Transcriptome analyses to investigate symbiotic relationships between marine protists

International audienceRhizaria are an important component of oceanic plankton communities worldwide. A number of species harbor eukaryotic microalgal symbionts, which are horizontally acquired in the environment at each generation. Although these photosymbioses are determinant for Rhizaria ability to thrive in oceanic ecosystems, the mechanisms for symbiotic interactions are unclear. Using high-throughput sequencing technology (i.e., 454), we generated large Expressed Sequence Tag (EST) datasets from four uncultured Rhizaria, an acantharian (Amphilonche elongata), two polycystines (Collozoum sp. and Spongosphaera streptacantha), and one phaeodarian (Aulacantha scolymantha). We assessed the main genetic features of the host/symbionts consortium (i.e., the holobiont) transcriptomes and found rRNA sequences affiliated to a wide range of bacteria and protists in all samples, suggesting that diverse microbial communities are associated with the holobionts. A particular focus was then carried out to search for genes potentially involved in symbiotic processes such as the presence of c-type lectins-coding genes, which are proteins that play a role in cell recognition among eukaryotes. Unigenes coding putative c-type lectin domains (CTLD) were found in the species bearing photosynthetic symbionts (A. elongata, Collozoum sp., and S. streptacantha) but not in the non-symbiotic one (A. scolymantha). More particularly, phylogenetic analyses group CTLDs from A. elongata and Collozoum sp. on a distinct branch from S. streptacantha CTLDs, which contained carbohydrate-binding motifs typically observed in other marine photosymbiosis. Our data suggest that similarly to other well-known marine photosymbiosis involving metazoans, the interactions of glycans with c-type lectins is likely involved in modulation of the host/symbiont specific recognition in Radiolaria

Cytoklepty in the plankton: A host strategy to optimize the bioenergetic machinery of endosymbiotic algae

Endosymbioses have shaped the evolutionary trajectory of life and remain ecologically important. Investigating oceanic photosymbioses can illuminate how algal endosymbionts are energetically exploited by their heterotrophic hosts and inform on putative initial steps of plastid acquisition in eukaryotes. By combining three-dimensional subcellular imaging with photophysiology, carbon flux imaging, and transcriptomics, we show that cell division of endosymbionts (Phaeocystis) is blocked within hosts (Acantharia) and that their cellular architecture and bioenergetic machinery are radically altered. Transcriptional evidence indicates that a nutrient-independent mechanism prevents symbiont cell division and decouples nuclear and plastid division. As endosymbiont plastids proliferate, the volume of the photosynthetic machinery volume increases 100-fold in correlation with the expansion of a reticular mitochondrial network in close proximity to plastids. Photosynthetic efficiency tends to increase with cell size, and photon propagation modeling indicates that the networked mitochondrial architecture enhances light capture. This is accompanied by 150-fold higher carbon uptake and up-regulation of genes involved in photosynthesis and carbon fixation, which, in conjunction with a ca.15-fold size increase of pyrenoids demonstrates enhanced primary production in symbiosis. Mass spectrometry imaging revealed major carbon allocation to plastids and transfer to the host cell. As in most photosymbioses, microalgae are contained within a host phagosome (symbiosome), but here, the phagosome invaginates into enlarged microalgal cells, perhaps to optimize metabolic exchange. This observation adds evidence that the algal metamorphosis is irreversible. Hosts, therefore, trigger and benefit from major bioenergetic remodeling of symbiotic microalgae with potential consequences for the oceanic carbon cycle. Unlike other photosymbioses, this interaction represents a so-called cytoklepty, which is a putative initial step toward plastid acquisition

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote Small Sub-Unit rRNA sequences with curated taxonomy

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 (PR2, 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 scientist

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote Small Sub-Unit rRNA sequences with curated taxonomy

International audienceThe 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 (PR2, 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

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

International audienceAlthough 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

Ecologie et évolution de la photosymbiose chez les acanthaires (radiolaires)

