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1.  Phylogenetic Affiliation of SSU rRNA Genes Generated by Massively Parallel Sequencing: New Insights into the Freshwater Protist Diversity 
PLoS ONE  2013;8(3):e58950.
Recent advances in next-generation sequencing (NGS) technologies spur progress in determining the microbial diversity in various ecosystems by highlighting, for example, the rare biosphere. Currently, high-throughput pyrotag sequencing of PCR-amplified SSU rRNA gene regions is mainly used to characterize bacterial and archaeal communities, and rarely to characterize protist communities. In addition, although taxonomic assessment through phylogeny is considered as the most robust approach, similarity and probabilistic approaches remain the most commonly used for taxonomic affiliation. In a first part of this work, a tree-based method was compared with different approaches of taxonomic affiliation (BLAST and RDP) of 18S rRNA gene sequences and was shown to be the most accurate for near full-length sequences and for 400 bp amplicons, with the exception of amplicons covering the V5-V6 region. Secondly, the applicability of this method was tested by running a full scale test using an original pyrosequencing dataset of 18S rRNA genes of small lacustrine protists (0.2–5 µm) from eight freshwater ecosystems. Our results revealed that i) fewer than 5% of the operational taxonomic units (OTUs) identified through clustering and phylogenetic affiliation had been previously detected in lakes, based on comparison to sequence in public databases; ii) the sequencing depth provided by the NGS coupled with a phylogenetic approach allowed to shed light on clades of freshwater protists rarely or never detected with classical molecular ecology approaches; and iii) phylogenetic methods are more robust in describing the structuring of under-studied or highly divergent populations. More precisely, new putative clades belonging to Mamiellophyceae, Foraminifera, Dictyochophyceae and Euglenida were detected. Beyond the study of protists, these results illustrate that the tree-based approach for NGS based diversity characterization allows an in-depth description of microbial communities including taxonomic profiling, community structuring and the description of clades of any microorganisms (protists, Bacteria and Archaea).
PMCID: PMC3597552  PMID: 23516585
2.  Impact of Toxic Cyanobacterial Blooms on Eurasian Perch (Perca fluviatilis): Experimental Study and In Situ Observations in a Peri-Alpine Lake 
PLoS ONE  2012;7(12):e52243.
Due to the importance of young-of-the-year (YOY) perch in the peri-alpine regions where they are consumed, the microcystin (MC) contamination of YOY perch was analysed both in field (Lake Bourget, France) and experimentally using force-feeding protocols with pure MCs. In-situ, schools of YOY perch present in the epilimnion of the lake were never found in direct contact with the P. rubescens blooms that were present in the metalimnion. However, MCs were detected in the muscles and liver of the fish and were thus assumed to reach YOY perch through dietary routes, particularly via the consumption of MC-containing Daphnia. Force-feeding experiment demonstrates the existence of MC detoxification/excretion processes and suggests that in situ, YOY perch could partly detoxify and excrete ingested MCs, thereby limiting the potential negative effects on perch populations under bloom conditions. However, because of chronic exposure these processes could not allow for the complete elimination of MCs. In both experimental and in situ studies, no histological change was observed in YOY perch, indicating that MC concentrations that occurred in Lake Bourget in 2009 were too low to cause histological damage prone to induce mortality. However, Deoxyribonucleic acid (DNA) damages were observed for both the high and low experimental MC doses, suggesting that similar effects could occur in situ and potentially result in perch population disturbance during cyanobacterial blooms. Our results indicate the presence of MCs in wild perch, the consumption of this species coming from Lake Bourget is not contested but more analyses are needed to quantify the risk.
PMCID: PMC3525550  PMID: 23272228
3.  Short-term responses of unicellular planktonic eukaryotes to increases in temperature and UVB radiation 
BMC Microbiology  2012;12:202.
Small size eukaryotes play a fundamental role in the functioning of coastal ecosystems, however, the way in which these micro-organisms respond to combined effects of water temperature, UVB radiations (UVBR) and nutrient availability is still poorly investigated.
