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Abstract Background Brown algae belong to the Stramenopiles phylum and are phylogenetically distant from plants and other multicellular organisms. This independent evolutionary history has shaped brown algae with numerous metabolic characteristics specific to this group, including the synthesis of peculiar polysaccharides contained in their extracellular matrix (ECM). Alginates and fucose-containing sulphated polysaccharides (FCSPs), the latter including fucans, are the main components of ECMs. However, the metabolic pathways of these polysaccharides remain poorly described due to a lack of genomic data. Results An extensive genomic dataset has been recently released for brown algae and their close sister species, for which we previously performed an expert annotation of key genes involved in ECM-carbohydrate metabolisms. Here we provide a deeper analysis of this set of genes using comparative genomics, phylogenetics analyses, and protein modelling. Two key gene families involved in both the synthesis and degradation of alginate were suggested to have been acquired by the common ancestor of brown algae and their closest sister species Schizocladia ischiensis. Our analysis indicates that this assumption can be extended to additional metabolic steps, and thus to the whole alginate metabolic pathway. The pathway for the biosynthesis of fucans still remains biochemically unresolved and we also investigate putative fucosyltransferase genes that may harbour a fucan synthase activity in brown algae. Conclusions Our analysis is the first extensive survey of carbohydrate-related enzymes in brown algae, and provides a valuable resource for future research into the glycome and ECM of brown algae. The expansion of specific families related to alginate metabolism may have represented an important prerequisite for the evolution of developmental complexity in brown algae. Our analysis questions the possible occurrence of FCSPs outside brown algae, notably within their closest sister taxon and in other Stramenopiles such as diatoms. Filling this knowledge gap in the future will help determine the origin and evolutionary history of fucan synthesis in eukaryotes.

In recent years micro- and nanoplastics and metal-oxide nanomaterials have been found in several environmental compartments. The Antarctic soft clam Laternula elliptica is an endemic Antarctic species having a wide distribution in the Southern Ocean. Being a filter-feeder, it could act as suitable bioindicator of pollution from nanoparticles also considering its sensitivity to various sources of stress. The present study aims to assess the impact of polystyrene nanoparticles (PS-NP) and the nanometal titanium-dioxide (n-TiO2) on genome-wide transcript expression of L. elliptica either alone and in combination and at two toxicological relevant concentrations (5 and 50 µg/L) during 96 h exposure. Transcript-target qRT-PCR was performed with the aim to identify suitable biomarkers of exposure and effects. As expected, at the highest concentration tested, the clustering was clearer between control and exposed clams. A total of 221 genes resulted differentially expressed in exposed clams and control ones, and 21 of them had functional annotation such as ribosomal proteins, antioxidant, ion transport (osmoregulation), acid-base balance, immunity, lipid metabolism, cell adhesion, cytoskeleton, apoptosis, chromatin condensation and cell signaling. At functional level, relevant transcripts were shared among some treatments and could be considered as general stress due to nanoparticle exposure. After applying transcript-target approach duplicating the number of clam samples, four ecologically relevant transcripts were revealed as biomarkers for PS-NP, n-TiO2 and their combination at 50 µg/L, that could be used for monitoring clams’ health status in different Antarctic localities.

Abstract Background The transient and fragmented nature of the deep-sea hydrothermal environment made of ridge subduction, plate collision and the emergence of new rifts is currently acting to separate of vent populations, promoting local adaptation and contributing to bursts of speciation and species specialization. The tube-dwelling worms Alvinella pompejana called the Pompeii worm and its sister species A. caudata live syntopically on the hottest part of deep-sea hydrothermal chimneys along the East Pacific Rise. They are exposed to extreme thermal and chemical gradients, which vary greatly in space and time, and thus represent ideal candidates for understanding the evolutionary mechanisms at play in the vent fauna evolution. Results We explored genomic patterns of divergence in the early and late stages of speciation of these emblematic worms using transcriptome assemblies and the first draft genome to better understand the relative role of geographic isolation and habitat preference in their genome evolution. Analyses were conducted on allopatric populations of Alvinella pompejana (early stage of separation) and between A. pompejana and its syntopic species Alvinella caudata (late stage of speciation). We first identified divergent genomic regions and targets of selection as well as their position in the genome over collections of orthologous genes and, then, described the speciation dynamics by documenting the annotation of the most divergent and/or positively selected genes involved in the isolation process. Gene mapping clearly indicated that divergent genes associated with the early stage of speciation, although accounting for nearly 30% of genes, are highly scattered in the genome without any island of divergence and not involved in gamete recognition or mito-nuclear incompatibilities. By contrast, genomes of A. pompejana and A. caudata are clearly separated with nearly all genes (96%) exhibiting high divergence. This congealing effect however seems to be linked to habitat specialization and still allows positive selection on genes involved in gamete recognition, as a possible long-duration process of species reinforcement. Conclusion Our analyses highlight the non-negligible role of natural selection on both the early and late stages of speciation in the iconic thermophilic worms living on the walls of deep-sea hydrothermal chimneys. They shed light on the evolution of gene divergence during the process of speciation and species specialization over a very long period of time.

ABSTRACT Marine Synechococcus comprise a numerically and ecologically prominent phytoplankton group, playing a major role in both carbon cycling and trophic networks in all oceanic regions except in the polar oceans. Despite their high abundance in coastal areas, our knowledge of Synechococcus communities in these environments is based on only a few local studies. Here, we use the global metagenome data set of the Ocean Sampling Day (June 21st, 2014) to get a snapshot of the taxonomic composition of coastal Synechococcus communities worldwide, by recruitment on a reference database of 141 picocyanobacterial genomes, representative of the whole Prochlorococcus, Synechococcus, and Cyanobium diversity. This allowed us to unravel drastic community shifts over small to medium scale gradients of environmental factors, in particular along European coasts. The combined analysis of the phylogeography of natural populations and the thermophysiological characterization of eight strains, representative of the four major Synechococcus lineages (clades I to IV), also brought novel insights about the differential niche partitioning of clades I and IV, which most often co-dominate the Synechococcus community in cold and temperate coastal areas. Altogether, this study reveals several important characteristics and specificities of the coastal communities of Synechococcus worldwide. IMPORTANCE Synechococcus is the second most abundant phytoplanktonic organism on Earth, and its wide genetic diversity allowed it to colonize all the oceans except for polar waters, with different clades colonizing distinct oceanic niches. In recent years, the use of global metagenomics data sets has greatly improved our knowledge of “who is where” by describing the distribution of Synechococcus clades or ecotypes in the open ocean. However, little is known about the global distribution of Synechococcus ecotypes in coastal areas, where Synechococcus is often the dominant phytoplanktonic organism. Here, we leverage the global Ocean Sampling Day metagenomics data set to describe Synechococcus community composition in coastal areas worldwide, revealing striking community shifts, in particular along the coasts of Europe. As temperature appears as an important driver of the community composition, we also characterize the thermal preferenda of 8 Synechococcus strains, bringing new insights into the adaptation to temperature of the dominant Synechococcus clades.

