricted to soil, rhizosphere, mangrove, and lotic ecosystems. In this study, we review the diversity and potential functions of microscopic fungi in aquatic ecosystems, with focus on the pelagic environments where they often are regarded as allochthonous material, of low ecological significance for food-web processes. Recent environmental 18S rDNA surveys of microbial eukaryotes have (1) unveiled a large reservoir of unexpected fungal diversity in pelagic systems, (2) emphasized their ecological potentials for ecosystem functioning, and (3) opened new perspectives in the context of food-web dynamics. In spite of persisting methodological difficulties, we conclude that a better documentation of the diversity and quantitative and functional importance of fungi will improve our understanding of pelagic processes and biogeochemical cycling.

Functional Effects of Parasites on Food Web Properties during the Spring Diatom Bloom in Lake Pavin: A Linear Inverse Modeling Analysis

This study is the first assessment of the quantitative impact of parasitic chytrids on a planktonic food web. We used a carbon-based food web model of Lake Pavin (Massif Central, France) to investigate the effects of chytrids during the spring diatom bloom by developing models with and without chytrids. Linear inverse modelling procedures were employed to estimate undetermined flows in the lake. The Monte Carlo Markov chain linear inverse modelling procedure provided estimates of the ranges of model-derived fluxes. Model results support recent theories on the probable impact of parasites on food web function. In the lake, during spring, when ‘inedible’ algae (unexploited by planktonic herbivores) were the dominant primary producers, the epidemic growth of chytrids significantly reduced the sedimentation loss of algal carbon to the detritus pool through the production of grazer-exploitable zoospores. We also review some theories about the potential influence of parasites on ecological network properties and argue that parasitism contributes to longer carbon path lengths, higher levels of activity and specialization, and lower recycling. Considering the “structural asymmetry” hypothesis as a stabilizing pattern, chytrids should contribute to the stability of aquatic food webs.

Molecular and morphological diversity of fungi and the associated functions in three European nearby lakes

This study presents an original rDNA PCR and microscopic survey of pelagic freshwater fungal communities, and was designed to unveil the diversity of true Fungi (i.e. the kingdom Eumycota) in three contrasting lake ecosystems (Lakes Pavin, Aydat and Vassivière) located in the French Massif Central. Three clone libraries were constructed from samples collected in the euphotic layers of the lakes during spring 2007. Phylogenetic analysis of the combined data from the three lakes clustered our sequences into thee divisions: Chytridiomycota (50% of total sequences), Ascomycota (40%) and Basidiomycota (10% in Pavin and Aydat only). Several sequences were assigned to a novel Chytridiomycota clade first recovered in Lake Pavin in 2005. Most of the sequences retrieved in the investigated lakes were affiliated with known fungal species, most of which were apparently well adapted to thrive in the pelagic realm. Their main functions (i.e. parasitism and saprophytism), putatively inferred from the closest relatives of the retrieved molecular sequences, were confirmed by microscopic approaches and by enrichment experiments with pollen grains. The occurrence of three fungal forms (zoosporic, yeast and mycelial) was associated with different trophic modes, establishing fungi as strong potential competitors for various niches in pelagic ecosystems, primarily in relation to the processing of particulate organic matter and the production of propagule food sources for grazers. For the first time, this study provides insight into the diversity and the associated functions of all members of the Kingdom Eumycota investigated in the whole plankton fraction of aquatic ecosystems.

Development of a Real-Time PCR assay for quantitative assessment of uncultured freshwater zoosporic fungi.

Recently, molecular environmental surveys of the eukaryotic microbial community in lakes have revealed a high diversity of sequences belonging to uncultured zoosporic fungi. Although they are known as saprobes and algal parasites in freshwater systems, zoosporic fungi have been neglected in microbial food web studies. Recently, it has been suggested that zoosporic fungi, via the consumption of their zoospores by zooplankters, could transfer energy from large inedible algae and particulate organic material to higher trophic levels. However, because of their small size and their lack of distinctive morphological features, traditional microscopy does not allow the detection of fungal zoospores in the field. Hence, quantitative data on fungal zoospores in natural environments is missing. We have developed a quantitative PCR (qPCR) assay for the quantification of fungal zoospores in lakes. Specific primers were designed and qPCR conditions were optimized using a range of target and non-target plasmids obtained from previous freshwater environmental 18S rDNA surveys. When optimal DNA extraction protocol and qPCR conditions were applied, the qPCR assay developed in this study demonstrated high specificity and sensitivity, with as low as 100 18S rDNA copies per reaction detected. Although the present work focuses on the design and optimization of a new qPCR assay, its application to natural samples indicated that qPCR offers a promising tool for quantitative assessment of fungal zoospores in natural environments. We conclude that this will contribute to a better understanding of the ecological significance of zoosporic fungi in microbial food webs of pelagic ecosystems.

