Publications by authors named "Richard Christen"

96 Publications

The many faced symbiotic snakelocks anemone (Anemonia viridis, Anthozoa): host and symbiont genetic differentiation among colour morphs.

Heredity (Edinb) 2020 02 16;124(2):351-366. Epub 2019 Sep 16.

UPMC Univ Paris 06, Univ Antilles, Univ Nice Sophia Antipolis, CNRS, Symbiose Marine, Evolution Paris Seine-Institut de Biologie Paris Seine (EPS-IBPS), Sorbonne Universités, 75005, Paris, France.

How can we explain morphological variations in a holobiont? The genetic determinism of phenotypes is not always obvious and could be circumstantial in complex organisms. In symbiotic cnidarians, it is known that morphology or colour can misrepresent a complex genetic and symbiotic diversity. Anemonia viridis is a symbiotic sea anemone from temperate seas. This species displays different colour morphs based on pigment content and lives in a wide geographical range. Here, we investigated whether colour morph differentiation correlated with host genetic diversity or associated symbiotic genetic diversity by using RAD sequencing and symbiotic dinoflagellate typing of 140 sea anemones from the English Channel and the Mediterranean Sea. We did not observe genetic differentiation among colour morphs of A. viridis at the animal host or symbiont level, rejecting the hypothesis that A. viridis colour morphs correspond to species level differences. Interestingly, we however identified at least four independent animal host genetic lineages in A. viridis that differed in their associated symbiont populations. In conclusion, although the functional role of the different morphotypes of A. viridis remains to be determined, our approach provides new insights on the existence of cryptic species within A. viridis.
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http://dx.doi.org/10.1038/s41437-019-0266-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972938PMC
February 2020

Intestinal Bacterial Communities of Trypanosome-Infected and Uninfected palpalis from Three Human African Trypanomiasis Foci in Cameroon.

Front Microbiol 2017 3;8:1464. Epub 2017 Aug 3.

UMR INTERTRYP, Institut de Recherche pour le Développement-CIRAD, CIRAD TA A-17/GMontpellier, France.

sp. the tsetse fly that transmits trypanosomes causing the Human or the Animal African Trypanosomiasis (HAT or AAT) can harbor symbiotic bacteria that are known to play a crucial role in the fly's vector competence. We hypothesized that other bacteria could be present, and that some of them could also influence the fly's vector competence. In this context the objectives of our work were: (a) to characterize the bacteria that compose the midgut bacteriome, (b) to evidence possible bacterial community differences between trypanosome-infected and non-infected fly individuals from a given AAT and HAT focus or from different foci using barcoded Illumina sequencing of the hypervariable V3-V4 region of the . Forty flies, either infected by or uninfected were sampled from three trypanosomiasis foci in Cameroon. A total of 143 OTUs were detected in the midgut samples. Most taxa were identified at the genus level, nearly 50% at the species level; they belonged to 83 genera principally within the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. Prominent representatives included (the fly's obligate symbiont), , and was identified for the first time in . The average number of bacterial species per tsetse sample was not significantly different regarding the fly infection status, and the hierarchical analysis based on the differences in bacterial community structure did not provide a clear clustering between infected and non-infected flies. Finally, the most important result was the evidence of the overall very large diversity of intestinal bacteria which, except for , were unevenly distributed over the sampled flies regardless of their geographic origin and their trypanosome infection status.
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http://dx.doi.org/10.3389/fmicb.2017.01464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5541443PMC
August 2017

Soil prokaryotic communities in Chernobyl waste disposal trench T22 are modulated by organic matter and radionuclide contamination.

FEMS Microbiol Ecol 2017 08;93(8)

CEA, CNRS, Aix-Marseille Université, UMR 7265 Biologie Végétale et Microbiologie Environnementale, LIPM, 13108 Saint-Paul-lez-Durance, France.

After the Chernobyl nuclear power plant accident in 1986, contaminated soils, vegetation from the Red Forest and other radioactive debris were buried within trenches. In this area, trench T22 has long been a pilot site for the study of radionuclide migration in soil. Here, we used 454 pyrosequencing of 16S rRNA genes to obtain a comprehensive view of the bacterial and archaeal diversity in soils collected inside and in the vicinity of the trench T22 and to investigate the impact of radioactive waste disposal on prokaryotic communities. A remarkably high abundance of Chloroflexi and AD3 was detected in all soil samples from this area. Our statistical analysis revealed profound changes in community composition at the phylum and OTUs levels and higher diversity in the trench soils as compared to the outside. Our results demonstrate that the total absorbed dose rate by cell and, to a lesser extent the organic matter content of the trench, are the principal variables influencing prokaryotic assemblages. We identified specific phylotypes affiliated to the phyla Crenarchaeota, Acidobacteria, AD3, Chloroflexi, Proteobacteria, Verrucomicrobia and WPS-2, which were unique for the trench soils.
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http://dx.doi.org/10.1093/femsec/fix079DOI Listing
August 2017

Characterization of glutathione peroxidase diversity in the symbiotic sea anemone Anemonia viridis.

Biochimie 2017 Jan 8;132:94-101. Epub 2016 Nov 8.