Threats to global biodiversity are increasingly recognised by scientists and the public as a critical challenge. Molecular sequencing technologies offer means to catalogue, explore, and monitor the richness and biogeography of life on Earth. However, exploiting their full potential requires tools that connect biodiversity infrastructures and resources. As a research infrastructure developing services and technical solutions that help integrate and coordinate life science resources across Europe, ELIXIR is a key player. To identify opportunities, highlight priorities, and aid strategic thinking, here we survey approaches by which molecular technologies help inform understanding of biodiversity. We detail example use cases to highlight how DNA sequencing is: resolving taxonomic issues; Increasing knowledge of marine biodiversity; helping understand how agriculture and biodiversity are critically linked; and playing an essential role in ecological studies. Together with examples of national biodiversity programmes, the use cases show where progress is being made but also highlight common challenges and opportunities for future enhancement of underlying technologies and services that connect molecular and wider biodiversity domains. Based on emerging themes, we propose key recommendations to guide future funding for biodiversity research: biodiversity and bioinformatic infrastructures need to collaborate closely and strategically; taxonomic efforts need to be aligned and harmonised across domains; metadata needs to be standardised and common data management approaches widely adopted; current approaches need to be scaled up dramatically to address the anticipated explosion of molecular data; bioinformatics support for biodiversity research needs to be enabled and sustained; training for end users of biodiversity research infrastructures needs to be prioritised; and community initiatives need to be proactive and focused on enabling solutions. For sequencing data to deliver their full potential they must be connected to knowledge: together, molecular sequence data collection initiatives and biodiversity research infrastructures can advance global efforts to prevent further decline of Earth’s biodiversity.

Abstract High‐throughput sequencing of amplicons (HTSA) has been proposed as an effective approach to evaluate taxonomic and genetic diversity at the same time. However, there are still uncertainties as to how the results produced by different bioinformatics treatments impact the conclusions drawn on biodiversity and population genetics indices. We evaluated the ability of six bioinformatics pipelines to recover taxonomic and genetic diversity from HTSA data obtained from controlled assemblages. To that end, 20 assemblages were produced using 354 colonies of Botrylloides spp., sampled in the wild in ten marinas around Brittany (France). We used DNA extracted from preservative ethanol (ebDNA) after various time of storage (3, 6, and 12 months), and from a bulk of preserved specimens (bulkDNA). DNA was amplified with primers designed for targeting this ascidian genus. Results obtained from HTSA data were compared with Sanger sequencing on individual zooids (i.e., individual barcoding). Species identification and relative abundance determined with HTSA data from either ebDNA or bulkDNA were similar to those obtained with traditional individual barcoding. However, after 12 months of storage, the correlation between HTSA and individual‐based data was lower than after shorter durations. The six bioinformatics pipelines were able to depict accurately the genetic diversity using standard population genetics indices (HS and FST), despite producing false positives and missing rare haplotypes. However, they did not perform equally and dada2 was the only pipeline able to retrieve all expected haplotypes. This study showed that ebDNA is a nondestructive alternative for both species identification and haplotype recovery, providing storage does not last more than 6 months before DNA extraction. Choosing the bioinformatics pipeline is a matter of compromise, aiming to retrieve all true haplotypes while avoiding false positives. We here recommend to process HTSA data using dada2, including a chimera‐removal step. Even if the possibility to use multiplexed primer sets deserves further investigation to expand the taxonomic coverage in future similar studies, we showed that primers targeting a particular genus allowed to reliably analyze this genus within a complex community.

Abstract Proton therapy allows to avoid excess radiation dose on normal tissues. However, there are some limitations. Indeed, passive delivery of proton beams results in an increase in the lateral dose upstream of the tumor and active scanning leads to strong differences in dose delivery. This study aims to assess possible differences in the transcriptomic response of skin in C57BL/6 mice after TBI irradiation by active or passive proton beams at the dose of 6 Gy compared to unirradiated mice. In that purpose, total RNA was extracted from skin samples 3 months after irradiation and RNA-Seq was performed. Results showed that active and passive delivery lead to completely different transcription profiles. Indeed, 140 and 167 genes were differentially expressed after active and passive scanning compared to unirradiated, respectively, with only one common gene corresponding to RIKEN cDNA 9930021J03. Moreover, protein–protein interactions performed by STRING analysis showed that 31 and 25 genes are functionally related after active and passive delivery, respectively, with no common gene between both types of proton delivery. Analysis showed that active scanning led to the regulation of genes involved in skin development which was not the case with passive delivery. Moreover, 14 ncRNA were differentially regulated after active scanning against none for passive delivery. Active scanning led to 49 potential mRNA-ncRNA pairs with one ncRNA mainly involved, Gm44383 which is a miRNA. The 43 genes potentially regulated by the miRNA Gm44393 confirmed an important role of active scanning on skin keratin pathway. Our results demonstrated that there are differences in skin gene expression still 3 months after proton irradiation versus unirradiated mouse skin. And strong differences do exist in late skin gene expression between scattered or scanned proton beams. Further investigations are strongly needed to understand this discrepancy and to improve treatments by proton therapy.

Abstract Background Dinoflagellates are aquatic protists particularly widespread in the oceans worldwide. Some are responsible for toxic blooms while others live in symbiotic relationships, either as mutualistic symbionts in corals or as parasites infecting other protists and animals. Dinoflagellates harbor atypically large genomes (~ 3 to 250 Gb), with gene organization and gene expression patterns very different from closely related apicomplexan parasites. Here we sequenced and analyzed the genomes of two early-diverging and co-occurring parasitic dinoflagellate Amoebophrya strains, to shed light on the emergence of such atypical genomic features, dinoflagellate evolution, and host specialization. Results We sequenced, assembled, and annotated high-quality genomes for two Amoebophrya strains (A25 and A120), using a combination of Illumina paired-end short-read and Oxford Nanopore Technology (ONT) MinION long-read sequencing approaches. We found a small number of transposable elements, along with short introns and intergenic regions, and a limited number of gene families, together contribute to the compactness of the Amoebophrya genomes, a feature potentially linked with parasitism. While the majority of Amoebophrya proteins (63.7% of A25 and 59.3% of A120) had no functional assignment, we found many orthologs shared with Dinophyceae. Our analyses revealed a strong tendency for genes encoded by unidirectional clusters and high levels of synteny conservation between the two genomes despite low interspecific protein sequence similarity, suggesting rapid protein evolution. Most strikingly, we identified a large portion of non-canonical introns, including repeated introns, displaying a broad variability of associated splicing motifs never observed among eukaryotes. Those introner elements appear to have the capacity to spread over their respective genomes in a manner similar to transposable elements. Finally, we confirmed the reduction of organelles observed in Amoebophrya spp., i.e., loss of the plastid, potential loss of a mitochondrial genome and functions. Conclusion These results expand the range of atypical genome features found in basal dinoflagellates and raise questions regarding speciation and the evolutionary mechanisms at play while parastitism was selected for in this particular unicellular lineage.

