Water-Soluble Saccharina latissima Polysaccharides and Relation of Their Structural Characteristics with In Vitro Immunostimulatory and Hypocholesterolemic Activities.

Brown macroalgae are an important source of polysaccharides, mainly fucose-containing sulphated polysaccharides (FCSPs), associated with several biological activities. However, the structural diversity and structure-function relationships for their bioactivities are still undisclosed. Thus, the aim of this work was to characterize the chemical structure of water-soluble Saccharina latissima polysaccharides and evaluate their immunostimulatory and hypocholesterolemic activities, helping to pinpoint a structure-activity relationship. Alginate, laminarans (F1, neutral glucose-rich polysaccharides), and two fractions (F2 and F3) of FCSPs (negatively charged) were studied. Whereas F2 is rich in uronic acids (45 mol%) and fucose (29 mol%), F3 is rich in fucose (59 mol%) and galactose (21 mol%). These two fractions of FCSPs showed immunostimulatory activity on B lymphocytes, which could be associated with the presence of sulphate groups. Only F2 exhibited a significant effect in reductions in in vitro cholesterol's bioaccessibility attributed to the sequestration of bile salts. Therefore, S. latissima FCSPs were shown to have potential as immunostimulatory and hypocholesterolemic functional ingredients, where their content in uronic acids and sulphation seem to be relevant for the bioactive and healthy properties.

Exhaustive reanalysis of barcode sequences from public repositories highlights ongoing misidentifications and impacts taxa diversity and distribution.

Accurate species identification often relies on public repositories to compare the barcode sequences of the investigated individual(s) with taxonomically assigned sequences. However, the accuracy of identifications in public repositories is often questionable, and the names originally given are rarely updated. For instance, species of the Sea Lettuce (Ulva spp.; Ulvophyceae, Ulvales, Ulvaceae) are frequently misidentified in public repositories, including herbaria and gene banks, making species identification based on traditional barcoding unreliable. We DNA barcoded 295 individual distromatic foliose strains of Ulva from the North-East Atlantic for three loci (rbcL, tufA, ITS1). Seven distinct species were found, and we compared our results with all worldwide Ulva spp. sequences present in the NCBI database for the three barcodes rbcL, tufA and the ITS1. Our results demonstrate a large degree of species misidentification, where we estimate that 24%-32% of the entries pertaining to foliose species are misannotated and provide an exhaustive list of NCBI sequences reannotations. An analysis of the global distribution of registered samples from foliose species also indicates possible geographical isolation for some species, and the absence of U. lactuca from Northern Europe. We extended our analytical framework to three other genera, Fucus, Porphyra and Pyropia and also identified erroneously labelled accessions and possibly new synonymies, albeit less than for Ulva spp. Altogether, exhaustive taxonomic clarification by aggregation of a library of barcode sequences highlights misannotations and delivers an improved representation of species diversity and distribution.

The genome of Ectocarpus subulatus - A highly stress-tolerant brown alga.

Brown algae are multicellular photosynthetic stramenopiles that colonize marine rocky shores worldwide. Ectocarpus sp. Ec32 has been established as a genomic model for brown algae. Here we present the genome and metabolic network of the closely related species, Ectocarpus subulatus Kützing, which is characterized by high abiotic stress tolerance. Since their separation, both strains show new traces of viral sequences and the activity of large retrotransposons, which may also be related to the expansion of a family of chlorophyll-binding proteins. Further features suspected to contribute to stress tolerance include an expanded family of heat shock proteins, the reduction of genes involved in the production of halogenated defence compounds, and the presence of fewer cell wall polysaccharide-modifying enzymes. Overall, E. subulatus has mainly lost members of gene families down-regulated in low salinities, and conserved those that were up-regulated in the same condition. However, 96% of genes that differed between the two examined Ectocarpus species, as well as all genes under positive selection, were found to encode proteins of unknown function. This underlines the uniqueness of brown algal stress tolerance mechanisms as well as the significance of establishing E. subulatus as a comparative model for future functional studies.