Sorbonne Universités, UPMC Univ Paris 06, Univ Antilles, Univ Nice Sophia Antipolis, CNRS, Evolution Paris Seine - Institut de Biologie Paris Seine (EPS - IBPS), 75005, Paris, France. Electronic address:

Cnidarians living in symbiosis with photosynthetic dinoflagellates (commonly named zooxanthellae) are exposed to high concentrations of reactive oxygen species (ROS) upon illumination. To quench ROS production, both the cnidarian host and zooxanthellae express a full suite of antioxidant enzymes. Studying antioxidative balance is therefore crucial to understanding how symbiotic cnidarians cope with ROS production. We characterized glutathione peroxidases (GPx) in the symbiotic cnidarian Anemonia viridis by analysis of their isoform diversity, their activity distribution in the three cellular compartments (ectoderm, endoderm and zooxanthellae) and their involvement in the response to thermal stress. We identified a GPx repertoire through a phylogenetic analysis showing 7 GPx transcripts belonging to the A. viridis host and 4 GPx transcripts strongly related to Symbiodinium sp. The biochemical approach, used for the first time with a cnidarian species, allowed the identification of GPx activity in the three cellular compartments and in the animal mitochondrial fraction, and revealed a high GPx electrophoretic diversity. The symbiotic lifestyle of zooxanthellae requires more GPx activity and diversity than that of free-living species. Heat stress induced no modification of GPx activities. We highlight a high GPx diversity in A. viridis tissues by genomic and biochemical approaches. GPx activities represent an overall constitutive enzymatic pattern inherent to symbiotic lifestyle adaptation. This work allows the characterization of the GPx family in a symbiotic cnidarian and establishes a foundation for future studies of GPx in symbiotic cnidarians.
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http://dx.doi.org/10.1016/j.biochi.2016.10.016DOI Listing
January 2017

Benthic protists: the under-charted majority.

FEMS Microbiol Ecol 2016 08 5;92(8). Epub 2016 Jun 5.

Ifremer, Centre de Brest DYNECO/Pelagos Technopôle Brest Iroise, BP 7029280 Plouzané, France.

Marine protist diversity inventories have largely focused on planktonic environments, while benthic protists have received relatively little attention. We therefore hypothesize that current diversity surveys have only skimmed the surface of protist diversity in marine sediments, which may harbor greater diversity than planktonic environments. We tested this by analyzing sequences of the hypervariable V4 18S rRNA from benthic and planktonic protist communities sampled in European coastal regions. Despite a similar number of OTUs in both realms, richness estimations indicated that we recovered at least 70% of the diversity in planktonic protist communities, but only 33% in benthic communities. There was also little overlap of OTUs between planktonic and benthic communities, as well as between separate benthic communities. We argue that these patterns reflect the heterogeneity and diversity of benthic habitats. A comparison of all OTUs against the Protist Ribosomal Reference database showed that a higher proportion of benthic than planktonic protist diversity is missing from public databases; similar results were obtained by comparing all OTUs against environmental references from NCBI's Short Read Archive. We suggest that the benthic realm may therefore be the world's largest reservoir of marine protist diversity, with most taxa at present undescribed.
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http://dx.doi.org/10.1093/femsec/fiw120DOI Listing
August 2016

Dynamics of Bacterial Community Composition in the Malaria Mosquito's Epithelia.

Front Microbiol 2015 5;6:1500. Epub 2016 Jan 5.

UMR Maladies Infectieuses Et Vecteurs Écologie, Génétique, Évolution Et Contrôle, IRD 224- Centre National de la Recherche Scientifique 5290- UM1- UM2Montpellier, France; Laboratoire d'entomologie médicale, OCEAC-IRDYaoundé, Cameroon.

The Anopheles midgut hosts diverse bacterial communities and represents a complex ecosystem. Several evidences indicate that mosquito midgut microbiota interferes with malaria parasite transmission. However, the bacterial composition of salivary glands and ovaries, two other biologically important tissues, has not been described so far. In this study, we investigated the dynamics of the bacterial communities in the mosquito tissues from emerging mosquitoes until 8 days after a blood meal containing Plasmodium falciparum gametocytes and described the temporal colonization of the mosquito epithelia. Bacterial communities were identified in the midgut, ovaries, and salivary glands of individual mosquitoes using pyrosequencing of the 16S rRNA gene. We found that the mosquito epithelia share a core microbiota, but some bacteria taxa were more associated with one or another tissue at a particular time point. The bacterial composition in the tissues of emerging mosquitoes varied according to the breeding site, indicating that some bacteria are acquired from the environment. Our results revealed temporal variations in the bacterial community structure, possibly as a result of the mosquito physiological changes. The abundance of Serratia significantly correlated with P. falciparum infection both in the midgut and salivary glands of malaria challenged mosquitoes, which suggests that interactions occur between microbes and parasites. These bacteria may represent promising targets for vector control strategies. Overall, this study points out the importance of characterizing bacterial communities in malaria mosquito vectors.
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http://dx.doi.org/10.3389/fmicb.2015.01500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700937PMC
January 2016

ERK1 and ERK2 present functional redundancy in tetrapods despite higher evolution rate of ERK1.

BMC Evol Biol 2015 Sep 3;15:179. Epub 2015 Sep 3.

Institute for Research on Cancer and Aging of Nice (IRCAN), University of Nice-Sophia Antipolis, CNRS UMR7284, INSERM, Centre A. Lacassagne, Nice, 06189, France.

Background: The Ras/Raf/MEK/ERK signaling pathway is involved in essential cell processes and it is abnormally activated in ~30 % of cancers and cognitive disorders. Two ERK isoforms have been described, ERK1 and ERK2; ERK2 being regarded by many as essential due to the embryonic lethality of ERK2 knock-out mice, whereas mice lacking ERK1 are viable and fertile. The controversial question of why we have two ERKs and whether they have differential functions or display functional redundancy has not yet been resolved.

Results: To investigate this question we used a novel approach based on comparing the evolution of ERK isoforms' sequences and protein expression across vertebrates. We gathered and cloned erk1 and erk2 coding sequences and we examined protein expression of isoforms in brain extracts in all major clades of vertebrate evolution. For the first time, we measured each isoforms' relative protein level in phylogenetically distant animals using anti-phospho antibodies targeting active ERKs. We demonstrate that squamates (lizards, snakes and geckos), despite having both genes, do not express ERK2 protein whereas other tetrapods either do not express ERK1 protein or have lost the erk1 gene. To demonstrate the unexpected squamates' lack of ERK2 expression, we targeted each ERK isoform in lizard primary fibroblasts by specific siRNA-mediated knockdown. We also found that undetectable expression of ERK2 in lizard is compensated by a greater strength of lizard's erk1 promoter. Finally, phylogenetic analysis revealed that ERK1 amino acids sequences evolve faster than ERK2's likely due to genomic factors, including a large difference in gene size, rather than from functional differences since amino acids essential for function are kept invariant.

