Emilie Lefèvre

The molecular diversity of freshwater picoeukaryotes reveals high occurrence of putative parasitoids in the plankton

Eukaryotic microorganisms have been undersampled in biodiversity studies in freshwater environments. We present an original 18S rDNA survey of freshwater picoeukaryotes sampled during spring/summer 2005, complementing an earlier study conducted in autumn 2004 in Lake Pavin (France). These studies were designed to detect the small unidentified heterotrophic flagellates (HF, 0.6–5 µm) which are considered the main bacterivores in aquatic systems. Alveolates, Fungi and Stramenopiles represented 65% of the total diversity and differed from the dominant groups known from microscopic studies. Fungi and Telonemia taxa were restricted to the oxic zone which displayed two fold more operational taxonomic units (OTUs) than the oxycline. Temporal forcing also appeared as a driving force in the diversification within targeted organisms. Several sequences were not similar to those in databases and were considered as new or unsampled taxa, some of which may be typical of freshwater environments. Two taxa known from marine systems, the genera Telonema and Amoebophrya, were retrieved for the first time in our freshwater study. The analysis of potential trophic strategies displayed among the targeted HF highlighted the dominance of parasites and saprotrophs, and provided indications that these organisms have probably been wrongfully regarded as bacterivores in previous studies. A theoretical exercise based on a new ‘parasite/saprotroph-dominated HF hypothesis’ demonstrates that the inclusion of parasites and saprotrophs may increase the functional role of the microbial loop as a link for carbon flows in pelagic ecosystems. New interesting perspectives in aquatic microbial ecology are thus opened.

A review of the environmental implications of in situ remediation by nanoscale zero valent iron (nZVI): Behavior, transport and impacts on microbial communities

The increasing use of strategies incorporating nanoscale zero valent iron (nZVI) for soil and groundwater in situ remediation is raising some concerns regarding the potential adverse effects nZVI could have on indigenous microbial communities and ecosystem functioning. This review provides an overview of the current literature pertaining to the impacts of nZVI applications on microbial communities. Toxicity studies suggest that cell membrane disruption and oxidative stress through the generation of Fe(2+) and reactive oxygen species by nZVI are the main mechanisms contributing to nZVI cytotoxicity. In addition, nZVI has been shown to substantially alter the taxonomic and functional composition of indigenous microbial communities. However, because the physico-chemical conditions encountered in situ highly modulate nZVI toxicity, a better understanding of the environmental factors affecting nZVI toxicity and transport in the environment is of primary importance in evaluating the ecological consequences that could result from a more extensive use of nZVI.

Hidden diversity among aquatic heterotrophic flagellates: ecological potentials of zoosporic fungi

Since the emergence of the ‘microbial loop’ concept, heterotrophic flagellates have received particular attention as grazers in aquatic ecosystems. These microbes have historically been regarded incorrectly as a homogeneous group of bacterivorous protists in aquatic systems. More recently, environmental rDNA surveys of small heterotrophic flagellates in the pelagic zone of freshwater ecosystems have provided new insights. (i) The dominant phyla found by molecular studies differed significantly from those known from morphological studies with the light microscope, (ii) the retrieved phylotypes generally belong to well-established eukaryotic clades, but there is a very large diversity within these clades and (iii) a substantial part of the retrieved sequences cannot be assigned to bacterivorous but can be assigned instead to parasitic and saprophytic organisms, such as zoosporic true fungi (chytrids), fungus-like organisms (stramenopiles), or virulent alveolate parasites (Perkinsozoa and Amoebophrya sp.). All these microorganisms are able to produce small zoospores to assure dispersal in water during their life-cycles. Based on the existing literature on true fungi and fungus-like organisms, and on the more recently published eukaryotic rDNA environmental studies and morphological observations, we conclude that previously overlooked microbial diversity and related ecological potentials require intensive investigation (i) for an improved understanding of the roles of heterotrophic flagellates in pelagic ecosystems and (ii) to properly integrate the concept of ‘the microbial loop’ into modern pelagic microbial ecology.

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.

A comparison of the community diversity of foliar fungal endophytes between seedling and adult loblolly pines (Pinus taeda)

Fungal endophytes represent one of the most ubiquitous plant symbionts on Earth and are phylogenetically diverse. The structure and diversity of endophyte communities have been shown to depend on host taxa and climate, but there have been relatively few studies exploring endophyte communities throughout host maturity. We compared foliar fungal endophyte communities between seedlings and adult trees of loblolly pines (Pinus taeda) at the same seasons and locations by culturing and culture-independent methods. We sequenced the internal transcribed spacer region and adjacent partial large subunit nuclear ribosomal RNA gene (ITS-LSU amplicon) to delimit operational taxonomic units and phylogenetically characterize the communities. Despite the lower infection frequency in seedlings compared to adult trees, seedling needles were receptive to a more diverse community of fungal endophytes. Culture-free method confirmed the presence of commonly cultured OTUs from adult needles but revealed several new OTUs from seedling needles that were not found with culturing methods. The two most commonly cultured OTUs in adults were rarely cultured from seedlings, suggesting that host age is correlated with a selective enrichment for specific endophytes. This shift in endophyte species dominance may be indicative of a functional change between these fungi and their loblolly pine hosts.