Marine Synechococcus cyanobacteria are ubiquitous in the ocean, a feature likely related to their extensive genetic diversity. Amongst the major lineages, clades I and IV preferentially thrive in temperate and cold, nutrient-rich waters, whilst clades II and III prefer warm, nitrogen or phosphorus-depleted waters. The existence of such cold (I/IV) and warm (II/III) thermotypes is corroborated by physiological characterization of representative strains. A fifth clade, CRD1, was recently shown to dominate the Synechococcus community in iron-depleted areas of the world ocean and to encompass three distinct ecologically significant taxonomic units (ESTUs CRD1A-C) occupying different thermal niches, suggesting that distinct thermotypes could also occur within this clade. Here, using comparative thermophysiology of strains representative of these three CRD1 ESTUs we show that the CRD1A strain MITS9220 is a warm thermotype, the CRD1B strain BIOS-U3-1 a cold temperate thermotype, and the CRD1C strain BIOS-E4-1 a warm temperate stenotherm. Curiously, the CRD1B thermotype lacks traits and/or genomic features typical of cold thermotypes. In contrast, we found specific physiological traits of the CRD1 strains compared to their clade I, II, III, and IV counterparts, including a lower growth rate and photosystem II maximal quantum yield at most temperatures and a higher turnover rate of the D1 protein. Together, our data suggests that the CRD1 clade prioritizes adaptation to low-iron conditions over temperature adaptation, even though the occurrence of several CRD1 thermotypes likely explains why the CRD1 clade as a whole occupies most iron-limited waters.

Abstract Background Common Pekin and Muscovy ducks and their intergeneric hinny and mule hybrids have different abilities for fatty liver production. RNA-Seq analyses from the liver of these different genetic types fed ad libitum or overfed would help to identify genes with different response to overfeeding between them. However RNA-seq analyses from different species and comparison is challenging. The goal of this study was develop a relevant strategy for transcriptome analysis and comparison between different species. Results Transcriptomes were first assembled with a reference-based approach. Important mapping biases were observed when heterologous mapping were conducted on common duck reference genome, suggesting that this reference-based strategy was not suited to compare the four different genetic types. De novo transcriptome assemblies were then performed using Trinity and Oases. Assemblies of transcriptomes were not relevant when more than a single genetic type was considered. Finally, single genetic type transcriptomes were assembled with DRAP in a mega-transcriptome. No bias was observed when reads from the different genetic types were mapped on this mega-transcriptome and differences in gene expression between the four genetic types could be identified. Conclusions Analyses using both reference-based and de novo transcriptome assemblies point out a good performance of the de novo approach for the analysis of gene expression in different species. It also allowed the identification of differences in responses to overfeeding between Pekin and Muscovy ducks and hinny and mule hybrids.

Abstract Background Under the threat of climate change populations can disperse, acclimatise or evolve in order to avoid fitness loss. In light of this, it is important to understand neutral gene flow patterns as a measure of dispersal potential, but also adaptive genetic variation as a measure of evolutionary potential. In order to assess genetic variation and how this relates to environment in the honeycomb worm (Sabellaria alveolata (L.)), a reef-building polychaete that supports high biodiversity, we carried out RAD sequencing using individuals from along its complete latitudinal range. Patterns of neutral population genetic structure were compared to larval dispersal as predicted by ocean circulation modelling, and outlier analyses and genotype-environment association tests were used to attempt to identify loci under selection in relation to local temperature data. Results We genotyped 482 filtered SNPs, from 68 individuals across nine sites, 27 of which were identified as outliers using BAYESCAN and ARLEQUIN. All outlier loci were potentially under balancing selection, despite previous evidence of local adaptation in the system. Limited gene flow was observed among reef-sites (FST = 0.28 ± 0.10), in line with the low dispersal potential identified by the larval dispersal models. The North Atlantic reef emerged as a distinct population and this was linked to high local larval retention and the effect of the North Atlantic Current on dispersal. Conclusions As an isolated population, with limited potential for natural genetic or demographic augmentation from other reefs, the North Atlantic site warrants conservation attention in order to preserve not only this species, but above all the crucial functional ecological roles that are associated with their bioconstructions. Our study highlights the utility of using seascape genomics to identify populations of conservation concern.

Abstract Background Despite a growing number of investigations on early diverging fungi, the corresponding lineages have not been as extensively characterized as Ascomycota or Basidiomycota ones. The Mucor genus, pertaining to one of these lineages is not an exception. To this date, a restricted number of Mucor annotated genomes is publicly available and mainly correspond to the reference species, Mucor circinelloides, and to medically relevant species. However, the Mucor genus is composed of a large number of ubiquitous species as well as few species that have been reported to specifically occur in certain habitats. The present study aimed to expand the range of Mucor genomes available and identify potential genomic imprints of adaptation to different environments and lifestyles in the Mucor genus. Results In this study, we report four newly sequenced genomes of Mucor isolates collected from non-clinical environments pertaining to species with contrasted lifestyles, namely Mucor fuscus and Mucor lanceolatus, two species used in cheese production (during ripening), Mucor racemosus, a recurrent cheese spoiler sometimes described as an opportunistic animal and human pathogen, and Mucor endophyticus, a plant endophyte. Comparison of these new genomes with those previously available for six Mucor and two Rhizopus (formerly identified as M. racemosus) isolates allowed global structural and functional description such as their TE content, core and species-specific genes and specialized genes. We proposed gene candidates involved in iron metabolism; some of these genes being known to be involved in pathogenicity; and described patterns such as a reduced number of CAZymes in the species used for cheese ripening as well as in the endophytic isolate that might be related to adaptation to different environments and lifestyles within the Mucor genus. Conclusions This study extended the descriptive data set for Mucor genomes, pointed out the complexity of obtaining a robust phylogeny even with multiple genes families and allowed identifying contrasting potentially lifestyle-associated gene repertoires. The obtained data will allow investigating further the link between genetic and its biological data, especially in terms of adaptation to a given habitat.

The Western Antarctic Peninsula (WAP) is among the areas of the planet showing some of the most significant increases in air and water temperature. It is projected that increasing temperature will modulate coastal ecosystems at species ecological performance and molecular composition. The main way that the organisms can cope with large thermal variation is by having a reversible phenotypic plasticity, which provides the organisms with a compensatory physiological response when facing challenging conditions. The giant Antarctic isopod Glyptonotus antarcticus is one of most common species in Antarctic waters. This species has a larval development inside of the maternal marsupium, where juveniles have a short period to acclimate to environmental conditions after birth. In this sense, we hypothesize that juveniles exposed to unusual temperature increases even for short periods, would not respond adequately showing a narrow phenotypic plasticity. We experimentally assessed if early juveniles of G. antarcticus have the molecular plasticity when exposed to increased temperature at 5°C during 1, 6, 12 and 24 h. Sequenced libraries were compared between control (0°C) and each experimental treatment to detect differentially expressed transcripts. The main molecular pathways affected by thermal stress were antioxidant, proteases, endopeptidases and ubiquination transcripts which were up-regulated and mitochondrial respiratory chain, cuticle, cytoskeleton and a molt transcript which were down-regulated. Regarding the HSP transcript, only 3 were up-regulated at least in two points of the stress kinetic, without classical Hsp70 and Hsp90 transcripts. This study shows that juveniles of G. antarcticus do not show molecular phenotypic plasticity to cope with acute short-term heat stress, even for one or few hours of exposure with an absence of an eco-physiological capacity to respond. This may have consequences at the ecological population level, showing a reduced individual ability to survive decreasing population recruitment.