The Unique Lipidomic Signatures of Saccharina latissima Can Be Used to Pinpoint Their Geographic Origin.

The aquaculture of macroalgae for human consumption and other high-end applications is experiencing unprecedented development in European countries, with the brown algae Saccharina latissima being the flag species. However, environmental conditions in open sea culture sites are often unique, which may impact the biochemical composition of cultured macroalgae. The present study compared the elemental compositions (CHNS), fatty acid profiles, and lipidomes of S. latissima originating from three distinct locations (France, Norway, and the United Kingdom). Significant differences were found in the elemental composition, with Norwegian samples displaying twice the lipid content of the others, and significantly less protein (2.6%, while French and UK samples contained 6.3% and 9.1%, respectively). The fatty acid profiles also differed considerably, with UK samples displaying a lower content of n-3 fatty acids (21.6%), resulting in a higher n-6/n-3 ratio. Regarding the lipidomic profile, samples from France were enriched in lyso lipids, while those from Norway displayed a particular signature of phosphatidylglycerol, phosphatidylinositol, and phosphatidylcholine. Samples from the UK featured higher levels of phosphatidylethanolamine and, in general, a lower content of galactolipids. These differences highlight the influence of site-specific environmental conditions in the shaping of macroalgae biochemical phenotypes and nutritional value. It is also important to highlight that differences recorded in the lipidome of S. latissima make it possible to pinpoint specific lipid species that are likely to represent origin biomarkers. This finding is relevant for future applications in the field of geographic origin traceability and food control.

"Ectrogella" Parasitoids of the Diatom Licmophora sp. are Polyphyletic.

The diatom genera Licmophora and Fragilaria are frequent epiphytes on marine macroalgae and can be infected by intracellular parasitoids traditionally assigned to the oomycete genus Ectrogella. Much debate and uncertainty remains about the taxonomy of these oomycetes, not least due to their morphological and developmental plasticity. Here, we used single-cell techniques to obtain partial sequences of the parasitoids 18S and cox2 genes. The former falls into two recently identified clades of Pseudo-nitzschia parasites temporarily named OOM_1_2 and OOM_2, closely related to the genera of brown and red algal pathogens Anisolpidium and Olpidiopsis. A third group of sequences falls at the base of the red algal parasites assigned to Olpidiopsis. In one instance, two oomycete parasitoids seemed to co-exist in a single diatom cell; this co-occurrence of distinct parasitoid taxa not only within a population of diatom epiphytes, but also within the same host cell, possibly explains the ongoing confusion in the taxonomy of these parasitoids. We demonstrate the polyphyly of Licmophora parasitoids previously assigned to Ectrogella (sensu Sparrow, 1960) and show that parasites of red algae assigned to the genus Olpidiopsis are most likely not monophyletic. We conclude that combining single-cell microscopy and molecular methods is necessary for their full characterisation.

Unusual Patterns of Mitochondrial Inheritance in the Brown Alga Ectocarpus.

Most eukaryotes inherit their mitochondria from only one of their parents. When there are different sexes, it is almost always the maternal mitochondria that are transmitted. Indeed, maternal uniparental inheritance has been reported for the brown alga Ectocarpus but we show in this study that different strains of Ectocarpus can exhibit different patterns of inheritance: Ectocarpus siliculosus strains showed maternal uniparental inheritance, as expected, but crosses using different Ectocarpus species 7 strains exhibited either paternal uniparental inheritance or an unusual pattern of transmission where progeny inherited either maternal or paternal mitochondria, but not both. A possible correlation between the pattern of mitochondrial inheritance and male gamete parthenogenesis was investigated. Moreover, in contrast to observations in the green lineage, we did not detect any change in the pattern of mitochondrial inheritance in mutant strains affected in life cycle progression. Finally, an analysis of field-isolated strains provided evidence of mitochondrial genome recombination in both Ectocarpus species.