Strategy to Find Molecular Signatures in a Small Series of Rare Cancers: Validation for Radiation-Induced Breast and Thyroid Tumors

Methods of classification using transcriptome analysis for case-by-case tumor diagnosis could be limited by tumor heterogeneity and masked information in the gene expression profiles, especially as the number of tumors is small. We propose a new strategy, EMts_2PCA, based on: 1) The identification of a gene expression signature with a great potential for discriminating subgroups of tumors (EMts stage), which includes: a) a learning step, based on an expectation-maximization (EM) algorithm, to select sets of candidate genes whose expressions discriminate two subgroups, b) a training step to select from the sets of candidate genes those with the highest potential to classify training tumors, c) the compilation of genes selected during the training step, and standardization of their levels of expression to finalize the signature. 2) The predictive classification of independent prospective tumors, according to the two subgroups of interest, by the definition of a validation space based on a two-step principal component analysis (2PCA). The present method was evaluated by classifying three series of tumors and its robustness, in terms of tumor clustering and prediction, was further compared with that of three classification methods (Gene expression bar code, Top-scoring pair(s) and a PCA-based method). Results showed that EMts_2PCA was very efficient in tumor classification and prediction, with scores always better that those obtained by the most common methods of tumor clustering. Specifically, EMts_2PCA permitted identification of highly discriminating molecular signatures to differentiate post-Chernobyl thyroid or post-radiotherapy breast tumors from their sporadic counterparts that were previously unsuccessfully classified or classified with errors.

Three new genera in Chytridiales from aquatic habitats in Argentina

Sampling for chytrids in a variety of habitats has resulted in pure cultures that when analyzed have yielded hypotheses of relationships based on molecular and zoospore ultrastructural markers. To extend our understanding of diversity of Chytridiales in eastern Argentina and USA, we isolated and examined the morphology, ultrastructure and 28S and ITS1-5.8S-ITS2 rDNA sequences of numerous chytrids from aquatic habitats from these two regions. Three family-level lineages (Chytridiaceae, Chytriomycetaceae, family incertae sedis) are represented in our molecular phylogeny, and three new genera (Avachytrium, Odontochytrium in Chytriomycetaceae, Delfinachytrium in family incertae sedis) are described. These findings of new genera and species emphasize the potential for discovery of additional diversity.

Characterization and Adaptation of Anaerobic Sludge Microbial Communities Exposed to Tetrabromobisphenol A.

The increasing occurrence of tetrabromobisphenol A (TBBPA) in the environment is raising questions about its potential ecological and human health impacts. TBBPA is microbially transformed under anaerobic conditions to bisphenol A (BPA). However, little is known about which taxa degrade TBBPA and the adaptation of microbial communities exposed to TBBPA. The objectives of this study were to characterize the effect of TBBPA on microbial community structure during the start-up phase of a bench-scale anaerobic sludge reactor, and identify taxa that may be associated with TBBPA degradation. TBBPA degradation was monitored using LC/MS-MS, and the microbial community was characterized using Ion Torrent sequencing and qPCR. TBBPA was nearly completely transformed to BPA via reductive debromination in 55 days. Anaerobic reactor performance was not negatively affected by the presence of TBBPA and the bulk of the microbial community did not experience significant shifts. Several taxa showed a positive response to TBBPA, suggesting they may be associated with TBBPA degradation. Some of these taxa had been previously identified as dehalogenating bacteria including Dehalococcoides, Desulfovibrio, Propionibacterium, and Methylosinus species, but most had not previously been identified as having dehalogenating capacities. This study is the first to provide in-depth information on the microbial dynamics of anaerobic microbial communities exposed to TBBPA.

Microbial community response to silver nanoparticles and Ag + in nitrifying activated sludge revealed by ion semiconductor sequencing

Silver nanoparticles (AgNPs), which are known to act as biocides, are incorporated into medical and consumer products including athletic clothing, stuffed animals, liquid dietary supplements, and more. The increasing use of AgNPs in these products is likely to lead to their entry into both natural and engineered systems, which has the potential to disrupt bacterial processes including those involved in nutrient cycling in wastewater treatment. In the present study, sequencing batch reactors (SBR) mimicking secondary wastewater treatment were operated to determine the effects of AgNPs on the microbial communities contained within activated sludge of wastewater treatment plants (WWTP). SBRs were treated with 0.2 and 2ppm of either gum Arabic (GA)-coated AgNPs, citrate (Ca)-coated AgNPs, or Ag+ as AgNO3. Cell samples were collected and DNA isolated periodically throughout SBR operation. DNA was used for Ion Torrent Next Gen Sequencing of the V3 region of the 16S rDNA gene. Subsequent analyses revealed that the microbial community both shifted and recovered quickly in response to Ag+. Both AgNP treatments resulted in slower initial community shifts than that observed with the Ag+ treatment. GA-AgNPs elicited the longest lasting effect. Additional examination of nitrogen removal bacteria suggested the possibility of an increase in sludge bulking species with increased concentrations of AgNPs in WWTPs. This study supports the hypothesis that Ag+ release from AgNPs is largely coating-dependent and thus a key driver in dictating AgNP toxicity.

Distance decay relationships in foliar fungal endophytes are driven by rare taxa

Foliar fungal endophytes represent a diverse and species-rich plant microbiome. Their biogeography provides essential clues to their cryptic relationship with hosts and the environment in which they disperse. We present species composition, diversity, and dispersal patterns of endophytic fungi associated with needles of Pinus taeda trees across regional scales in the absence of strong environmental gradients as well as within individual trees. An empirical designation of rare and abundant taxa enlightens us on the structure of endophyte communities. We report multiple distance-decay patterns consistent with effects of dispersal limitation, largely driven by community changes in rare taxa, those taxonomic units that made up less than 0.31% of reads per sample on average. Distance-decay rates and community structure also depended on specific classes of fungi and were predominantly influenced by rare members of Dothideomycetes. Communities separated by urban areas also revealed stronger effects of distance on community similarity, confirming that host density and diversity plays an important role in symbiont biogeography, which may ultimately lead to a mosaic of functional diversity as well as rare species diversity across landscapes.