Les relations symbiotiques sont répandues dans la nature et sont fondamentales pour le fonctionnement des écosystèmes et l'évolution de la biodiversité. Dans le milieu marin, la symbiose mutualiste avec des microalgues (photosymbiose) a été très étudiée dans les écosystèmes récifaux, mais reste incomprise dans l'océan ouvert malgré le rôle majeur du plancton dans l'écologie et la biogéochimie globale. Les acanthaires (radiolaires), unicellulaires eucaryotes caractérisés par un squelette minéral en sulfate de strontium, sont des acteurs clés dans le milieu pélagique. Bien que découverte au 19ème siècle, leur relation photosymbiotique a été très peu étudiée depuis. Les acanthaires sont un modèle prometteur pour explorer l importance évolutive et écologique de la photosymbiose dans le domaine planctonique. Le principal objectif de ma thèse était de dévoiler l'identité et la diversité des microalgues symbiotiques, et d'étudier l'influence de la symbiose sur l'évolution, l'écologie et le cycle de vie des acanthaires à différentes échelles, de la cellule aux écosystèmes. Pour mettre en évidence le contexte évolutif de la symbiose, des analyses phylogénétiques ont été réalisées avec 78 et 107 séquences partielles de l'ADNr 18S et l ADNr 28S, respectivement. Chacune de ces séquences a été obtenue à partir de cellule unique d acanthaire. Cette phylogénie, composée de dix clades, a révélé des conflits avec la taxonomie traditionnelle qui date de 100 ans. Les clades les plus récents et les plus divers génétiquement (clades E et F) sont représentés par les acanthaires symbiotiques, qui ont un squelette complexe par rapport aux non-symbiotiques. Cela suggère que la symbiose a été une source d innovation pour la complexité phénotypique de ces microorganismes. Les analyses phylogénétiques ont également été menées sur les microalgues symbiotiques à partir de différentes espèces hôtes et de régions géographiques distinctes. Nous avons constaté que tous les acanthaires des clades E et F vivent constamment avec la microalgue Phaeocystis (haptophyte), une espèce phytoplanctonique clé dans les écosystèmes marins, dont le mode symbiotique était inconnu. Une horloge moléculaire et des analyses cophylogénétiques ont montré que l origine de cette symbiose date du Mésozoïque (~175 Mya) dans des conditions sévères d oligotrophie, et que la biogéographie, plutôt que la taxonomie de l hôte, est le principal déterminant de l'association. Chez une espèce d acanthaire d un clade basal, une autre association a été caractérisée avec des symbiotes de différentes lignées de microalgues (haptophytes et dinoflagellés) au sein d'une seule cellule hôte. Comme Phaeocystis, ces microalgues sont abondantes et ubiquitaires en phase libre, formant parfois de grandes efflorescences. Cela contraste avec les symbioses terrestres et marines, où le symbiote est généralement rare en dehors de son hôte. Ce mode original d'interaction serait une stratégie pour assurer la capture récurrente d'un partenaire approprié dans l océan ouvert, un monde vaste et moins dense en organismes. Enfin, la distribution géographique et l'abondance relative des groupes d acanthaires ont été explorées par une approche de metabarcoding. La base de données morpho-génétique a été utilisée pour lier les séquences environnementales V9 de l ADNr 18S à un morphotype. Les acanthaires symbiotiques étaient prédominants dans la zone photique sous toutes les latitudes, soulignant le succès écologique et la flexibilité de cette association. Au contraire, les groupes non-symbiotiques se situaient principalement dans les eaux méso- et bathypélagiques. Nous avons montré que ces acanthaires ont une partie de leur cycle de vie dans ces eaux profondes via la formation de kystes très denses, contribuant au cycle biogéochimique du carbone et du strontium. Ce travail de thèse soulève des hypothèses sur l'évolution morphologique des acanthaires, ainsi que sur le fonctionnement et l'évolution de la photosymbiose planctonique, mettant en évidence les forces évolutives intrinsèques de l immense milieu pélagiquePARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Intracellular Diversity of the V4 and V9 Regions of the 18S rRNA in Marine Protists (Radiolarians) Assessed by High-Throughput Sequencing

International audienceMetabarcoding is a powerful tool for exploring microbial diversity in the environment, but its accurate interpretation is impeded by diverse technical (e.g. PCR and sequencing errors) and biological biases (e.g. intra-individual polymorphism) that remain poorly understood. To help interpret environmental metabarcoding datasets, we investigated the intracellular diversity of the V4 and V9 regions of the 18S rRNA gene from Acantharia and Nassellaria (radiolarians) using 454 pyrosequencing. Individual cells of radiolarians were isolated, and PCRs were performed with generalist primers to amplify the V4 and V9 regions. Different denoising procedures were employed to filter the pyrosequenced raw amplicons (Acacia, AmpliconNoise, Linkage method). For each of the six isolated cells, an average of 541 V4 and 562 V9 amplicons assigned to radiolarians were obtained, from which one numerically dominant sequence and several minor variants were found. At the 97% identity, a diversity metrics commonly used in environmental surveys, up to 5 distinct OTUs were detected in a single cell. However, most amplicons grouped within a single OTU whereas other OTUs contained very few amplicons. Different analytical methods provided evidence that most minor variants forming different OTUs correspond to PCR and sequencing artifacts. Duplicate PCR and sequencing from the same DNA extract of a single cell had only 9 to 16% of unique amplicons in common, and alignment visualization of V4 and V9 amplicons showed that most minor variants contained substitutions in highly-conserved regions. We conclude that intracellular variability of the 18S rRNA in radiolarians is very limited despite its multi-copy nature and the existence of multiple nuclei in these protists. Our study recommends some technical guidelines to conservatively discard artificial amplicons from metabarcoding datasets, and thus properly assess the diversity and richness of protists in the environment