Chytrid infecting the bloom-forming marine diatom Skeletonema sp.: Morphology, phylogeny and distribution of a novel species within the Rhizophydiales.

Chytrids have long been recognised as important parasites of microalgae in freshwater systems, able to shape the dynamics of blooms, the gene pool of their host and phytoplankton succession. In the sea however, where the presence of these organisms is erratic and ephemeral, studies concerning chytrids are sparse and confined to metabarcoding surveys or microscopy observations. Despite the scarcity of data, chytrid epidemics are supposed to play an important role in marine biogeochemical cycles, being one of the drivers of phytoplankton dynamics. Here we combine microscopy observations and in silico mining of a single-cell whole genome to molecularly and morphologically characterise a novel chytrid parasite of the dominant diatom genus Skeletonema. Morphological observations highlight features of the thallus and ascertain the parasitic nature of the interaction whilst the genetic markers obtained allows for a phylogenetic reconstruction, placing the new species in the order Rhizophydiales. Thanks to the molecular data obtained we are also able to provide a first investigation of the global distribution of this organism by screening the Ocean Sampling Day (OSD) dataset, highlighting a northern transatlantic dissemination.

The Ectocarpus IMMEDIATE UPRIGHT gene encodes a member of a novel family of cysteine-rich proteins with an unusual distribution across the eukaryotes.

The sporophyte generation of the brown alga Ectocarpus sp. exhibits an unusual pattern of development compared with the majority of brown algae. The first cell division is symmetrical and the apical-basal axis is established late in development. In the immediate upright (imm) mutant, the initial cell undergoes an asymmetric division to immediately establish the apical-basal axis. We provide evidence which suggests that this phenotype corresponds to the ancestral state of the sporophyte. The IMM gene encodes a protein of unknown function that contains a repeated motif also found in the EsV-1-7 gene of the Ectocarpus virus EsV-1. Brown algae possess large families of EsV-1-7 domain genes but these genes are rare in other stramenopiles, suggesting that the expansion of this family might have been linked with the emergence of multicellular complexity. EsV-1-7 domain genes have a patchy distribution across eukaryotic supergroups and occur in several viral genomes, suggesting possible horizontal transfer during eukaryote evolution.

Re-annotation, improved large-scale assembly and establishment of a catalogue of noncoding loci for the genome of the model brown alga Ectocarpus.

The genome of the filamentous brown alga Ectocarpus was the first to be completely sequenced from within the brown algal group and has served as a key reference genome both for this lineage and for the stramenopiles. We present a complete structural and functional reannotation of the Ectocarpus genome. The large-scale assembly of the Ectocarpus genome was significantly improved and genome-wide gene re-annotation using extensive RNA-seq data improved the structure of 11 108 existing protein-coding genes and added 2030 new loci. A genome-wide analysis of splicing isoforms identified an average of 1.6 transcripts per locus. A large number of previously undescribed noncoding genes were identified and annotated, including 717 loci that produce long noncoding RNAs. Conservation of lncRNAs between Ectocarpus and another brown alga, the kelp Saccharina japonica, suggests that at least a proportion of these loci serve a function. Finally, a large collection of single nucleotide polymorphism-based markers was developed for genetic analyses. These resources are available through an updated and improved genome database. This study significantly improves the utility of the Ectocarpus genome as a high-quality reference for the study of many important aspects of brown algal biology and as a reference for genomic analyses across the stramenopiles.

Recent mobility of plastid encoded group II introns and twintrons in five strains of the unicellular red alga Porphyridium.