We coupled molecular tools (18S rRNA gene sequencing and fingerprinting) with microscope-based identification and counting to experimentally investigate the short-term responses of small eukaryotes (<6 μm; from a coastal Mediterranean lagoon) to a warming treatment (+3°C) and UVB radiation increases (+20%) at two different nutrient levels. Interestingly, the increase in temperature resulted in higher pigmented eukaryotes abundances and in community structure changes clearly illustrated by molecular analyses. For most of the phylogenetic groups, some rearrangements occurred at the OTUs level even when their relative proportion (microscope counting) did not change significantly. Temperature explained almost 20% of the total variance of the small eukaryote community structure (while UVB explained only 8.4%). However, complex cumulative effects were detected. Some antagonistic or non additive effects were detected between temperature and nutrients, especially for Dinophyceae and Cryptophyceae.
This multifactorial experiment highlights the potential impacts, over short time scales, of changing environmental factors on the structure of various functional groups like small primary producers, parasites and saprotrophs which, in response, can modify energy flow in the planktonic food webs.
PMCID: PMC3478981  PMID: 22966751
Small eukaryotes; Molecular diversity; Temperature; UVB radiation; Microcosms experiment; Mediterranean lagoon
4.  Quantitative PCR Enumeration of Total/Toxic Planktothrix rubescens and Total Cyanobacteria in Preserved DNA Isolated from Lake Sediments▿† 
Applied and Environmental Microbiology  2011;77(24):8744-8753.
The variability of spatial distribution and the determinism of cyanobacterial blooms, as well as their impact at the lake scale, are still not understood, partly due to the lack of long-term climatic and environmental monitoring data. The paucity of these data can be alleviated by the use of proxy data from high-resolution sampling of sediments. Coupling paleolimnological and molecular tools and using biomarkers such as preserved DNA are promising approaches, although they have not been performed often enough so far. In our study, a quantitative PCR (qPCR) technique was applied to enumerate total cyanobacterial and total and toxic Planktothrix communities in preserved DNA derived from sediments of three lakes located in the French Alps (Lake Geneva, Lake Bourget, and Lake Annecy), containing a wide range of cyanobacterial species. Preserved DNA from lake sediments was analyzed to assess its quality, quantity, and integrity, with further application for qPCR. We applied the qPCR assay to enumerate the total cyanobacterial community, and multiplex qPCR assays were applied to quantify total and microcystin-producing Planktothrix populations in a single reaction tube. These methods were optimized, calibrated, and applied to sediment samples, and the specificity and reproducibility of qPCR enumeration were tested. Accurate estimation of potential inhibition within sediment samples was performed to assess the sensitivity of such enumeration by qPCR. Some precautions needed for interpreting qPCR results in the context of paleolimnological approaches are discussed. We concluded that the qPCR assay can be used successfully for the analysis of lake sediments when DNA is well preserved in order to assess the presence and dominance of cyanobacterial and Planktothrix communities.
PMCID: PMC3233095  PMID: 21984244
5.  Effect of grazers and viruses on bacterial community structure and production in two contrasting trophic lakes 
BMC Microbiology  2011;11:88.
Over the last 30 years, extensive studies have revealed the crucial roles played by microbes in aquatic ecosystems. It has been shown that bacteria, viruses and protozoan grazers are dominant in terms of abundance and biomass. The frequent interactions between these microbiological compartments are responsible for strong trophic links from dissolved organic matter to higher trophic levels, via heterotrophic bacteria, which form the basis for the important biogeochemical roles of microbial food webs in aquatic ecosystems. To gain a better understanding of the interactions between bacteria, viruses and flagellates in lacustrine ecosystems, we investigated the effect of protistan bacterivory on bacterial abundance, production and structure [determined by 16S rRNA PCR-DGGE], and viral abundance and activity of two lakes of contrasting trophic status. Four experiments were conducted in the oligotrophic Lake Annecy and the mesotrophic Lake Bourget over two seasons (early spring vs. summer) using a fractionation approach. In situ dark vs. light incubations were performed to consider the effects of the different treatments in the presence and absence of phototrophic activity.
The presence of grazers (i.e. < 5-μm small eukaryotes) affected viral production positively in all experiments, and the stimulation of viral production (compared to the treatment with no eukaryotic predators) was more variable between lakes than between seasons, with the highest value having been recorded in the mesotrophic lake (+30%). Viral lysis and grazing activities acted additively to sustain high bacterial production in all experiments. Nevertheless, the stimulation of bacterial production was more variable between seasons than between lakes, with the highest values obtained in summer (+33.5% and +37.5% in Lakes Bourget and Annecy, respectively). The presence of both predators (nanoflagellates and viruses) did not seem to have a clear influence upon bacterial community structure according to the four experiments.