Conclusions: ERK isoforms appeared by a single gene duplication at the onset of vertebrate evolution at least 400 Mya. Our results demonstrate that tetrapods can live by expressing either one or both ERK isoforms, supporting the notion that ERK1/2 act interchangeably. Substrate recognition sites and catalytic cleft are nearly invariant in all vertebrate ERKs further suggesting functional redundancy. We suggest that future ERK research should shift towards understanding the role and regulation of total ERK quantity, especially in light of newly described erk2 gene amplification identified in tumors.
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http://dx.doi.org/10.1186/s12862-015-0450-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4559367PMC
September 2015

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing.

Environ Microbiol 2015 Oct 4;17(10):4035-49. Epub 2015 Aug 4.

Ifremer DYNECO/Pelagos, BP 7029280, Plouzané, France.

Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date.
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http://dx.doi.org/10.1111/1462-2920.12955DOI Listing
October 2015

PhytoREF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy.

Mol Ecol Resour 2015 Nov 6;15(6):1435-45. Epub 2015 Apr 6.

CNRS, UMR 7138, Systématique Adaptation Evolution, Parc Valrose, BP71, Nice, F06108, France.

Photosynthetic eukaryotes have a critical role as the main producers in most ecosystems of the biosphere. The ongoing environmental metabarcoding revolution opens the perspective for holistic ecosystems biological studies of these organisms, in particular the unicellular microalgae that often lack distinctive morphological characters and have complex life cycles. To interpret environmental sequences, metabarcoding necessarily relies on taxonomically curated databases containing reference sequences of the targeted gene (or barcode) from identified organisms. To date, no such reference framework exists for photosynthetic eukaryotes. In this study, we built the PhytoREF database that contains 6490 plastidial 16S rDNA reference sequences that originate from a large diversity of eukaryotes representing all known major photosynthetic lineages. We compiled 3333 amplicon sequences available from public databases and 879 sequences extracted from plastidial genomes, and generated 411 novel sequences from cultured marine microalgal strains belonging to different eukaryotic lineages. A total of 1867 environmental Sanger 16S rDNA sequences were also included in the database. Stringent quality filtering and a phylogeny-based taxonomic classification were applied for each 16S rDNA sequence. The database mainly focuses on marine microalgae, but sequences from land plants (representing half of the PhytoREF sequences) and freshwater taxa were also included to broaden the applicability of PhytoREF to different aquatic and terrestrial habitats. PhytoREF, accessible via a web interface (http://phytoref.fr), is a new resource in molecular ecology to foster the discovery, assessment and monitoring of the diversity of photosynthetic eukaryotes using high-throughput sequencing.
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http://dx.doi.org/10.1111/1755-0998.12401DOI Listing
November 2015

Composition of Anopheles coluzzii and Anopheles gambiae microbiota from larval to adult stages.

Infect Genet Evol 2014 Dec 2;28:715-24. Epub 2014 Oct 2.

UMR MIVEGEC (IRD 224-CNRS 5290-UM1-UM2), Institut de Recherche pour le Développement, Montpellier, France; Laboratoire d'Entomologie Médicale, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale, Yaoundé, Cameroon. Electronic address:

During their immature life stages, malaria mosquitoes are exposed to a wide array of microbes and contaminants from the aquatic habitats. Although prior studies have suggested that environmental exposure shapes the microbial community structure in the adult mosquito, most reports have focused on laboratory-based experiments and on a single mosquito epithelium, the gut. In this study, we investigated the influence of the breeding site on the development of the Anopheles coluzzii and Anopheles gambiae microbiota in natural conditions. We characterized bacterial communities from aquatic habitats, at surface microlayer and subsurface water levels, to freshly emerge adult mosquitoes using multiplexed 16S rRNA gene pyrosequencing and we separately analyzed the microbiota associated with the different epithelia of adult individual, midguts, ovaries and salivary glands. We found that the distribution of bacterial communities in the aquatic habitats differed according to the depth of water collections. Inter-individual variation of bacterial composition was large in larvae guts but adult mosquitoes from a same breeding site shared quite similar microbiota. Although some differences in bacterial abundances were highlighted between the different epithelia of freshly emerged An. coluzzii and An. gambiae, an intriguing feature from our study is the particular similarity of the overall bacterial communities. Our results call for further investigations on the bacterial population dynamics in the different tissues to determine the distinctive characteristics of each microbiota during the mosquito lifespan and to identify specific interactions between certain key phyla or species and the insect life history traits.
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http://dx.doi.org/10.1016/j.meegid.2014.09.029DOI Listing
December 2014

Patterns of rare and abundant marine microbial eukaryotes.

Curr Biol 2014 Apr 3;24(8):813-21. Epub 2014 Apr 3.

Institut de Ciències del Mar (ICM), CSIC, Passeig Marítim de la Barceloneta 37-49, 08003 Barcelona, Spain.

Background: Biological communities are normally composed of a few abundant and many rare species. This pattern is particularly prominent in microbial communities, in which most constituent taxa are usually extremely rare. Although abundant and rare subcommunities may present intrinsic characteristics that could be crucial for understanding community dynamics and ecosystem functioning, microbiologists normally do not differentiate between them. Here, we investigate abundant and rare subcommunities of marine microbial eukaryotes, a crucial group of organisms that remains among the least-explored biodiversity components of the biosphere. We surveyed surface waters of six separate coastal locations in Europe, independently considering the picoplankton, nanoplankton, and microplankton/mesoplankton organismal size fractions.