The filamentous algal endophyte Laminarionema elsbetiae is highly prevalent in European populations of the brown alga Saccharina latissima, but has also been found occasionally in the other kelp species Laminaria digitata. The presence of L. elsbetiae coincides with morphological changes in the hosts such as twisted stipes and deformed blades, however, little is known about the molecular bases of these algal host-endophyte interactions. Using a co-cultivation experiment, we showed that physiological and gene regulation responses, and later endophyte prevalences are different between the main and the occasional host. The contact with the endophyte L. elsbetiae induced a stronger and faster transcriptomic regulation in the occasional host L. digitata after 24 h, from which growth rate was later affected. During the first two days of co-cultivation, only 21 differentially expressed genes (DEGs) were common in both kelps, indicating a crucial difference between the molecular responses of the two hosts. By functional annotation, we identified DEGs related to host-endophyte recognition, defense response and cell wall modification. Our results suggest that expression pattern differences between the two kelps related to the recognition of the endophyte and later defense reactions could explain the variability of observed physiological responses and host-endophyte specificity in kelp natural populations.

Abstract Background Kelps (Laminariales, Phaeophyceae) are brown macroalgae of utmost ecological, and increasingly economic, importance on temperate to polar rocky shores. Omics approaches in brown algae are still scarce and knowledge of their acclimation mechanisms to the changing conditions experienced in coastal environments can benefit from the application of RNA-sequencing. Despite evidence of ecotypic differentiation, transcriptomic responses from distinct geographical locations have, to our knowledge, never been studied in the sugar kelp Saccharina latissima so far. Results In this study we investigated gene expression responses using RNA-sequencing of S. latissima from environments with contrasting temperature and salinity conditions – Roscoff, in temperate eastern Atlantic, and Spitsbergen in the Arctic. Juvenile sporophytes derived from uniparental stock cultures from both locations were pre-cultivated at 8 °C and SA 30. Sporophytes acclimated to 0 °C, 8 °C and 15 °C were exposed to a low salinity treatment (SA 20) for 24 h. Hyposalinity had a greater impact at the transcriptomic level than the temperature alone, and its effects were modulated by temperature. Namely, photosynthesis and pigment synthesis were extensively repressed by low salinity at low temperatures. Although some responses were shared among sporophytes from the different sites, marked differences were revealed by principal component analysis, differential expression and GO enrichment. The interaction between low temperature and low salinity drove the largest changes in gene expression in sporophytes from Roscoff while specimens from Spitsbergen required more metabolic adjustment at higher temperatures. Moreover, genes related to cell wall adjustment were differentially expressed between Spitsbergen and Roscoff control samples. Conclusions Our study reveals interactive effects of temperature and salinity on transcriptomic profiles in S. latissima. Moreover, our data suggest that under identical culture conditions sporophytes from different locations diverge in their transcriptomic responses. This is probably connected to variations in temperature and salinity in their respective environment of origin. The current transcriptomic results support the plastic response pattern in sugar kelp which is a species with several reported ecotypes. Our data provide the baseline for a better understanding of the underlying processes of physiological plasticity and may help in the future to identify strains adapted to specific environments and its genetic control.

The cuttlefish (Sepia officinalis) is a Cephalopod mollusk that lives in the English Channel and breeds in coastal spawning grounds in spring. A previous work showed that the control of egg-laying is monitored by different types of regulators, among which neuropeptides play a major role. They are involved in the integration of environmental cues, and participate in the transport of oocytes in the genital tract and in the secretion of capsular products. This study addresses a family of neuropeptides recently identified and suspected to be involved in the control of the reproduction processes. Detected by mass spectrometry and immunocytochemistry in the nerve endings of the accessory sex glands of the females and ovary, these neuropeptides are also identified in the hemolymph of egg-laying females demonstrating that they also have a hormone-like role. Released in the hemolymph by the sub-esophageal mass, a region that innervates the genital tract and the neurohemal area of the vena cava, in in vitro conditions these neuropeptides modulated oocyte transport and capsular secretion. Finally, in silico analyses indicated that these neuropeptides, initially called FLGamide, had extensive structural homology with orcokinin B, which motivated their name change.

Animals, plants, and algae rely on symbiotic microorganisms for their development and functioning. Genome sequencing and genomic analyses of these microorganisms provide opportunities to construct metabolic networks and to analyze the metabolism of the symbiotic communities they constitute. Genome-scale metabolic network reconstructions rest on information gained from genome annotation. As there are multiple annotation pipelines available, the question arises to what extent differences in annotation pipelines impact outcomes of these analyses. Here, we compare five commonly used pipelines (Prokka, MaGe, IMG, DFAST, RAST) from predicted annotation features (coding sequences, Enzyme Commission numbers, hypothetical proteins) to the metabolic network-based analysis of symbiotic communities (biochemical reactions, producible compounds, and selection of minimal complementary bacterial communities). While Prokka and IMG produced the most extensive networks, RAST and DFAST networks produced the fewest false positives and the most connected networks with the fewest dead-end metabolites. Our results underline differences between the outputs of the tested pipelines at all examined levels, with small differences in the draft metabolic networks resulting in the selection of different microbial consortia to expand the metabolic capabilities of the algal host. However, the consortia generated yielded similar predicted producible compounds and could therefore be considered functionally interchangeable. This contrast between selected communities and community functions depending on the annotation pipeline needs to be taken into consideration when interpreting the results of metabolic complementarity analyses. In the future, experimental validation of bioinformatic predictions will likely be crucial to both evaluate and refine the pipelines and needs to be coupled with increased efforts to expand and improve annotations in reference databases.

In a context of ongoing biodiversity erosion, obtaining genomic resources from wildlife is essential for conservation. The thousands of yearly mammalian roadkill provide a useful source material for genomic surveys. To illustrate the potential of this underexploited resource, we used roadkill samples to study the genomic diversity of the bat-eared fox (Otocyon megalotis) and the aardwolf (Proteles cristatus), both having subspecies with similar disjunct distributions in Eastern and Southern Africa. First, we obtained reference genomes with high contiguity and gene completeness by combining Nanopore long reads and Illumina short reads. Then, we showed that the two subspecies of aardwolf might warrant species status (P. cristatus and P. septentrionalis) by comparing their genome-wide genetic differentiation to pairs of well-defined species across Carnivora with a new Genetic Differentiation index (GDI) based on only a few resequenced individuals. Finally, we obtained a genome-scale Carnivora phylogeny including the new aardwolf species.