Molecular Phylogeny and Morphological Evolution of the Acantharia (Radiolaria)

Acantharia are ubiquitous and abundant rhizarian protists in the world ocean. The skeleton made of strontium sulphate and the fact that certain harbour microalgal endosymbionts make them key planktonic players for the ecology of marine ecosystems. Based on morphological criteria, the current taxonomy of Acantharia was established by W.T. Schewiakoff in 1926, since when no major revision has been undertaken. Here, we established the first comprehensive molecular phylogeny from single morphologically-identified acantharian cells, isolated from various oceans. Our phylogenetic analyses based on 78 18S rDNA and 107 partial 28S rDNA revealed the existence of 6 main clades, sub-divided into 13 sub-clades. The polyphyletic nature of acantharian families and genera demonstrates the need for revision of the current taxonomy. This molecular phylogeny, which highlights the taxonomic relevance of specific morphological criteria, such as the presence of a shell and the organisation of the central junction, provides a robust phylogenetic framework for future taxonomic emendation. Finally, mapping all the existing environmental sequences available to date from different marine ecosystems onto our reference phylogeny unveiled another 3 clades and improved the understanding of the biogeography and ecology of Acantharia. © 2011 Elsevier GmbH. All rights reserved. Key words: Acantharia; strontium sulfate; molecular phylogeny; Radiolaria; Rhizaria; single-cells. Introduction Acantharia are marine protists taxonomically affiliated to the super-group Rhizaria, the phylum Retaria and to the first rank taxon Radiolaria (Adl et al. 2005; Moreira et al. 2007). Their characteristic star-shaped morphology consists of a skeleton of 10 or 20 spicules made of celestite (strontium sulphate; Odum 1951; Suzuki and Aita, 2011), arranged according to the geometric law of Müller 1 Corresponding author; fax +33 2 98 29 23 23 e-mail [email protected] (J. Decelle). (1859). The skeleton supports a typical rhizarian amoeboid cell whose shape and motility are controlled by-characteristic axopods and myonemes (Febvre, 1981). The Acantharia are the only known organisms able to biomineralize strontium sulfate as the principal component of the skeleton. In surface waters of marine ecosystems, Acantharia consistently outnumber their rhizarian counterparts, such as Foraminifera and Polycystinea (Caron and Swanberg 1990; Michaels et al. 1995; Stoecker et al. 2009 Andreoli 1982), and they can form blooms at certain periods of the year, reaching densities up to 500 000 individuals.m -2 (Massera Bottazzi and Andreoli 1978, 1981). Acantharian cells have also been found hundreds and even thousands of meters deep in the water column (Antia et al. 1993; Bernstein et al. 1987; Martin et al. 2010). Within the marine food web, the Acantharia are active predators (Swanberg and Caron 1991), and they significantly contribute to carbon flux and biogeochemical cycles of strontium and barium in the oceans (Bernstein et al., 1987; Martin et al. 2010). Furthermore, they indirectly contribute to primary production through endosymbiotic relationships with tens to hundreds of microalgae per cell (Michaels 1988(Michaels , 1991. Despite holding a key position in marine ecosystems, the Acantharia have been largely overlooked in ecological studies, essentially due to dissolution of the skeleton in classical fixatives (Beers and Stewart 1970) and the lack of success in culturing them. Our knowledge of their biology and diversity is therefore still in its infancy. The first classification of Acantharia was initiated by Johannes Müller (1856Müller ( , 1859, and completed by his student Ernst Haeckel (1887, 1888). This classification initially comprised 372 species, and further studies added around 80 more (Mielck 1907; Popofsky 1904a Popofsky , b, 1906. All of the diagnostic characters used by these authors were exclusively based on the morphology of the skeleton, such as the length, form and central junction of the spicules. Working on living specimens, W.T. Schewiakoff emended this classification in 1926 by taking into account various features of the cell body (e.g. structure and colour of the cytoplasm, absence or presence of the central capsule, myonemes). In his remarkable monograph based on accurate observations of 500 living cells, he confirmed a total of 130 species, and erected the main taxonomic framework for the Acantharia (Schewiakoff 1926). Minor modifications have since been made to this classification (Bernstein et al. 1999; Febvre et al. 2000; Reshetnyak 1981; Tan 1998; Trégouboff 1953). The class Acantharia currently comprises around 50 genera