Repeated co-option of HMG-box genes for sex determination in brown algae and animals.

In many eukaryotes, genetic sex determination is not governed by XX/XY or ZW/ZZ systems but by a specialized region on the poorly studied U (female) or V (male) sex chromosomes. Previous studies have hinted at the existence of a dominant male-sex factor on the V chromosome in brown algae, a group of multicellular eukaryotes distantly related to animals and plants. The nature of this factor has remained elusive. Here, we demonstrate that an HMG-box gene acts as the male-determining factor in brown algae, mirroring the role HMG-box genes play in sex determination in animals. Over a billion-year evolutionary timeline, these lineages have independently co-opted the HMG box for male determination, representing a paradigm for evolution's ability to recurrently use the same genetic "toolkit" to accomplish similar tasks.

Diverse eukaryotic phytoplankton from around the Marquesas Islands documented by combined microscopy and molecular techniques.

Oceanic phytoplankton serve as a base for the food webs within the largest planetary ecosystem. Despite this, surprisingly little is known about species composition, function and ecology of phytoplankton communities, especially for vast areas of the open ocean. In this study we focus on the marine phytoplankton microflora from the vicinity of the Marquesas Islands in the Southern Pacific Ocean collected during the Tara Oceans expedition. Multiple samples from four sites and two depths were studied in detail using light microscopy, scanning electron microscopy, and automated confocal laser scanning microscopy. In total 289 taxa were identified, with Dinophyceae and Bacillariophyceae contributing 60% and 32% of taxa, respectively, to phytoplankton community composition. Notwithstanding, a large number of cells could not be assigned to any known species. Coccolithophores and other flagellates together contributed less than 8% to the species list. Observed cell densities were generally low, but at sites of high autotrophic biomass, diatoms reached the highest cell densities (1.26 × 104 cells L-1). Overall, 18S rRNA metabarcode-based community compositions matched microscopy-based estimates, particularly for the main diatom taxa, indicating consistency and complementarity between different methods, while the wide range of microscopy-based methods permitted several unknown and poorly studied taxa to be revealed and identified.

The baseless mutant links protein phosphatase 2A with basal cell identity in the brown alga Ectocarpus.

The first mitotic division of the initial cell is a key event in all multicellular organisms and is associated with the establishment of major developmental axes and cell fates. The brown alga Ectocarpus has a haploid-diploid life cycle that involves the development of two multicellular generations: the sporophyte and the gametophyte. Each generation deploys a distinct developmental programme autonomously from an initial cell, the first cell division of which sets up the future body pattern. Here, we show that mutations in the BASELESS (BAS) gene result in multiple cellular defects during the first cell division and subsequent failure to produce basal structures during both generations. BAS encodes a type B″ regulatory subunit of protein phosphatase 2A (PP2A), and transcriptomic analysis identified potential effector genes that may be involved in determining basal cell fate. The bas mutant phenotype is very similar to that observed in distag (dis) mutants, which lack a functional Tubulin-binding co-factor Cd1 (TBCCd1) protein, indicating that TBCCd1 and PP2A are two essential components of the cellular machinery that regulates the first cell division and mediates basal cell fate determination.

A robust approach to estimate relative phytoplankton cell abundances from metagenomes.

Phytoplankton account for >45% of global primary production, and have an enormous impact on aquatic food webs and on the entire Earth System. Their members are found among prokaryotes (cyanobacteria) and multiple eukaryotic lineages containing chloroplasts. Genetic surveys of phytoplankton communities generally consist of PCR amplification of bacterial (16S), nuclear (18S) and/or chloroplastic (16S) rRNA marker genes from DNA extracted from environmental samples. However, our appreciation of phytoplankton abundance or biomass is limited by PCR-amplification biases, rRNA gene copy number variations across taxa, and the fact that rRNA genes do not provide insights into metabolic traits such as photosynthesis. Here, we targeted the photosynthetic gene psbO from metagenomes to circumvent these limitations: the method is PCR-free, and the gene is universally and exclusively present in photosynthetic prokaryotes and eukaryotes, mainly in one copy per genome. We applied and validated this new strategy with the size-fractionated marine samples collected by Tara Oceans, and showed improved correlations with flow cytometry and microscopy than when based on rRNA genes. Furthermore, we revealed unexpected features of the ecology of these ecosystems, such as the high abundance of picocyanobacterial aggregates and symbionts in the ocean, and the decrease in relative abundance of phototrophs towards the larger size classes of marine dinoflagellates. To facilitate the incorporation of psbO in molecular-based surveys, we compiled a curated database of >18,000 unique sequences. Overall, psbO appears to be a promising new gene marker for molecular-based evaluations of entire phytoplankton communities.