Dinoflagellates are major components of phytoplankton that play critical roles in many microbial food webs, many of them being hosts of countless intracellular parasites. The phototrophic dinoflagellate Scrippsiella acuminata (Dinophyceae) can be infected by the microeukaryotic parasitoids Amoebophrya spp. (Syndiniales), some of which primarily target and digest the host nucleus. Early digestion of the nucleus at the beginning of the infection is expected to greatly impact the host metabolism, inducing the knockout of the organellar machineries that highly depend upon nuclear gene expression, such as the mitochondrial OXPHOS pathway and the plastid photosynthetic carbon fixation. However, previous studies have reported that chloroplasts remain functional in swimming host cells infected by Amoebophrya. We report here a multi-approach monitoring study of S. acuminata organelles over a complete infection cycle by nucleus-targeting Amoebophrya sp. strain A120. Our results show sustained and efficient photosystem II activity as a hallmark of functional chloroplast throughout the infection period despite the complete digestion of the host nucleus. We also report the importance played by light on parasite production, i.e., the amount of host biomass converted to parasite infective propagules. Using a differential gene expression analysis, we observed an apparent increase of all 3 mitochondrial and 9 out of the 11 plastidial genes involved in the electron transport chains (ETC) of the respiration pathways during the first stages of the infection. The longer resilience of organellar genes compared to those encoded by the nucleus suggests that both mitochondria and chloroplasts remain functional throughout most of the infection. This extended organelle functionality, along with higher parasite production under light conditions, suggests that host bioenergetic organelles likely benefit the parasite Amoebophrya sp. A120 and improve its fitness during the intracellular infective stage.

Kelps in the Arctic region are facing challenging natural conditions. They experience over 120 days of darkness during the polar night surviving on storage compounds without conducting photosynthesis. Furthermore, the Arctic is experiencing continuous warming as a consequence of climate change. Such temperature increase may enhance the metabolic activity of kelps, using up storage compounds faster. As the survival strategy of kelps during darkness in the warming Arctic is poorly understood, we studied the physiological and transcriptomic responses of Saccharina latissima, one of the most common kelp species in the Arctic, after a 2-week dark exposure at two temperatures (0 and 4°C) versus the same temperatures under low light conditions. Growth rates were decreased in darkness but remained stable at two temperatures. Pigments had higher values in darkness and at 4°C. Darkness had a greater impact on the transcriptomic performance of S. latissima than increased temperature according to the high numbers of differentially expressed genes between dark and light treatments. Darkness generally repressed the expression of genes coding for glycolysis and metabolite biosynthesis, as well as some energy-demanding processes, such as synthesis of photosynthetic components and transporters. Moreover, increased temperature enhanced these repressions, while the expression of some genes encoding components of the lipid and laminaran catabolism, glyoxylate cycle and signaling were enhanced in darkness. Our study helps to understand the survival strategy of kelp in the early polar night and its potential resilience to the warming Arctic.

Heterotis niloticus is an African species of Osteoglossiformes that presents biological peculiarities and zootechnical performances favorable for fish farming. However, the absence of a sexual dimorphism hinders the optimization of its reproduction in captivity and limits the understanding of its reproductive behavior. This study is aimed at developing a minimally invasive and reliable sexing method to detect vitellogenin (Vtg) in female plasma. A commercial sexing kit (Acobium, Montpellier, France) for Arapaima gigas—a phylogenetically sister species of H. niloticus—successfully identified only 20% of mature H. niloticus females. Enzyme-linked immunosorbent assays (ELISA) were carried out using three Vtg antibodies. The A. gigas Vtg1 antibody cross-reacted significantly with plasma dilutions of female H. niloticus ranging from 1:1000 to 1:10,000, but with relatively low intensity. The Vtg antibody from Osteoglossum bicirrhosum, another species of Osteoglossiformes, showed non-specific binding with the Vtg of H. niloticus female plasma. Finally, an antibody for H. niloticus Vtg developed in this study allowed us to differentiate the two sexes with plasma coating dilutions ranging from 1:1000 to 1:10,000. The results of the assay were validated by a proteomic approach showing that Vtg-targeted mass spectrometry analysis of H. niloticus blood protein extracts could be used to accurately determine the presence of Vtg in the plasma of mature females. The final validation of the ELISA technique using the H. niloticus Vtg antibody was confirmed by visual sexing of a significant number of blood-sampled fish gonads; 100% of the fish were correctly sexed by the ELISA method.

Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus are the most abundant photosynthetic organisms on Earth, an ecological success thought to be linked to the differential partitioning of distinct ecotypes into specific ecological niches. However, the underlying processes that governed the diversification of these microorganisms and the appearance of niche-related phenotypic traits are just starting to be elucidated. Here, by comparing 81 genomes, including 34 new Synechococcus, we explored the evolutionary processes that shaped the genomic diversity of picocyanobacteria. Time-calibration of a core-protein tree showed that gene gain/loss occurred at an unexpectedly low rate between the different lineages, with for instance 5.6 genes gained per million years (My) for the major Synechococcus lineage (sub-cluster 5.1), among which only 0.71/My have been fixed in the long term. Gene content comparisons revealed a number of candidates involved in nutrient adaptation, a large proportion of which are located in genomic islands shared between either closely or more distantly related strains, as identified using an original network construction approach. Interestingly, strains representative of the different ecotypes co-occurring in phosphorus-depleted waters (Synechococcus clades III, WPC1, and sub-cluster 5.3) were shown to display different adaptation strategies to this limitation. In contrast, we found few genes potentially involved in adaptation to temperature when comparing cold and warm thermotypes. Indeed, comparison of core protein sequences highlighted variants specific to cold thermotypes, notably involved in carotenoid biosynthesis and the oxidative stress response, revealing that long-term adaptation to thermal niches relies on amino acid substitutions rather than on gene content variation. Altogether, this study not only deciphers the respective roles of gene gains/losses and sequence variation but also uncovers numerous gene candidates likely involved in niche partitioning of two key members of the marine phytoplankton.

Eyes morphologies may differ but those differences are not reflected at the molecular level. Indeed, the ability to perceive light is thought to come from the same conserved gene families: opsins and cryptochromes. Even though cuttlefish (Cephalopoda) are known for their visually guided behaviors, there is a lack of data about the different opsins and cryptochromes orthologs represented in the genome and their expressions. Here we studied the evolutionary history of opsins, cryptochromes but also visual arrestins in molluscs with an emphasis on cephalopods. We identified 6 opsins, 2 cryptochromes and 1 visual arrestin in Sepia officinalis and we showed these families undergo several duplication events in Mollusca: one duplication in the arrestin family and two in the opsin family. In cuttlefish, we studied the temporal expression of these genes in the eyes of embryos from stage 23 to hatching and their expression in two extraocular tissues, skin and central nervous system (CNS = brain + optic lobes). We showed in embryos that some of these genes (Sof_CRY6, Sof_reti-1, Sof_reti-2, Sof_r-opsin1 and Sof_v-arr) are expressed in the eyes and not in the skin or CNS. By looking at a juvenile and an adult S. officinalis, it seems that some of these genes (Sof_r-opsin1 and Sof_reti1) are used for light detection in these extraocular tissues but that they set-up later in development than in the eyes. We also showed that their expression (except for Sof_CRY6) undergoes an increase in the eyes from stage 25 to 28 thus confirming their role in the ability of the cuttlefish embryos to perceive light through the egg capsule. This study raises the question of the role of Sof_CRY6 in the developing eyes in cuttlefish embryos and the role and localization of xenopsins and r-opsin2. Consequently, the diversity of molecular actors involved in light detection both in the eyes and extraocular tissues is higher than previously known. These results open the way for studying new molecules such as those of the signal transduction cascade.