and 150 species, which are grouped into 18 families distributed in 4 orders: Holacanthida, Chaunacanthida, Symphiacanthida and Arthracanthida (Bernstein et al. 1999). The distinction between the four orders is mainly based on the way the spicules cross the cytoplasm. In the Holacanthida, which was considered by Schewiakoff to be the most basal order, 10 diametral spicules loosely cross the centre of the cell, where they tangle to form a central body (Acanthocollidae) or do not join at all (Acantochiasmidae and Acanthoplegmidae). The Chaunacanthida are characterized by having 20 spicules that are more or less joined at the cell centre and that can be easily dissociated. The Symphiacanthida and the Arthracanthida have 20 tightly joined spicules. The spicules in the Symphiacanthida are attached to each other at the centre by their basal parts, forming a uniform central body. The Arthracanthida, which are characterized by the presence of a thick central capsule, were suggested to represent the most derived forms of Acantharia (Schewiakoff 1926), and are divided into two suborders, the Sphaenacantha and the Phyllacantha. Hitherto, there has been little effort to validate this morphology-based acantharian taxonomy using molecular phylogenetics. Because of the elusive nature of Acantharia and the difficulty to perform accurate morphological identification on living specimens, very few cells have been isolated, morphologically identified and sequenced (about 20 18S rDNA sequences are publicly available to date). For instance, only 4 of the 30 genera of Arthracanthida are represented in GenBank from isolated specimens (Gilg et al. 2009; Oka et al. 2005; Zettler et al. 1997). Molecular phylogenies including these sequences have nevertheless highlighted inconsistencies within the existing morphological classification. The orders Symphiacanthida and Arthracanthida are mixed, the Chaunacanthida includes specimens identified as Symphiacanthida, and the Holacanthida is simply missing in these analyses (Gilg et al. 2009; Oka et al. 2005). Yet, together with more recent investigations (Krabberød et al. 2011), these studies demonstrated the monophyly of the Acantharia among the Radiolaria. In addition to sequences from isolates, numerous sequences assigned to Acantharia have been retrieved from environmental surveys of genetic diversity in various environments, including coastal López-García et al. 2003 and Edgcomb et al. 2002 respectively). This considerable diversity of environmental 18S rDNA sequences from Acantharia has no associated morphological information. This phenomenon will undoubtedly be further amplified with the advent of environmental surveys using high-throughput DNA sequencing technologies. As for many protist groups, reference sequences (from Molecular Phylogeny of Acantharia 437 morphologically identified organisms) are fundamental anchors to taxonomically characterize the coming profusion of environmental data. The current vision of the systematics of Acantharia is very obscure. Molecular tools can be helpful to examine the relationships among Acantharia, for which morphology-based classification is unstable (Febvre, 1989). The present study aims at producing a comprehensive molecular phylogeny of Acantharia, and shedding light on the morphological evolution of this ecologically key group of marine protists. To do so, we isolated, morphologically identified and sequenced ribosomal DNA markers (18S rDNA and partial 28S rDNA) for more than 100 acantharian specimens collected worldwide. This morpho-molecular approach on single-cells allowed to assess the validity of the current morphological taxonomy, and to explore the evolution of the group. Results Molecular Phylogeny of the Acantharia The entire 18S rDNA and the D1 and D2 regions of the 28S rDNA were sequenced from acantharian cells isolated in the Mediterranean sea, the Red sea, the English Channel and the West Pacific ocean. In total, 107 partial 28S rDNA and 78 18S rDNA sequences were obtained From the 28S rDNA phylogeny Comparison between Molecular Phylogeny and Morphological Taxonomy Our sampling included representatives of the 4 orders of Acantharia, including the first sequence data for Holacanthida. Based on morphological criteria, we identified 24 (of the 49 described) genera and 14 (of 18) families. Clades A, B, C and D encompass the two acantharian orders Holacanthida and Chaunacanthida, except for Phyllostaurus echinoides (Ei 68) and Acanthostaurus purpurascens (Vil 45) in clade B Clades E and F mainly contain representatives from the Arthracanthida, and a few from the Symphiacanthida. The specimens forming clade E are easier to identify compared to other Acantharia, due to the presence of a shel