A primary small cell neuroendocrine carcinoma (SCNC) of the oral cavity (cheek mucosa): Description of a case report.

BACKGROUND: Small cell neuroendocrine carcinoma (SCNC) of the oral cavity is a poorly differentiated, high-grade and very aggressive tumor with a poor prognosis. CASE DESCRIPTION: A 64-year-old, Caucasian, smoker man consulted for an ulcero-necrotic, exophytic, lesion of the right retromolar trigone. Haed&neck CT scan showed a right tonsillar tumor lesion. The 18F-PET scan confirmed the presence of a right, highly hypermetabolic tonsillar lesion and two homolateral, cervical lymph nodes. Histology and immunohistochemistry were consisted with the diagnosis of a primary SCNC of the oral cavity. As the tumor was locally advanced and unresectable, the patient underwent a definitive radio-chemotherapy with a cisplatin/etoposide combined regimen (4 cycles). The treatment was well tolerated and led to a complete tumor response. CONCLUSION: The particularity of this case relies on the rarity of the oral SCNC, its difficult and challenging diagnosis, and the complexity of its management that is not validated by large clinical trials, data being extrapolated from small cell lung cancer. In our case, the patient presenting a locally advanced tumor was treated by a combined radio-chemiotherapy leading to a complete tumor regression. The patient's follow up is too short to assess the real benefit of this treatment on overall survival.

A fatal primary gastric melanoma treated by a double immunotherapy with ipilimumab/nivolumab: description of a case report.

Background: Primary gastric melanoma (GM) is a very uncommon tumor with a poor prognosis. Until now, only a few cases have been reported in the literature. Case Description: A 70-year-old, Caucasian, ex-smoker man, presented with asthenia, anorexia, and weight loss of 5 kg during the last 2 months. Biological test showed high levels of transaminases and a microcytic, hypochromic anemia. Whole body CT-scan documented a gastric tumor lesion with concomitant loco-regional lymph node and hepatic metastases. Histology was consisted with the diagnosis of a primary GM. A double immunotherapy with nivolumab and ipilimumab was started but, 2 weeks later, the patient presented an acute hepatic failure quickly leading to his death despite a high dose corticotherapy. Conclusions: The particularity of this case relies on the rarity of GM, its difficult diagnosis representing a clinical challenge, and the complexity of its management that is not validated by large clinical trials, data being extrapolated from the treatment protocols routinely used in cutaneous melanoma. In our case, the patient died 2 weeks after the first cycle of a nivolumab/ipilimumab combined treatment for an acute hepatic failure that could be related to a treatment toxicity or a tumor hyperprogression. The patient's survival was very short not allowing any accurate evaluation of the efficacy of this therapy.

Global distribution patterns of marine nitrogen-fixers by imaging and molecular methods.

Nitrogen fixation has a critical role in marine primary production, yet our understanding of marine nitrogen-fixers (diazotrophs) is hindered by limited observations. Here, we report a quantitative image analysis pipeline combined with mapping of molecular markers for mining >2,000,000 images and >1300 metagenomes from surface, deep chlorophyll maximum and mesopelagic seawater samples across 6 size fractions (<0.2-2000 µm). We use this approach to characterise the diversity, abundance, biovolume and distribution of symbiotic, colony-forming and particle-associated diazotrophs at a global scale. We show that imaging and PCR-free molecular data are congruent. Sequence reads indicate diazotrophs are detected from the ultrasmall bacterioplankton (<0.2 µm) to mesoplankton (180-2000 µm) communities, while images predict numerous symbiotic and colony-forming diazotrophs (>20 µm). Using imaging and molecular data, we estimate that polyploidy can substantially affect gene abundances of symbiotic versus colony-forming diazotrophs. Our results support the canonical view that larger diazotrophs (>10 µm) dominate the tropical belts, while unicellular cyanobacterial and non-cyanobacterial diazotrophs are globally distributed in surface and mesopelagic layers. We describe co-occurring diazotrophic lineages of different lifestyles and identify high-density regions of diazotrophs in the global ocean. Overall, we provide an update of marine diazotroph biogeographical diversity and present a new bioimaging-bioinformatic workflow.