Our results highlight the importance of a synergistic effect, i.e. the positive influence of grazers on viral activities in sustaining (directly and indirectly) bacterial production and affecting composition, in both oligotrophic and mesotrophic lakes.
PMCID: PMC3114703  PMID: 21527043
Lakes; microcosm; spring-summer variations; bacterial production; viral production; bacterial community structure; grazers
6.  Community Structure and Dynamics of Small Eukaryotes Targeted by New Oligonucleotide Probes: New Insight into the Lacustrine Microbial Food Web▿  
Applied and Environmental Microbiology  2009;75(19):6373-6381.
The seasonal dynamics of the small eukaryotic fraction (cell diameter, 0.2 to 5 μm) was investigated in a mesotrophic lake by tyramide signal amplification-fluorescence in situ hybridization targeting seven different phylogenetic groups: Chlorophyceae, Chrysophyceae, Cryptophyceae, Cercozoa, LKM11, Perkinsozoa (two clades), and Fungi. The abundance of small eukaryotes ranged from 1,692 to 10,782 cells ml−1. The dominant groups were the Chrysophyceae and the Chlorophyceae, which represented 19.6% and 17.9% of small eukaryotes, respectively. The results also confirmed the quantitative importance of putative parasites, Fungi and Perkinsozoa, in the small heterotrophic eukaryotic assemblage. The relative abundances recorded for the Perkinsozoa group reached as much as 31.6% of total targeted eukaryotes during the summer. The dynamics of Perkinsozoa clade 1 coincided with abundance variations in Peridinium and Ceratium spp. (Dinoflagellates), while the dynamics of Perkinsozoa clade 2 was linked to the presence of Dinobryon spp. (Chrysophyceae). Fungi, represented by chytrids, reached maximal abundance in December (569 cells ml−1) and were mainly correlated with the dynamics of diatoms, especially Melosira varians. A further new finding of this study is the recurrent presence of Cercozoa (6.2%) and LKM11 (4.5%) cells. This quantitative approach based on newly designed probes offers a promising means of in-depth analysis of microbial food webs in lakes, especially by revealing the phylogenetic composition of the small heterotrophic flagellate assemblage, for which an important fraction of cells are generally unidentified by classical microscopy (on average, 96.8% of the small heterotrophic flagellates were identified by the specific probes we used in this study).
PMCID: PMC2753085  PMID: 19666727
7.  Unexpected Importance of Potential Parasites in the Composition of the Freshwater Small-Eukaryote Community▿  
Applied and Environmental Microbiology  2008;74(10):2940-2949.
The diversity of small eukaryotes (0.2 to 5 μm) 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.
PMCID: PMC2394938  PMID: 18359836
8.  Succession and Regulation Factors of Small Eukaryote Community Composition in a Lacustrine Ecosystem (Lake Pavin) 
The structure and dynamics of small eukaryotes (cells with a diameter less than 5 μm) were studied over two consecutive years in an oligomesotrophic lake (Lake Pavin in France). Water samples were collected at 5 and 30 m below the surface; when the lake was stratified, these depths corresponded to the epilimnion and hypolimnion. Changes in small-eukaryote structure were analyzed using terminal restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of the 18S rRNA genes. Terminal restriction fragments from clones were used to reveal the dominant taxa in T-RFLP profiles of the environmental samples. Spumella-like cells (Chrysophyceae) did not dominate the small eukaryote community identified by molecular techniques in lacustrine ecosystems. Small eukaryotes appeared to be dominated by heterotrophic cells, particularly Cercozoa, which represented nearly half of the identified phylotypes, followed by the Fungi-LKM11 group (25%), choanoflagellates (10.3%) and Chrysophyceae (8.9%). Bicosoecida, Cryptophyta, and ciliates represented less than 9% of the community studied. No seasonal reproducibility in temporal evolution of the small-eukaryote community was observed from 1 year to the next. The T-RFLP patterns were related to bottom-up (resources) and top-down (grazing) variables using canonical correspondence analysis. The results showed a strong top-down regulation of small eukaryotes by zooplankton, more exactly, by cladocerans at 5 m and copepods at 30 m. Among bottom-up factors, temperature had a significant effect at both depths. The concentrations of nitrogenous nutrients and total phosphorus also had an effect on small-eukaryote dynamics at 5 m, whereas bacterial abundance and dissolved oxygen played a more important structuring role in the deeper zone.
PMCID: PMC1449018  PMID: 16598004

Results 1-8 (8)