Results: Deep Illumina sequencing of the 18S rRNA indicated that the abundant regional community was mostly structured by organismal size fraction, whereas the rare regional community was mainly structured by geographic origin. However, some abundant and rare taxa presented similar biogeography, pointing to spatiotemporal structure in the rare microeukaryote biosphere. Abundant and rare subcommunities presented regular proportions across samples, indicating similar species-abundance distributions despite taxonomic compositional variation. Several taxa were abundant in one location and rare in other locations, suggesting large oscillations in abundance. The substantial amount of metabolically active lineages found in the rare biosphere suggests that this subcommunity constitutes a diversity reservoir that can respond rapidly to environmental change.

Conclusions: We propose that marine planktonic microeukaryote assemblages incorporate dynamic and metabolically active abundant and rare subcommunities, with contrasting structuring patterns but fairly regular proportions, across space and time.
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http://dx.doi.org/10.1016/j.cub.2014.02.050DOI Listing
April 2014

Updating the Vibrio clades defined by multilocus sequence phylogeny: proposal of eight new clades, and the description of Vibrio tritonius sp. nov.

Front Microbiol 2013 27;4:414. Epub 2013 Dec 27.

Division of Genomics and Bioenvironmental Science, Frontier Science Research Center, University of Miyazaki Miyazaki, Japan.

To date 142 species have been described in the Vibrionaceae family of bacteria, classified into seven genera; Aliivibrio, Echinimonas, Enterovibrio, Grimontia, Photobacterium, Salinivibrio and Vibrio. As vibrios are widespread in marine environments and show versatile metabolisms and ecologies, these bacteria are recognized as one of the most diverse and important marine heterotrophic bacterial groups for elucidating the correlation between genome evolution and ecological adaptation. However, on the basis of 16S rRNA gene phylogeny, we could not find any robust monophyletic lineages in any of the known genera. We needed further attempts to reconstruct their evolutionary history based on multilocus sequence analysis (MLSA) and/or genome wide taxonomy of all the recognized species groups. In our previous report in 2007, we conducted the first broad multilocus sequence analysis (MLSA) to infer the evolutionary history of vibrios using nine housekeeping genes (the 16S rRNA gene, gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA), and we proposed 14 distinct clades in 58 species of Vibrionaceae. Due to the difficulty of designing universal primers that can amplify the genes for MLSA in every Vibrionaceae species, some clades had yet to be defined. In this study, we present a better picture of an updated molecular phylogeny for 86 described vibrio species and 10 genome sequenced Vibrionaceae strains, using 8 housekeeping gene sequences. This new study places special emphasis on (1) eight newly identified clades (Damselae, Mediterranei, Pectenicida, Phosphoreum, Profundum, Porteresiae, Rosenbergii, and Rumoiensis); (2) clades amended since the 2007 proposal with recently described new species; (3) orphan clades of genomospecies F6 and F10; (4) phylogenetic positions defined in 3 genome-sequenced strains (N418, EX25, and EJY3); and (5) description of V. tritonius sp. nov., which is a member of the "Porteresiae" clade.
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http://dx.doi.org/10.3389/fmicb.2013.00414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873509PMC
January 2014

Modulation of malaria infection in Anopheles gambiae mosquitoes exposed to natural midgut bacteria.

PLoS One 2013 6;8(12):e81663. Epub 2013 Dec 6.

UMR MIVEGEC (IRD 224- CNRS 5290- UM1- UM2), Institut de Recherche pour le Développement, Montpellier, France ; Laboratoire d'entomologie médicale, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale, Yaoundé, Cameroon.

The development of Plasmodium falciparum within the Anopheles gambiae mosquito relies on complex vector-parasite interactions, however the resident midgut microbiota also plays an important role in mediating parasite infection. In natural conditions, the mosquito microbial flora is diverse, composed of commensal and symbiotic bacteria. We report here the isolation of culturable midgut bacteria from mosquitoes collected in the field in Cameroon and their identification based on the 16S rRNA gene sequencing. We next measured the effect of selected natural bacterial isolates on Plasmodium falciparum infection prevalence and intensity over multiple infectious feedings and found that the bacteria significantly reduced the prevalence and intensity of infection. These results contrast with our previous study where the abundance of Enterobacteriaceae positively correlated with P. falciparum infection (Boissière et al. 2012). The oral infection of bacteria probably led to the disruption of the gut homeostasis and activated immune responses, and this pinpoints the importance of studying microbe-parasite interactions in natural conditions. Our results indicate that the effect of bacterial exposure on P. falciparum infection varies with factors from the parasite and the human host and calls for deeper dissection of these parameters for accurate interpretation of bacterial exposure results in laboratory settings.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081663PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3855763PMC
September 2014

Exploration of Deinococcus-Thermus molecular diversity by novel group-specific PCR primers.

Microbiologyopen 2013 Oct 29;2(5):862-72. Epub 2013 Aug 29.

CEA, DSV, IBEB, SBVME, LIPM, F-13108, Saint-Paul-lez-Durance, France; CNRS, UMR 7265, F-13108, Saint-Paul-lez-Durance, France; Université d'Aix-Marseille, F-13108, Saint-Paul-lez-Durance, France; IRSN, PRP-ENV, SERIS, L2BT, F-13115, Saint Paul-lez-Durance, France.

The deeply branching Deinococcus-Thermus lineage is recognized as one of the most extremophilic phylum of bacteria. In previous studies, the presence of Deinococcus-related bacteria in the hot arid Tunisian desert of Tataouine was demonstrated through combined molecular and culture-based approaches. Similarly, Thermus-related bacteria have been detected in Tunisian geothermal springs. The present work was conducted to explore the molecular diversity within the Deinococcus-Thermus phylum in these extreme environments. A set of specific primers was designed in silico on the basis of 16S rRNA gene sequences, validated for the specific detection of reference strains, and used for the polymerase chain reaction (PCR) amplification of metagenomic DNA retrieved from the Tataouine desert sand and Tunisian hot spring water samples. These analyses have revealed the presence of previously undescribed Deinococcus-Thermus bacterial sequences within these extreme environments. The primers designed in this study thus represent a powerful tool for the rapid detection of Deinococcus-Thermus in environmental samples and could also be applicable to clarify the biogeography of the Deinococcus-Thermus phylum.
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http://dx.doi.org/10.1002/mbo3.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831646PMC
October 2013

Microbacterium lemovicicum sp. nov., a bacterium isolated from a natural uranium-rich soil.