The epibiotic life of the cosmopolitan diatom Fragilariopsis doliolus on heterotrophic ciliates in the open ocean.

Diatoms are a diverse and ecologically important group of phytoplankton. Although most species are considered free living, several are known to interact with other organisms within the plankton. Detailed imaging and molecular characterization of any such partnership is, however, limited, and an appraisal of the large-scale distribution and ecology of such consortia was never attempted. Here, observation of Tara Oceans samples from the Benguela Current led to the detection of an epibiotic association between a pennate diatom and a tintinnid ciliate. We identified the diatom as Fragilariopsis doliolus that possesses a unique feature to form barrel-shaped chains, associated with seven different genera of tintinnids including five previously undescribed associations. The organisms were commonly found together in the Atlantic and Pacific Ocean basins, and live observations of the interaction have been recorded for the first time. By combining confocal and scanning electron microscopy of individual consortia with the sequencing of high-resolution molecular markers, we analyzed their distribution in the global ocean, revealing morpho-genetically distinct tintinnid haplotypes and biogeographically structured diatom haplotypes. The diatom was among the most abundant in the global ocean. We show that the consortia were particularly prevalent in nutrient-replete conditions, rich in potential predators. These observations support the hypothesis of a mutualistic symbiosis, wherein diatoms acquire increased motility and tintinnids benefit from silicification through increased protection, and highlight that such associations may be more prevalent than currently appreciated.

DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus.

Brown algae are one of the most developmentally complex groups within the eukaryotes. As in many land plants and animals, their main body axis is established early in development, when the initial cell gives rise to two daughter cells that have apical and basal identities, equivalent to shoot and root identities in land plants, respectively. We show here that mutations in the Ectocarpus DISTAG (DIS) gene lead to loss of basal structures during both the gametophyte and the sporophyte generations. Several abnormalities were observed in the germinating initial cell in dis mutants, including increased cell size, disorganization of the Golgi apparatus, disruption of the microtubule network, and aberrant positioning of the nucleus. DIS encodes a TBCCd1 protein, which has a role in internal cell organization in animals, Chlamydomonas reinhardtii, and trypanosomes. Our study highlights the key role of subcellular events within the germinating initial cell in the determination of apical/basal cell identities in a brown alga and emphasizes the remarkable functional conservation of TBCCd1 in regulating internal cell organization across extremely distant eukaryotic groups.

Quantitative 3D-imaging for cell biology and ecology of environmental microbial eukaryotes.

We present a 3D-fluorescence imaging and classification tool for high throughput analysis of microbial eukaryotes in environmental samples. It entails high-content feature extraction that permits accurate automated taxonomic classification and quantitative data about organism ultrastructures and interactions. Using plankton samples from the Tara Oceans expeditions, we validate its applicability to taxonomic profiling and ecosystem analyses, and discuss its potential for future integration of eukaryotic cell biology into evolutionary and ecological studies.

The symbiotic life of Symbiodinium in the open ocean within a new species of calcifying ciliate (Tiarina sp.).