Group II introns are closely linked to eukaryote evolution because nuclear spliceosomal introns and the small RNAs associated with the spliceosome are thought to trace their ancient origins to these mobile elements. Therefore, elucidating how group II introns move, and how they lose mobility can potentially shed light on fundamental aspects of eukaryote biology. To this end, we studied five strains of the unicellular red alga Porphyridium purpureum that surprisingly contain 42 group II introns in their plastid genomes. We focused on a subset of these introns that encode mobility-conferring intron-encoded proteins (IEPs) and found them to be distributed among the strains in a lineage-specific manner. The reverse transcriptase and maturase domains were present in all lineages but the DNA endonuclease domain was deleted in vertically inherited introns, demonstrating a key step in the loss of mobility. P. purpureum plastid intron RNAs had a classic group IIB secondary structure despite variability in the DIII and DVI domains. We report for the first time the presence of twintrons (introns-within-introns, derived from the same mobile element) in Rhodophyta. The P. purpureum IEPs and their mobile introns provide a valuable model for the study of mobile retroelements in eukaryotes and offer promise for biotechnological applications.

Using natural selection to explore the adaptive potential of Chlamydomonas reinhardtii.

Improving feedstock is critical to facilitate the commercial utilization of algae, in particular in open pond systems where, due to the presence of competitors and pests, high algal growth rates and stress tolerance are beneficial. Here we raised laboratory cultures of the model alga Chlamydomonas reinhardtii under serial dilution to explore the potential of crop improvement using natural selection. The alga was evolved for 1,880 generations in liquid medium under continuous light (EL population). At the end of the experiment, EL cells had a growth rate that was 35% greater than the progenitor population (PL). The removal of acetate from the medium demonstrated that EL growth enhancement largely relied on efficient usage of this organic carbon source. Genome re-sequencing uncovered 1,937 polymorphic DNA regions in the EL population with 149 single nucleotide polymorphisms resulting in amino acid substitutions. Transcriptome analysis showed, in the EL population, significant up regulation of genes involved in protein synthesis, the cell cycle and cellular respiration, whereas the DNA repair pathway and photosynthesis were down regulated. Like other algae, EL cells accumulated neutral lipids under nitrogen depletion. Our work demonstrates transcriptome and genome-wide impacts of natural selection on algal cells and points to a useful strategy for strain improvement.

Evolution of salt tolerance in a laboratory reared population of Chlamydomonas reinhardtii.

Understanding the genetic underpinnings of adaptive traits in microalgae is important for the study of evolution and for applied uses. We used long-term selection under a regime of serial transfers with haploid populations of the green alga Chlamydomonas reinhardtii raised in liquid TAP medium containing 200 mM NaCl. After 1255 generations, evolved salt (ES) populations could grow as rapidly in high salt medium as progenitor cells (progenitor light [PL]). Transcriptome data were analysed to elucidate the basis of salt tolerance in ES cells when compared with PL cells and to cells incubated for 48 h in high salt medium (progenitor salt [PS], the short-term acclimation response). These data demonstrate that evolved and short-term acclimation responses to salt stress differ fundamentally from each other. Progenitor salt cells exhibit well-known responses to salt stress such as reduction in photosynthesis, upregulation of glycerophospholipid signaling, and upregulation of the transcription and translation machinery. In contrast, ES cells show downregulation of genes involved in the stress response and in transcription/translation. Our results suggest that gene-rich mixotrophic lineages such as C. reinhardtii may be able to adapt rapidly to abiotic stress engendered either by a rapidly changing climate or physical vicariance events that isolate populations in stressful environments.

Genome of the red alga Porphyridium purpureum.

The limited knowledge we have about red algal genomes comes from the highly specialized extremophiles, Cyanidiophyceae. Here, we describe the first genome sequence from a mesophilic, unicellular red alga, Porphyridium purpureum. The 8,355 predicted genes in P. purpureum, hundreds of which are likely to be implicated in a history of horizontal gene transfer, reside in a genome of 19.7 Mbp with 235 spliceosomal introns. Analysis of light-harvesting complex proteins reveals a nuclear-encoded phycobiliprotein in the alga. We uncover a complex set of carbohydrate-active enzymes, identify the genes required for the methylerythritol phosphate pathway of isoprenoid biosynthesis, and find evidence of sexual reproduction. Analysis of the compact, function-rich genome of P. purpureum suggests that ancestral lineages of red algae acted as mediators of horizontal gene transfer between prokaryotes and photosynthetic eukaryotes, thereby significantly enriching genomes across the tree of photosynthetic life.