Int J Syst Evol Microbiol 2013 Jul 21;63(Pt 7):2600-2606. Epub 2012 Dec 21.

Université d'Aix-Marseille, 13108 Saint-Paul-lez-Durance, France.

An actinobacterial strain, designated ViU22(T), was isolated from a natural uranium-rich soil and was studied using a polyphasic approach. Cells formed orange-pigmented colonies, were rod-shaped, Gram-positive (non-staining method), non-motile and non-spore-forming. This organism grew in 0-4.5 % (w/v) NaCl and at 15-37 °C, with optimal growth occurring in 0.5 % (w/v) NaCl and at 30 °C. Comparative 16S rRNA gene sequence analysis revealed that the strain ViU22(T) belonged to the genus Microbacterium. It exhibited highest 16S rRNA gene sequence similarity with the type strains of Microbacterium testaceum (98.14 %) and Microbacterium binotii (98.02 %). The DNA-DNA relatedness of strains ViU22(T) with the most closely related type strains Microbacterium testaceum and Microbacterium binotii DSM 19164(T) was 20.10 % (± 0.70) and 28.05 % (± 0.35), respectively. Strain ViU22(T) possessed a type B2β peptidoglycan with partial substitution of glutamic acid by 3-hydroxy glutamic acid. The major menaquinones were MK-11 and MK-12. Major polar lipids detected in the strain ViU22(T) were diphosphatidylglycerol, phosphatidylglycerol, an unknown phospholipid and unknown glycolipids. The predominant fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0, a pattern reported for other Microbacterium species. The major cell-wall sugars were galactose, xylose and mannose and the DNA G+C content was 71 mol%. Together, the DNA-DNA hybridization results and the differentiating phenotypic characteristics, showed that strain ViU22(T) should be classified as the type strain of a novel species within the genus Microbacterium, for which the name Microbacterium lemovicicum sp. nov. is proposed. The type strain is ViU22(T) ( = ATCC BAA-2396(T) = CCUG 62198(T) = DSM 25044(T)).
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http://dx.doi.org/10.1099/ijs.0.048454-0DOI Listing
July 2013

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy.

Nucleic Acids Res 2013 Jan 27;41(Database issue):D597-604. Epub 2012 Nov 27.

CNRS, UMR 7144, Adaptation et Diversité en Milieu Marin, 29682 Roscoff, France.

The interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR(2), http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientists.
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http://dx.doi.org/10.1093/nar/gks1160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531120PMC
January 2013

Significant and persistent impact of timber harvesting on soil microbial communities in Northern coniferous forests.

ISME J 2012 Dec 2;6(12):2199-218. Epub 2012 Aug 2.

Department of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.

Forest ecosystems have integral roles in climate stability, biodiversity and economic development. Soil stewardship is essential for sustainable forest management. Organic matter (OM) removal and soil compaction are key disturbances associated with forest harvesting, but their impacts on forest ecosystems are not well understood. Because microbiological processes regulate soil ecology and biogeochemistry, microbial community structure might serve as indicator of forest ecosystem status, revealing changes in nutrient and energy flow patterns before they have irreversible effects on long-term soil productivity. We applied massively parallel pyrosequencing of over 4.6 million ribosomal marker sequences to assess the impact of OM removal and soil compaction on bacterial and fungal communities in a field experiment replicated at six forest sites in British Columbia, Canada. More than a decade after harvesting, diversity and structure of soil bacterial and fungal communities remained significantly altered by harvesting disturbances, with individual taxonomic groups responding differentially to varied levels of the disturbances. Plant symbionts, like ectomycorrhizal fungi, and saprobic taxa, such as ascomycetes and actinomycetes, were among the most sensitive to harvesting disturbances. Given their significant ecological roles in forest development, the fate of these taxa might be critical for sustainability of forest ecosystems. Although abundant bacterial populations were ubiquitous, abundant fungal populations often revealed a patchy distribution, consistent with their higher sensitivity to the examined soil disturbances. These results establish a comprehensive inventory of bacterial and fungal community composition in northern coniferous forests and demonstrate the long-term response of their structure to key disturbances associated with forest harvesting.
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http://dx.doi.org/10.1038/ismej.2012.84DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3504969PMC
December 2012

Midgut microbiota of the malaria mosquito vector Anopheles gambiae and interactions with Plasmodium falciparum infection.

PLoS Pathog 2012 31;8(5):e1002742. Epub 2012 May 31.

UMR MIVEGEC (IRD 224- CNRS 5290- UM1- UM2), Montpellier, France.

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.
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http://dx.doi.org/10.1371/journal.ppat.1002742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364955PMC
October 2012

Patho-Genes.org: a website dedicated to gene sequences of potential bioterror bacteria and PCR primers used to amplify them.

Microb Biotechnol 2012 Sep 10;5(5):594-8. Epub 2012 Jun 10.

CNRS UMR 7138 Systématique Adaptation et Evolution, Université de Nice-Sophia Antipolis, Parc Valrose BP71, F06108, Nice cedex, 02, France.