Understanding how microorganisms adjust their metabolism to maintain their ability to cope with short-term environmental variations constitutes one of the major current challenges in microbial ecology. Here, the best physiologically characterized marine Synechococcus strain, WH7803, was exposed to modulated light/dark cycles or acclimated to continuous high-light (HL) or low-light (LL), then shifted to various stress conditions, including low (LT) or high temperature (HT), HL and ultraviolet (UV) radiations. Physiological responses were analyzed by measuring time courses of photosystem (PS) II quantum yield, PSII repair rate, pigment ratios and global changes in gene expression. Previously published membrane lipid composition were also used for correlation analyses. These data revealed that cells previously acclimated to HL are better prepared than LL-acclimated cells to sustain an additional light or UV stress, but not a LT stress. Indeed, LT seems to induce a synergic effect with the HL treatment, as previously observed with oxidative stress. While all tested shift conditions induced the downregulation of many photosynthetic genes, notably those encoding PSI, cytochrome b6/f and phycobilisomes, UV stress proved to be more deleterious for PSII than the other treatments, and full recovery of damaged PSII from UV stress seemed to involve the neo-synthesis of a fairly large number of PSII subunits and not just the reassembly of pre-existing subunits after D1 replacement. In contrast, genes involved in glycogen degradation and carotenoid biosynthesis pathways were more particularly upregulated in response to LT. Altogether, these experiments allowed us to identify responses common to all stresses and those more specific to a given stress, thus highlighting genes potentially involved in niche acclimation of a key member of marine ecosystems. Our data also revealed important specific features of the stress responses compared to model freshwater cyanobacteria.

Summary: Inferring genome-scale metabolic networks in emerging model organisms is challenged by incomplete biochemical knowledge and partial conservation of biochemical pathways during evolution. Therefore, specific bioinformatic tools are necessary to infer biochemical reactions and metabolic structures that can be checked experimentally. Using an integrative approach combining genomic and metabolomic data in the red algal model Chondrus crispus, we show that, even metabolic pathways considered as conserved, like sterols or mycosporine-like amino acid synthesis pathways, undergo substantial turnover. This phenomenon, here formally defined as “metabolic pathway drift,” is consistent with findings from other areas of evolutionary biology, indicating that a given phenotype can be conserved even if the underlying molecular mechanisms are changing. We present a proof of concept with a methodological approach to formalize the logical reasoning necessary to infer reactions and molecular structures, abstracting molecular transformations based on previous biochemical knowledge. : Bioinformatics; Genomics; Metabolomics Subject Areas: Bioinformatics, Genomics, Metabolomics

Brown algae are key components of marine ecosystems and live in association with bacteria that are essential for their growth and development. Ectocarpus siliculosus is a genetic and genomic model for brown algae. Here we use this model to start disentangling the complex interactions that may occur between the algal host and its associated bacteria. We report the genome-sequencing of 10 alga-associated bacteria and the genome-based reconstruction of their metabolic networks. The predicted metabolic capacities were then used to identify metabolic complementarities between the algal host and the bacteria, highlighting a range of potentially beneficial metabolite exchanges between them. These putative exchanges allowed us to predict consortia consisting of a subset of these ten bacteria that would best complement the algal metabolism. Finally, co-culture experiments were set up with a subset of these consortia to monitor algal growth as well as the presence of key algal metabolites. Although we did not fully control but only modified bacterial communities in our experiments, our data demonstrated a significant increase in algal growth in cultures inoculated with the selected consortia. In several cases, we also detected, in algal extracts, the presence of key metabolites predicted to become producible via an exchange of metabolites between the alga and the microbiome. Thus, although further methodological developments will be necessary to better control and understand microbial interactions in Ectocarpus, our data suggest that metabolic complementarity is a good indicator of beneficial metabolite exchanges in the holobiont.

Saccharina latissima is an economically and ecologically relevant kelp species in Europe and North America. In kelps, the sexuality is expressed during the haploid life stage and the microscopic gametophytes exhibit significant sexual dimorphism. To understand the sex-dependent impact of temperature on the gametophyte stage, we analyzed for the first time, gene expression profiles of male and female gametophytes at three different temperatures (4, 12, and 20°C) characteristic for the species distribution range by using RNA-sequencing. We identified several differentially expressed genes (DEGs) between sexes; while female biased genes were enriched in general metabolism and energy production, male biased genes function within cell cycle and signaling. In our study, temperature modulated sex-biased gene expression, with only a small percentage of DEGs consistently male (7%) or female-biased (12%) at the three temperatures. Female gametophytes responded stronger to higher temperatures than males, suggesting that males are more heat tolerant. Differences between S. latissima and other brown algal gender-dependent gene expression might mirror the different evolutionary and ecological contexts. Genomic information on kelp gametophyte is still scarce and thus this study adds to our knowledge on sex differences in abiotic stress responses in macroalgae at the transcriptomic level.

Abstract Background Study of meta-transcriptomic datasets involving non-model organisms represents bioinformatic challenges. The production of chimeric sequences and our inability to distinguish the taxonomic origins of the sequences produced are inherent and recurrent difficulties in de novo assembly analyses. As the study of holobiont meta-transcriptomes is affected by challenges invoked above, we propose an innovative bioinformatic approach to tackle such difficulties and tested it on marine models as a proof of concept. Results We considered three holobiont models, of which two transcriptomes were previously published and a yet unpublished transcriptome, to analyze and sort their raw reads using Short Read Connector, a k-mer based similarity method. Before assembly, we thus defined four distinct categories for each holobiont meta-transcriptome: host reads, symbiont reads, shared reads, and unassigned reads. Afterwards, we observed that independent de novo assemblies for each category led to a diminution of the number of chimeras compared to classical assembly methods. Moreover, the separation of each partner’s transcriptome offered the independent and comparative exploration of their functional diversity in the holobiont. Finally, our strategy allowed to propose new functional annotations for two well-studied holobionts (a Cnidaria-Dinophyta, a Porifera-Bacteria) and a first meta-transcriptome from a planktonic Radiolaria-Dinophyta system forming widespread symbiotic association for which our knowledge is considerably limited. Conclusions In contrast to classical assembly approaches, our bioinformatic strategy generates less de novo assembled chimera and allows biologists to study separately host and symbiont data from a holobiont mixture. The pre-assembly separation of reads using an efficient tool as Short Read Connector is an effective way to tackle meta-transcriptomic challenges and offers bright perpectives to study holobiont systems composed of either well-studied or poorly characterized symbiotic lineages and ultimately expand our knowledge about these associations.