High Lytic Infection Rates but Low Abundances of Prokaryote Viruses in a Humic Lake (Vassivière, Massif Central, France)

ABSTRACT We explored the abundance and infection rates of viruses on a time series scale in the euphotic zone of the humic mesotrophic Lake Vassivière (Massif Central, France) and compared them to nonhumic lakes of contrasting trophy (i.e., the oligomesotrophic Lake Pavin and the eutrophic Lake Aydat) located in the same geographical region and sampled during the same period. In Lake Vassivière, the abundances of virus-like particles (range, 1.7 × 1010 to 2.6 × 1010 liter−1) were significantly (P < 0.001) lower than in Lakes Pavin and Aydat. The percentage of virus-infected prokaryotic cells (mean, 18.0%) was significantly higher (P < 0.001) in Vassivière than in Pavin (mean, 11.5%) and Aydat (mean, 9.7%). In Vassivière, the abundance of prokaryotes was a good predictor (r = 0.78, P < 0.001) of the number of virus-like particles, while the potential grazing rate from heterotrophic nanoflagellates was positively correlated to the viral infection rate (r = 0.75, P < 0.001; n = 20), indicating the prevalence of cycling interactions among viruses, prokaryotes, and grazers, which is in agreement with past experiments. The absence of correlation between chlorophyll a concentrations (Chl) and viral parameters suggested that the resources for the lytic activity of viruses in Vassivière were mainly under allochthonous control, through host activity. Indeed, compilation of data obtained from several nonhumic lakes in the French Massif Central revealed that Chl was positively correlated to the abundance of virus-like particles at concentrations above 0.5 μg Chl liter−1 and negatively at concentrations below 0.5 μg Chl liter−1, suggesting that phytoplankton-derived resources could force prokaryotic growth to attain a certain threshold level when the host availability is sufficient to boost the proliferation of viruses. Therefore, based on the high level of lytic infection rates in Lake Vassivière, we conclude that viruses are key agents for prokaryotic mortality and could influence the food web dynamics in humic lakes, which may ultimately depend on the internal cycling of resources and, perhaps, mainly on the allochthonous inputs and the associated humic substances.

Microbial diversity supporting dark fermentation of waste.

Microbial diversity is essential for human well-being and ecosystem services. Use of microorganisms in biomolecule production is common, but involves single-strain cultures. Microbial consortia provide advantages in the process of degrading organic waste to yield biomolecules of biotechnological interest. Microbial diversity in consortia can be an asset in the context of bioenergy and chemical production, which are key concerns for global energy demands. Improving knowledge of microbial consortia will therefore be important for biotechnology.

Genetic diversity along the life cycle of the cyanobacterium Microcystis: highlight on the complexity of benthic and planktonic interactions

Microcystis is a toxic freshwater cyanobacterium with an annual life cycle characterized by the alternation of a planktonic proliferation stage in summer and a benthic resting stage in winter. Given the importance of both stages for the development and the survival of the population, we investigated the genotypic composition of the planktonic and benthic Microcystis subpopulations from the Grangent reservoir (France) during two distinct proliferation periods. Our results showed a succession of different dominant genotypes in the sediment as well as in the water all along the study periods with some common genotypes to both compartments. Analysis of molecular variance and UniFrac analysis confirmed the similarity between some benthic and planktonic samples, thus evidencing exchanges of genotypes between water and sediment. Thanks to these data, recruitment and sedimentation were proven not to be restricted to spring and autumn, contrary to what was previously thought. Finally, genetic diversity was significantly higher in the sediment than in the water (P < 0.01; Students t-test). Taken together, our results shed light on the hidden contribution of the benthic compartment in maintaining the genetic diversity of Microcystis populations throughout their annual cycle, which could explain their ecological success in aquatic ecosystems.

Microbial diversity in support of anaerobic biomass valorization

Abstract Microbial diversity provides an immense reservoir of functions and supports key steps in maintaining ecosystem balance through matter decomposition processes and nutrient recycling. The use of microorganisms for biomolecule production is now common, but often involves single-strain cultures. In this review, we highlight the significance of using ecosystem-derived microbial diversity for biotechnological researches. In the context of organic matter mineralization, diversity of microorganisms is essential and enhances the degradation processes. We focus on anaerobic production of biomolecules of interest from discarded biomass, which is an important issue in the context of organic waste valorization and processing. Organic waste represents an important and renewable raw material but remains underused. It is commonly accepted that anaerobic mineralization of organic waste allows the production of diverse interesting molecules within several fields of application. We provide evidence that complex and diversified microbial communities isolated from ecosystems, i.e. microbial consortia, offer considerable advantages in degrading complex organic waste, to yield biomolecules of interest. We defend our opinion that this approach is more efficient and offers enhanced potential compared to the approaches that use single strain cultures.