Pathogenic agents can be very hard to detect, and usually they do not cause illness for several hours or days. To improve the speed and the accuracy of detection tests and satisfy the needs of early diagnosis, molecular biology methods such as PCR are now used. However, selecting a proper target gene and designing good primers is often not easy. We present a dedicated website, http://patho-genes.org, where we provide every sequence, functional annotation, published primer and relevant article for every annotated gene of major pathogenic bacterial species listed as key agents to be used for a bioterrorism attack. Each published primer was analysed to determine its melting temperature, its specificity and its coverage (i.e. its sensitivity against every allele of its target gene). Data generated have been organized in the form of data sheet for each gene, which are available through multiple browser panels and query systems.
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http://dx.doi.org/10.1111/j.1751-7915.2012.00353.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3815871PMC
September 2012

In silico analyses of primers used to detect the pathogenicity genes of Vibrio cholerae.

Microbes Environ 2012 17;27(3):250-6. Epub 2012 May 17.

Centre de Biochimie, Université de Nice Sophia-Antipolis, Parc Valrose, F 06108 Nice, France.

In Vibrio cholerae, the etiological agent of cholera, most of the virulence genes are located in two pathogenicity islands, named TCP (Toxin-Co-regulated Pilus) and CTX (Cholera ToXins). For each V. cholerae pathogenicity gene, we retrieved every primer published since 1990 and every known allele in order to perform a complete in silico survey and assess the quality of the PCR primers used for amplification of these genes. Primers with a melting temperature in the range 55-60°C against any target sequence were considered valid. Our survey clearly revealed that two thirds of the published primers are not able to properly detect every genetic variant of the target genes. Moreover, the quality of primers did not improve with time. Their lifetime, i.e. the number of times they were cited in the literature, is also not a factor allowing the selection of valid primers. We were able to improve some primers or design new primers for the few cases where no valid primer was found. In conclusion, many published primers should be avoided or improved for use in molecular detection tests, in order to improve and perfect specificity and coverage. This study suggests that bioinformatic analyses are important to validate the choice of primers.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036039PMC
http://dx.doi.org/10.1264/jsme2.me11317DOI Listing
January 2013

Influence of uranium on bacterial communities: a comparison of natural uranium-rich soils with controls.

PLoS One 2011 5;6(10):e25771. Epub 2011 Oct 5.

CEA, DSV, IBEB, Laboratoire Interactions Protéine Métal, Saint-Paul-lez-Durance, France.

This study investigated the influence of uranium on the indigenous bacterial community structure in natural soils with high uranium content. Radioactive soil samples exhibiting 0.26% - 25.5% U in mass were analyzed and compared with nearby control soils containing trace uranium. EXAFS and XRD analyses of soils revealed the presence of U(VI) and uranium-phosphate mineral phases, identified as sabugalite and meta-autunite. A comparative analysis of bacterial community fingerprints using denaturing gradient gel electrophoresis (DGGE) revealed the presence of a complex population in both control and uranium-rich samples. However, bacterial communities inhabiting uraniferous soils exhibited specific fingerprints that were remarkably stable over time, in contrast to populations from nearby control samples. Representatives of Acidobacteria, Proteobacteria, and seven others phyla were detected in DGGE bands specific to uraniferous samples. In particular, sequences related to iron-reducing bacteria such as Geobacter and Geothrix were identified concomitantly with iron-oxidizing species such as Gallionella and Sideroxydans. All together, our results demonstrate that uranium exerts a permanent high pressure on soil bacterial communities and suggest the existence of a uranium redox cycle mediated by bacteria in the soil.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0025771PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3187815PMC
February 2012

Molecular biomass and MetaTaxogenomic assessment of soil microbial communities as influenced by soil DNA extraction procedure.

Microb Biotechnol 2012 Jan 12;5(1):135-41. Epub 2011 Oct 12.

INRA-Université de Bourgogne, UMR Microbiologie du Sol et de l'Environnement, CMSE, 17, rue Sully, B.V. 86510, 21065 Dijon Cedex, France.

Three soil DNA extraction procedures (homemade protocols and commercial kit) varying in their practicability were applied to contrasting soils to evaluate their efficiency in recovering: (i) soil DNA and (ii) bacterial diversity estimated by 16S rDNA pyrosequencing. Significant differences in DNA yield were systematically observed between tested procedures. For certain soils, 10 times more DNA was recovered with one protocol than with the others. About 15,000 sequences of 16S rDNA were obtained for each sample which were clustered to draw rarefaction curves. These curves, as well as the PCA ordination of community composition based on OTU clustering, did not reveal any significant difference between procedures. Nevertheless, significant differences between procedures were highlighted by the taxonomic identification of sequences obtained at the phylum to genus levels. Depending on the soil, differences in the number of genera detected ranged from 1% to 26% between the most and least efficient procedures, mainly due to a poorer capacity to recover populations belonging to Actinobacteria, Firmicutes or Crenarchaeota. This study enabled us to rank the relative efficiencies of protocols for their recovery of soil molecular microbial biomass and bacterial diversity and to help choosing an appropriate soil DNA extraction procedure adapted to novel sequencing technologies.
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http://dx.doi.org/10.1111/j.1751-7915.2011.00307.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3815280PMC
January 2012

Validation and application of a PCR primer set to quantify fungal communities in the soil environment by real-time quantitative PCR.

PLoS One 2011 8;6(9):e24166. Epub 2011 Sep 8.

INRA-Université de Bourgogne, UMR Microbiologie du Sol et de l'Environnement, CMSE, Dijon, France.