Symbiotic partnerships between heterotrophic hosts and intracellular microalgae are common in tropical and subtropical oligotrophic waters of benthic and pelagic marine habitats. The iconic example is the photosynthetic dinoflagellate genus Symbiodinium that establishes mutualistic symbioses with a wide diversity of benthic hosts, sustaining highly biodiverse reef ecosystems worldwide. Paradoxically, although various species of photosynthetic dinoflagellates are prevalent eukaryotic symbionts in pelagic waters, Symbiodinium has not yet been reported in symbiosis within oceanic plankton, despite its high propensity for the symbiotic lifestyle. Here we report a new pelagic photosymbiosis between a calcifying ciliate host and the microalga Symbiodinium in surface ocean waters. Confocal and scanning electron microscopy, together with an 18S rDNA-based phylogeny, showed that the host is a new ciliate species closely related to Tiarina fusus (Colepidae). Phylogenetic analyses of the endosymbionts based on the 28S rDNA gene revealed multiple novel closely related Symbiodinium clade A genotypes. A haplotype network using the high-resolution internal transcribed spacer-2 marker showed that these genotypes form eight divergent, biogeographically structured, subclade types that do not seem to associate with any benthic hosts. Ecological analyses using the Tara Oceans metabarcoding data set (V9 region of the 18S rDNA) and contextual oceanographic parameters showed a global distribution of the symbiotic partnership in nutrient-poor surface waters. The discovery of the symbiotic life of Symbiodinium in the open ocean provides new insights into the ecology and evolution of this pivotal microalga and raises new hypotheses about coastal pelagic connectivity.

Ocean plankton. Eukaryotic plankton diversity in the sunlit ocean.

Marine plankton support global biological and geochemical processes. Surveys of their biodiversity have hitherto been geographically restricted and have not accounted for the full range of plankton size. We assessed eukaryotic diversity from 334 size-fractionated photic-zone plankton communities collected across tropical and temperate oceans during the circumglobal Tara Oceans expedition. We analyzed 18S ribosomal DNA sequences across the intermediate plankton-size spectrum from the smallest unicellular eukaryotes (protists, >0.8 micrometers) to small animals of a few millimeters. Eukaryotic ribosomal diversity saturated at ~150,000 operational taxonomic units, about one-third of which could not be assigned to known eukaryotic groups. Diversity emerged at all taxonomic levels, both within the groups comprising the ~11,200 cataloged morphospecies of eukaryotic plankton and among twice as many other deep-branching lineages of unappreciated importance in plankton ecology studies. Most eukaryotic plankton biodiversity belonged to heterotrophic protistan groups, particularly those known to be parasites or symbiotic hosts.

Ocean plankton. Determinants of community structure in the global plankton interactome.

Species interaction networks are shaped by abiotic and biotic factors. Here, as part of the Tara Oceans project, we studied the photic zone interactome using environmental factors and organismal abundance profiles and found that environmental factors are incomplete predictors of community structure. We found associations across plankton functional types and phylogenetic groups to be nonrandomly distributed on the network and driven by both local and global patterns. We identified interactions among grazers, primary producers, viruses, and (mainly parasitic) symbionts and validated network-generated hypotheses using microscopy to confirm symbiotic relationships. We have thus provided a resource to support further research on ocean food webs and integrating biological components into ocean models.

An original mode of symbiosis in open ocean plankton.

Symbiotic relationships are widespread in nature and are fundamental for ecosystem functioning and the evolution of biodiversity. In marine environments, photosymbiosis with microalgae is best known for sustaining benthic coral reef ecosystems. Despite the importance of oceanic microbiota in global ecology and biogeochemical cycles, symbioses are poorly characterized in open ocean plankton. Here, we describe a widespread symbiotic association between Acantharia biomineralizing microorganisms that are abundant grazers in plankton communities, and members of the haptophyte genus Phaeocystis that are cosmopolitan bloom-forming microalgae. Cophylogenetic analyses demonstrate that symbiont biogeography, rather than host taxonomy, is the main determinant of the association. Molecular dating places the origin of this photosymbiosis in the Jurassic (ca. 175 Mya), a period of accentuated marine oligotrophy. Measurements of intracellular dimethylated sulfur indicate that the host likely profits from antioxidant protection provided by the symbionts as an adaptation to life in transparent oligotrophic surface waters. In contrast to terrestrial and marine symbioses characterized to date, the symbiont reported in this association is extremely abundant and ecologically active in its free-living phase. In the vast and barren open ocean, partnership with photosymbionts that have extensive free-living populations is likely an advantageous strategy for hosts that rely on such interactions. Discovery of the Acantharia-Phaeocystis association contrasts with the widely held view that symbionts are specialized organisms that are rare and ecologically passive outside the host.

Degradation of lambda-carrageenan by Pseudoalteromonas carrageenovora lambda-carrageenase: a new family of glycoside hydrolases unrelated to kappa- and iota-carrageenases.