Abstract Background Aquaculture production is expected to double by 2030, and demands for aquafeeds and raw materials are expected to increase accordingly. Sustainable growth of aquaculture will require the development of highly nutritive and functional raw materials to efficiently replace fish meal. Enzymatic hydrolysis of marine and aquaculture raw materials could bring new functionalities to finished products. The aim of this study was to determine the zootechnical and transcriptomic performances of protein hydrolysates of different origins (tilapia, shrimp, and a combination of the two) in European seabass (Dicentrarchux labrax) fed a low fish meal diet (5%), for 65 days. Results Results were compared to a positive control fed with 20% of fish meal. Growth performances, anterior intestine histological organization and transcriptomic responses were monitored and analyzed. Dietary inclusion of protein hydrolysates in the low fish meal diet restored similar growth performances to those of the positive control. Inclusion of dietary shrimp hydrolysate resulted in larger villi and more goblet cells, even better than the positive control. Transcriptomic analysis of the anterior intestine showed that dietary hydrolysate inclusion restored a pattern of intestinal gene expression very close to the pattern of the positive control. However, as compared to the low fish meal diet and depending on their origin, the different hydrolysates did not modulate metabolic pathways in the same way. Dietary shrimp hydrolysate inclusion modulated more metabolic pathways related to immunity, while nutritional metabolism was more impacted by dietary tilapia hydrolysate. Interestingly, the combination of the two hydrolysates enhanced the benefits of hydrolysate inclusion in diets: more genes and metabolic pathways were regulated by the combined hydrolysates than by each hydrolysate tested independently. Conclusions Protein hydrolysates manufactured from aquaculture by-products are promising candidates to help replace fish meal in aquaculture feeds without disrupting animal metabolism and performances.

Antimicrobial peptides (AMPs) participate in the immune system to avoid infection, are present in all living organisms and can be used as drugs. Fish express numerous AMP families including defensins, cathelicidins, liver-expressed antimicrobial peptides (LEAPs), histone-derived peptides, and piscidins (a fish-specific AMP family). The present study demonstrates for the first time the occurrence of several AMPs in lionfish (Pterois volitans). Using the lionfish transcriptome, we identified four transcript sequences encoding cysteine-rich AMPs and two new transcripts encoding piscidin-like peptides. These AMPs are described for the first time in a species of the Scorpaenidae family. A functional approach on new pteroicidins was carried out to determine antimicrobial sequences and potential uses, with a view to using some of these AMPs for human health or in aquaculture.

The marine diatom Guinardia delicatula is a cosmopolitan species that dominates seasonal blooms in the English Channel and the North Sea. Several eukaryotic parasites are known to induce the mortality of this species. Here, we report the isolation and characterization of the first viruses that infect G. delicatula. Viruses were isolated from the Western English Channel (SOMLIT-Astan station) during the late summer bloom decline of G. delicatula. A combination of laboratory approaches revealed that these lytic viruses (GdelRNAV) are small tailless particles of 35–38 nm in diameter that replicate in the host cytoplasm where both unordered particles and crystalline arrays are formed. GdelRNAV display a linear single-stranded RNA genome of ~9 kb, including two open reading frames encoding for replication and structural polyproteins. Phylogenetic relationships based on the RNA-dependent-RNA-polymerase gene marker showed that GdelRNAV are new members of the Bacillarnavirus, a monophyletic genus belonging to the order Picornavirales. GdelRNAV are specific to several strains of G. delicatula. They were rapidly and largely produced (

The fungal phytopathogen Colletotrichum lupini is responsible for lupin anthracnose, resulting in significant yield losses worldwide. The molecular mechanisms underlying this infectious process are yet to be elucidated. This study proposes to evaluate C. lupini gene expression and protein synthesis during lupin infection, using, respectively, an RNAseq-based transcriptomic approach and a mass spectrometry-based proteomic approach. Patterns of differentially-expressed genes in planta were evaluated from 24 to 84 hours post-inoculation, and compared to in vitro cultures. A total of 897 differentially-expressed genes were identified from C. lupini during interaction with white lupin, of which 520 genes were predicted to have a putative function, including carbohydrate active enzyme, effector, protease or transporter-encoding genes, commonly described as pathogenicity factors for other Colletotrichum species during plant infection, and 377 hypothetical proteins. Simultaneously, a total of 304 proteins produced during the interaction were identified and quantified by mass spectrometry. Taken together, the results highlight that the dynamics of symptoms, gene expression and protein synthesis shared similarities to those of hemibiotrophic pathogens. In addition, a few genes with unknown or poorly-described functions were found to be specifically associated with the early or late stages of infection, suggesting that they may be of importance for pathogenicity. This study, conducted for the first time on a species belonging to the Colletotrichum acutatum species complex, presents an opportunity to deepen functional analyses of the genes involved in the pathogenicity of Colletotrichum spp. during the onset of plant infection.

Global change scenarios in the Mediterranean basin predict a precipitation reduction within the coming hundred years. Therefore, increased drought will affect forests both in terms of adaptive ecology and ecosystemic services. However, how vegetation might adapt to drought is poorly understood. In this report, four years of climate change was simulated by excluding 35% of precipitation above a downy oak forest. RNASeq data allowed us to assemble a genome-guided transcriptome. This led to the identification of differentially expressed features, which was supported by the characterization of target metabolites using a metabolomics approach. We provided 2.5 Tb of RNASeq data and the assembly of the first genome guided transcriptome of Quercus pubescens. Up to 5724 differentially expressed transcripts were obtained; 42 involved in plant response to drought. Transcript set enrichment analysis showed that drought induces an increase in oxidative pressure that is mitigated by the upregulation of ubiquitin-like protein protease, ferrochelatase, oxaloacetate decarboxylase and oxo-acid-lyase activities. Furthermore, the downregulation of auxin biosynthesis and transport, carbohydrate storage metabolism were observed as well as the concomitant accumulation of metabolites, such as oxalic acid, malate and isocitrate. Our data suggest that early metabolic changes in the resistance of Q. pubescens to drought involve a tricarboxylic acid (TCA) cycle shunt through the glyoxylate pathway, galactose metabolism by reducing carbohydrate storage and increased proteolytic activity.

Cuttlefish (Sepia officinalis) haemocytes are potential sources of antimicrobial peptides (AMPs). To study the immune response to Vibrio splendidus and identify new AMPs, an original approach was developed based on a differential transcriptomic study and an in-depth in silico analysis using multiple tools. Two de novo transcriptomes were retrieved from cuttlefish haemocytes following challenge by V. splendidus or not. A first analysis of the annotated transcripts revealed the presence of Toll/NF-κB pathway members, including newly identified factors such as So-TLR-h, So-IKK-h and So-Rel/NF-κB-h. Out of the eight Toll/NF-κB pathway members, seven were found up-regulated following V. splendidus challenge. Besides, immune factors involved in the immune response were also identified and up-regulated. However, no AMP was identified based on annotation or conserved pattern searches. We therefore performed an in-depth in silico analysis of unannotated transcripts based on differential expression and sequence characteristics, using several tools available like PepTraq, a homemade software program. Finally, five AMP candidates were synthesized. Among them, NF19, AV19 and GK28 displayed antibacterial activity against Gram-negative bacteria. Each peptide had a different spectrum of activity, notably against Vibrio species. GK28—the most active peptide—was not haemolytic, whereas NF19 and AV19 were haemolytic at concentrations between 50 and 100 µM, 5 to 10 times higher than their minimum inhibitory concentration.