Fungi constitute an important group in soil biological diversity and functioning. However, characterization and knowledge of fungal communities is hampered because few primer sets are available to quantify fungal abundance by real-time quantitative PCR (real-time Q-PCR). The aim in this study was to quantify fungal abundance in soils by incorporating, into a real-time Q-PCR using the SYBRGreen® method, a primer set already used to study the genetic structure of soil fungal communities. To satisfy the real-time Q-PCR requirements to enhance the accuracy and reproducibility of the detection technique, this study focused on the 18S rRNA gene conserved regions. These regions are little affected by length polymorphism and may provide sufficiently small targets, a crucial criterion for enhancing accuracy and reproducibility of the detection technique. An in silico analysis of 33 primer sets targeting the 18S rRNA gene was performed to select the primer set with the best potential for real-time Q-PCR: short amplicon length; good fungal specificity and coverage. The best consensus between specificity, coverage and amplicon length among the 33 sets tested was the primer set FR1/FF390. This in silico analysis of the specificity of FR1/FF390 also provided additional information to the previously published analysis on this primer set. The specificity of the primer set FR1/FF390 for Fungi was validated in vitro by cloning--sequencing the amplicons obtained from a real time Q-PCR assay performed on five independent soil samples. This assay was also used to evaluate the sensitivity and reproducibility of the method. Finally, fungal abundance in samples from 24 soils with contrasting physico-chemical and environmental characteristics was examined and ranked to determine the importance of soil texture, organic carbon content, C∶N ratio and land use in determining fungal abundance in soils.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024166PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3169588PMC
March 2012

Ultra-deep sequencing of foraminiferal microbarcodes unveils hidden richness of early monothalamous lineages in deep-sea sediments.

Proc Natl Acad Sci U S A 2011 Aug 25;108(32):13177-82. Epub 2011 Jul 25.

Department of Genetics and Evolution, University of Geneva, CH-1211 Geneva 4, Switzerland.

Deep-sea floors represent one of the largest and most complex ecosystems on Earth but remain essentially unexplored. The vastness and remoteness of this ecosystem make deep-sea sampling difficult, hampering traditional taxonomic observations and diversity assessment. This problem is particularly true in the case of the deep-sea meiofauna, which largely comprises small-sized, fragile, and difficult-to-identify metazoans and protists. Here, we introduce an ultra-deep sequencing-based metagenetic approach to examine the richness of benthic foraminifera, a principal component of deep-sea meiofauna. We used Illumina sequencing technology to assess foraminiferal richness in 31 unsieved deep-sea sediment samples from five distinct oceanic regions. We sequenced an extremely short fragment (36 bases) of the small subunit ribosomal DNA hypervariable region 37f, which has been shown to accurately distinguish foraminiferal species. In total, we obtained 495,978 unique sequences that were grouped into 1,643 operational taxonomic units, of which about half (841) could be reliably assigned to foraminifera. The vast majority of the operational taxonomic units (nearly 90%) were either assigned to early (ancient) lineages of soft-walled, single-chambered (monothalamous) foraminifera or remained undetermined and yet possibly belong to unknown early lineages. Contrasting with the classical view of multichambered taxa dominating foraminiferal assemblages, our work reflects an unexpected diversity of monothalamous lineages that are as yet unknown using conventional micropaleontological observations. Although we can only speculate about their morphology, the immense richness of deep-sea phylotypes revealed by this study suggests that ultra-deep sequencing can improve understanding of deep-sea benthic diversity considered until now as unknowable based on a traditional taxonomic approach.
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http://dx.doi.org/10.1073/pnas.1018426108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3156150PMC
August 2011

Analysis of bacterial core communities in the central Baltic by comparative RNA-DNA-based fingerprinting provides links to structure-function relationships.

ISME J 2012 Jan 23;6(1):195-212. Epub 2011 Jun 23.

Department of Vaccinology and Applied Microbiology, Helmholtz Centre of Infection Research (HZI), Braunschweig, Germany.

Understanding structure-function links of microbial communities is a central theme of microbial ecology since its beginning. To this end, we studied the spatial variability of the bacterioplankton community structure and composition across the central Baltic Sea at four stations, which were up to 450 km apart and at a depth profile representative for the central part (Gotland Deep, 235 m). Bacterial community structure was followed by 16S ribosomal RNA (rRNA)- and 16S rRNA gene-based fingerprints using single-strand conformation polymorphism (SSCP) electrophoresis. Species composition was determined by sequence analysis of SSCP bands. High similarities of the bacterioplankton communities across several hundred kilometers were observed in the surface water using RNA- and DNA-based fingerprints. In these surface communities, the RNA- and DNA-based fingerprints resulted in very different pattern, presumably indicating large difference between the active members of the community as represented by RNA-based fingerprints and the present members represented by the DNA-based fingerprints. This large discrepancy changed gradually over depth, resulting in highly similar RNA- and DNA-based fingerprints in the anoxic part of the water column below 130 m depth. A conceivable mechanism explaining this high similarity could be the reduced oxidative stress in the anoxic zone. The stable communities on the surface and in the anoxic zone indicate the strong influence of the hydrography on the bacterioplankton community structure. Comparative analysis of RNA- and DNA-based community structure provided criteria for the identification of the core community, its key members and their links to biogeochemical functions.
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http://dx.doi.org/10.1038/ismej.2011.80DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3246236PMC
January 2012

Defining reference sequences for Nocardia species by similarity and clustering analyses of 16S rRNA gene sequence data.

PLoS One 2011 8;6(6):e19517. Epub 2011 Jun 8.

Sydney Medical School, The University of Sydney, Sydney, New South Wales, Australia.

Background: The intra- and inter-species genetic diversity of bacteria and the absence of 'reference', or the most representative, sequences of individual species present a significant challenge for sequence-based identification. The aims of this study were to determine the utility, and compare the performance of several clustering and classification algorithms to identify the species of 364 sequences of 16S rRNA gene with a defined species in GenBank, and 110 sequences of 16S rRNA gene with no defined species, all within the genus Nocardia.

Methods: A total of 364 16S rRNA gene sequences of Nocardia species were studied. In addition, 110 16S rRNA gene sequences assigned only to the Nocardia genus level at the time of submission to GenBank were used for machine learning classification experiments. Different clustering algorithms were compared with a novel algorithm or the linear mapping (LM) of the distance matrix. Principal Components Analysis was used for the dimensionality reduction and visualization.