Bradyrhizobia nodulating the Acacia mangium x A. auriculiformis interspecific hybrid are specific and differ from those associated with both parental species.

In the context of an increasing utilization of the interspecific hybrid Acacia mangium x A. auriculiformis as a plantation tree in the tropical humid zone, its symbiotic characterization was carried out in comparison with that of its two parental species. Rhizobium strains of diverse geographical origins were isolated from root nodules of the hybrid and its parents. Almost all Acacia hybrid isolates were fast growing on yeast extract-mannitol medium, in contrast to those isolated from both parental species, which were mostly slow growing. The rhizobium strains were characterized through partial sequencing of the rRNA operon. In the phylogenetic tree, almost all strains isolated from the hybrid were grouped together in a clade close to Bradyrhizobium japonicum, while all strains isolated from both parental species were close to Bradyrhizobium elkanii. Inoculation experiments performed under in vitro or greenhouse conditions showed that all strains were infective with their original hosts but exhibited very variable degrees of effectivity according to the host plant tested. Thus, homologous strain-host associations were more effective than heterologous ones. This shows that there is still a high potential for isolating and testing new strains from hybrids to be used as inoculants in the context of large-scale afforestation programs.

Effects of Medicago truncatula genetic diversity, rhizobial competition, and strain effectiveness on the diversity of a natural sinorhizobium species community.

We investigated the genetic diversity and symbiotic efficiency of 223 Sinorhizobium sp. isolates sampled from a single Mediterranean soil and trapped with four Medicago truncatula lines. DNA molecular polymorphism was estimated by capillary electrophoresis-single-stranded conformation polymorphism and restriction fragment length polymorphism on five loci (IGS(NOD), typA, virB11, avhB11, and the 16S rRNA gene). More than 90% of the rhizobia isolated belonged to the Sinorhizobium medicae species (others belonged to Sinorhizobium meliloti), with different proportions of the two species among the four M. truncatula lines. The S. meliloti population was more diverse than that of S. medicae, and significant genetic differentiation among bacterial populations was detected. Single inoculations performed in tubes with each bacterial genotype and each plant line showed significant bacterium-plant line interactions for nodulation and N(2) fixation levels. Competition experiments within each species highlighted either strong or weak competition among genotypes within S. medicae and S. meliloti, respectively. Interspecies competition experiments showed S. meliloti to be more competitive than S. medicae for nodulation. Although not highly divergent at a nucleotide level, isolates collected from this single soil sample displayed wide polymorphism for both nodulation and N(2) fixation. Each M. truncatula line might influence Sinorhizobium soil population diversity differently via its symbiotic preferences. Our data suggested that the two species did not evolve similarly, with S. meliloti showing polymorphism and variable selective pressures and S. medicae showing traces of a recent demographic expansion. Strain effectiveness might have played a role in the species and genotype proportions, but in conjunction with strain adaptation to environmental factors.

Active dispersal by wild Triatoma infestans in the Bolivian Andes.

Triatoma infestans is the main vector of Chagas disease and target of control programmes in the Southern Cone countries. So far Bolivia is the only country where true T. infestans wild foci are documented. The dispersal ability for wild T. infestans was studied at microgeographical scale in Bolivian Andes, to assess the possibility for wild populations to actively recolonize insecticide-treated villages. Nine microsatellite loci were used to detect the extent of gene flow between neighbouring collecting sites. The detection of restricted gene flow between close but distinct sylvatic sites supports the hypothesis that wild T. infestans does not disperse by flying at high altitude (2,750 m asl). It gradually disperses over small distances by walking within a 'patch' of continuous land cover. The genetic differentiation detected between sylvatic and domestic populations suggests a limited short-term role of wild insects in the process of recolonization of insecticide-treated houses in the Andes.