Carrageenans are sulfated galactans found in the cell walls of red seaweeds. They are classified according to the number and the position of sulfate ester groups. lambda-Carrageenan is the most sulfated carrageenan and carries at least three sulfates per disaccharide unit. The sole known depolymerizing enzyme of lambda-carrageenan, the lambda-carrageenase from Pseudoalteromonas carrageenovora, has been purified, cloned and sequenced. Sequence analyses have revealed that the lambda-carrageenase, referred to as CglA, is the first member of a new family of GHs (glycoside hydrolases), which is unrelated to families GH16, that contains kappa-carrageenases, and GH82, that contains iota-carrageenases. This large enzyme (105 kDa) features a low-complexity region, suggesting the presence of a linker connecting at least two independent modules. The N-terminal region is predicted to fold as a beta-propeller. The main degradation products have been purified and characterized as neo-lambda-carratetraose [DP (degree of polymerization) 4] and neo-lambda-carrahexaose (DP6), indicating that CglA hydrolyses the beta-(1-->4) linkage of lambda-carrageenan. LC-MALLS (liquid chromatography-multi-angle laser light scattering) and (1)H-NMR monitoring of the enzymatic degradation of lambda-carrageenan indicate that CglA proceeds according to an endolytic mode of action and a mechanism of inversion of the anomeric configuration. Using 2-aminoacridone-labelled neo-lambda-carrabiose oligosaccharides, in the present study we demonstrate that the active site of CglA comprises at least 8 subsites (-4 to +4) and that a DP6 oligosaccharide binds in the subsites -4 to +2 and can be hydrolysed into DP4 and DP2.

Cloning and biochemical characterization of the fucanase FcnA: definition of a novel glycoside hydrolase family specific for sulfated fucans.

Sulfated fucans are matrix polysaccharides from marine brown algae, consisting of an alpha-L-fucose backbone substituted by sulfate-ester groups, masked with ramifications, and containing other monosaccharide residues. We here report on the characterization of a novel glycoside hydrolase (FcnA) specific for the degradation of sulfated fucans. This glycoside hydrolase was purified to electrophoretic homogeneity from a Flavobacteriaceae referred to as SW5. The gene fcnA was cloned and sequenced (3021 nucleotides), and the protein (1007 amino acids) was produced in Escherichia coli. FcnA exhibited a modular architecture consisting of a 400-residue-long N-terminal domain followed by three repeated domains predicted to adopt an immunoglobulin fold and by an 80-amino acid-long C-terminal domain. A truncated recombinant protein encompassing the N-terminal domain and the immunoglobulin-like repeats was shown to retain the enzyme activity. The N-terminal catalytic domain shared approximately 25% of sequence identity with two patented fucanase genes, and these three fucanases delineate a new family of glycoside hydrolases. As shown by size-exclusion chromatography (SEC) and 1H-NMR analyses, the fucanase FcnA proceeds according to an endolytic mode of action and cleaves the alpha-(1-->4) glycosidic linkages within the blocks of repeating motifs [-->4)-alpha-L-fucopyranosyl-2,3-disulfate-(1-->3)-alpha-L-fucopyranosyl-2-sulfate-(1-->]n.

Isolation and culture of a marine bacterium degrading the sulfated fucans from marine brown algae.

Fucoidans are matrix polysaccharides from marine brown algae, consisting of an alpha-L-fucose backbone substituted by sulfate-ester groups and masked with ramifications containing other monosaccharide residues. In spite of their interest as biologically active compounds in a number of homologous and heterologous systems, no convenient sources with fucanase activity are available yet for the degradation of the fucalean algae. We here report on the isolation, characterization, and culture conditions of a bacterial strain capable of degrading various brown algal fucoidans. This bacterium, a member of the family Flavobacteriaceae, was shown to secrete fucoidan endo-hydrolase activity. An extracellular enzyme preparation was used to degrade the fucoidan from the brown alga Pelvetia canaliculata. End products included a tetrasaccharide and a hexasaccharide made of the repetition of disaccharidic units consisting of alpha-1-->3-L-fucopyranose-2-sulfate-alpha-1-->4-L-fucopyranose-2,3-disulfate, with the 3-linked residues at the nonreducing end.