Results: The LM algorithm achieved the highest performance and classified the set of 364 16S rRNA sequences into 80 clusters, the majority of which (83.52%) corresponded with the original species. The most representative 16S rRNA sequences for individual Nocardia species have been identified as 'centroids' in respective clusters from which the distances to all other sequences were minimized; 110 16S rRNA gene sequences with identifications recorded only at the genus level were classified using machine learning methods. Simple kNN machine learning demonstrated the highest performance and classified Nocardia species sequences with an accuracy of 92.7% and a mean frequency of 0.578.

Conclusion: The identification of centroids of 16S rRNA gene sequence clusters using novel distance matrix clustering enables the identification of the most representative sequences for each individual species of Nocardia and allows the quantitation of inter- and intra-species variability.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019517PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3110597PMC
October 2011

Eukaryotic richness in the abyss: insights from pyrotag sequencing.

PLoS One 2011 Apr 4;6(4):e18169. Epub 2011 Apr 4.

Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland.

Background: The deep sea floor is considered one of the most diverse ecosystems on Earth. Recent environmental DNA surveys based on clone libraries of rRNA genes confirm this observation and reveal a high diversity of eukaryotes present in deep-sea sediment samples. However, environmental clone-library surveys yield only a modest number of sequences with which to evaluate the diversity of abyssal eukaryotes.

Methodology/principal Findings: Here, we examined the richness of eukaryotic DNA in deep Arctic and Southern Ocean samples using massively parallel sequencing of the 18S ribosomal RNA (rRNA) V9 hypervariable region. In very small volumes of sediments, ranging from 0.35 to 0.7 g, we recovered up to 7,499 unique sequences per sample. By clustering sequences having up to 3 differences, we observed from 942 to 1756 Operational Taxonomic Units (OTUs) per sample. Taxonomic analyses of these OTUs showed that DNA of all major groups of eukaryotes is represented at the deep-sea floor. The dinoflagellates, cercozoans, ciliates, and euglenozoans predominate, contributing to 17%, 16%, 10%, and 8% of all assigned OTUs, respectively. Interestingly, many sequences represent photosynthetic taxa or are similar to those reported from the environmental surveys of surface waters. Moreover, each sample contained from 31 to 71 different metazoan OTUs despite the small sample volume collected. This indicates that a significant faction of the eukaryotic DNA sequences likely do not belong to living organisms, but represent either free, extracellular DNA or remains and resting stages of planktonic species.

Conclusions/significance: In view of our study, the deep-sea floor appears as a global DNA repository, which preserves genetic information about organisms living in the sediment, as well as in the water column above it. This information can be used for future monitoring of past and present environmental changes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0018169PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3070721PMC
April 2011

Protistan microbial observatory in the Cariaco Basin, Caribbean. I. Pyrosequencing vs Sanger insights into species richness.

ISME J 2011 Aug 10;5(8):1344-56. Epub 2011 Mar 10.

Department of Geology and Geophysics, Woods Hole Oceanographic Institution, Woods Hole, MA, USA.

Microbial diversity and distribution are topics of intensive research. In two companion papers in this issue, we describe the results of the Cariaco Microbial Observatory (Caribbean Sea, Venezuela). The Basin contains the largest body of marine anoxic water, and presents an opportunity to study protistan communities across biogeochemical gradients. In the first paper, we survey 18S ribosomal RNA (rRNA) gene sequence diversity using both Sanger- and pyrosequencing-based approaches, employing multiple PCR primers, and state-of-the-art statistical analyses to estimate microbial richness missed by the survey. Sampling the Basin at three stations, in two seasons, and at four depths with distinct biogeochemical regimes, we obtained the largest, and arguably the least biased collection of over 6000 nearly full-length protistan rRNA gene sequences from a given oceanographic regime to date, and over 80,000 pyrosequencing tags. These represent all major and many minor protistan taxa, at frequencies globally similar between the two sequence collections. This large data set provided, via the recently developed parametric modeling, the first statistically sound prediction of the total size of protistan richness in a large and varied environment, such as the Cariaco Basin: over 36,000 species, defined as almost full-length 18S rRNA gene sequence clusters sharing over 99% sequence homology. This richness is a small fraction of the grand total of known protists (over 100,000-500,000 species), suggesting a degree of protistan endemism.
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http://dx.doi.org/10.1038/ismej.2011.6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3146274PMC
August 2011

Depicting more accurate pictures of protistan community complexity using pyrosequencing of hypervariable SSU rRNA gene regions.

Environ Microbiol 2011 Feb 8;13(2):340-9. Epub 2010 Sep 8.

Department of Ecology, University of Kaiserslautern, D-67653 Kaiserslautern, Germany.

Initial environmental pyrosequencing studies suggested highly complex protistan communities with phylotype richness decisively higher than previously estimated. However, recent studies on individual bacteria or artificial bacterial communities evidenced that pyrosequencing errors may skew our view of the true complexity of microbial communities. We pyrosequenced two diversity markers (hypervariable regions V4 and V9 of the small-subunit rDNA) of an intertidal protistan model community, using the Roche GS-FLX and the most recent GS-FLX Titanium sequencing systems. After pyrosequencing 24 reference sequences we obtained up to 2039 unique tags (from 3879 V4 GS-FLX Titanium reads), 77% of which were singletons. Even binning sequences that share 97% similarity still emulated a pseudodiversity exceeding the true complexity of the model community up to three times (V9 GS-FLX). Pyrosequencing error rates were higher for V4 fragments compared with the V9 domain and for the GS-FLX Titanium compared with the GS-FLX system. Furthermore, this experiment revealed that error rates are taxon-specific. As an outcome of this study we suggest a fast and efficient strategy to discriminate pyrosequencing signals from noise in order to more realistically depict the structure of protistan communities using simple tools that are implemented in standard tag data-processing pipelines.
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http://dx.doi.org/10.1111/j.1462-2920.2010.02332.xDOI Listing
February 2011
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