Biological control using bacteriophages is a promising approach for mitigating the devastating effects of coral diseases. Several phages that infect Vibrio coralliilyticus, a widespread coral pathogen, have been isolated, suggesting that this bacterium is permissive to viral infection and is, therefore, a suitable candidate for treatment by phage therapy. In this study, we combined functional and genomic approaches to evaluate the therapeutic potential of BONAISHI, a novel V. coralliilyticus phage, which was isolated from the coral reef in Van Phong Bay (Vietnam). BONAISHI appears to be strictly lytic for several pathogenic strains of V. coralliilyticus and remains infectious over a broad range of environmental conditions. This candidate has an unusually large dsDNA genome (303 kb), with no genes that encode known toxins or implicated in lysogeny control. We identified several proteins involved in host lysis, which may offer an interesting alternative to the use of whole bacteriophages for controlling V. coralliilyticus. A preliminary therapy test showed that adding BONAISHI to an infected culture of Symbiodinium sp. cells reduced the impact of V. coralliilyticus on Symbiodinium sp. photosynthetic activity. This study showed that BONAISHI is able to mitigate V. coralliilyticus infections, making it a good candidate for phage therapy for coral disease.

Understanding factors that generate, maintain, and constrain host-parasite associations is of major interest to biologists. Although little studied, many extremely virulent micro-eukaryotic parasites infecting microalgae have been reported in the marine plankton. This is the case for Amoebophrya, a diverse and highly widespread group of Syndiniales infecting and potentially controlling dinoflagellate populations. Here, we analyzed the time-scale gene expression of a complete infection cycle of two Amoebophrya strains infecting the same host (the dinoflagellate Scrippsiella acuminata), but diverging by their host range (one infecting a single host, the other infecting more than one species). Over two-thirds of genes showed two-fold differences in expression between at least two sampled stages of the Amoebophrya life cycle. Genes related to carbohydrate metabolism as well as signaling pathways involving proteases and transporters were overexpressed during the free-living stage of the parasitoid. Once inside the host, all genes related to transcription and translation pathways were actively expressed, suggesting the rapid and extensive protein translation needed following host-cell invasion. Finally, genes related to cellular division and components of the flagellum organization were overexpressed during the sporont stage. In order to gain a deeper understanding of the biological basis of the host-parasitoid interaction, we screened proteins involved in host-cell recognition, invasion, and protection against host-defense identified in model apicomplexan parasites. Very few of the genes encoding critical components of the parasitic lifestyle of apicomplexans could be unambiguously identified as highly expressed in Amoebophrya. Genes related to the oxidative stress response were identified as highly expressed in both parasitoid strains. Among them, the correlated expression of superoxide dismutase/ascorbate peroxidase in the specialist parasite was consistent with previous studies on Perkinsus marinus defense. However, this defense process could not be identified in the generalist Amoebophrya strain, suggesting the establishment of different strategies for parasite protection related to host specificity.

Modern genome sequencing strategies are highly sensitive to contamination making the detection of foreign DNA sequences an important part of analysis pipelines. Here we use Taxoblast, a simple pipeline with a graphical user interface, for the post-assembly detection of contaminating sequences in the published genome of the kelp Saccharina japonica. Analyses were based on multiple blastn searches with short sequence fragments. They revealed a number of probable bacterial contaminations as well as hybrid scaffolds that contain both bacterial and algal sequences. This or similar types of analysis, in combination with manual curation, may thus constitute a useful complement to standard bioinformatics analyses prior to submission of genomic data to public repositories. Our analysis pipeline is open-source and freely available at http://sdittami.altervista.org/taxoblast and via SourceForge (https://sourceforge.net/projects/taxoblast).

Kelps are founding species of temperate marine ecosystems, living in intertidal coastal areas where they are often challenged by generalist and specialist herbivores. As most sessile organisms, kelps develop defensive strategies to restrain grazing damage and preserve their own fitness during interactions with herbivores. To decipher some inducible defense and signaling mechanisms, we carried out metabolome and transcriptome analyses in two emblematic kelp species, Lessonia spicata from South Pacific coasts and Laminaria digitata from North Atlantic, when challenged with their main specialist herbivores. Mass spectrometry based metabolomics revealed large metabolic changes induced in these two brown algae following challenges with their own specialist herbivores. Targeted metabolic profiling of L. spicata further showed that free fatty acid (FFA) and amino acid (AA) metabolisms were particularly regulated under grazing. An early stress response was illustrated by the accumulation of Sulphur containing amino acids in the first twelve hours of herbivory pressure. At latter time periods (after 24 hours), we observed FFA liberation and eicosanoid oxylipins synthesis likely representing metabolites related to stress. Global transcriptomic analysis identified sets of candidate genes specifically induced by grazing in both kelps. qPCR analysis of the top candidate genes during a 48-hours time course validated the results. Most of these genes were particularly activated by herbivore challenge after 24 hours, suggesting that transcriptional reprogramming could be operated at this time period. We demonstrated the potential utility of these genes as molecular markers for herbivory by measuring their inductions in grazed individuals of field harvested L. digitata and L. spicata. By unravelling the regulation of some metabolites and genes following grazing pressure in two kelps representative of the two hemispheres, this work contributes to provide a set of herbivore-induced chemical and molecular responses in kelp species, showing similar inducible responses upon specialist herbivores in their respective ecosystems.

Understanding growth mechanisms in brown algae is a current scientific and economic challenge that can benefit from the modeling of their metabolic networks. The sequencing of the genomes of Saccharina japonica and Cladosiphon okamuranus has provided the necessary data for the reconstruction of Genome−Scale Metabolic Networks (GSMNs). The same in silico method deployed for the GSMN reconstruction of Ectocarpus siliculosus to investigate the metabolic capabilities of these two algae, was used. Integrating metabolic profiling data from the literature, we provided functional GSMNs composed of an average of 2230 metabolites and 3370 reactions. Based on these GSMNs and previously published work, we propose a model for the biosynthetic pathways of the main carotenoids in these two algae. We highlight, on the one hand, the reactions and enzymes that have been preserved through evolution and, on the other hand, the specificities related to brown algae. Our data further indicate that, if abscisic acid is produced by Saccharina japonica, its biosynthesis pathway seems to be different in its final steps from that described in land plants. Thus, our work illustrates the potential of GSMNs reconstructions for formalizing hypotheses that can be further tested using targeted biochemical approaches.

Amelotin (AMTN) is an ameloblast-secreted protein that belongs to the secretory calcium-binding phosphoprotein family, which also includes the enamel matrix proteins amelogenin, ameloblastin and enamelin. Although AMTN is supposed to play an important role in enamel formation, data were long limited to the rodents, in which it is expressed during the maturation stage. Recent comparative studies in sauropsids and amphibians revealed that (i) AMTN was expressed earlier, i.e. as soon as ameloblasts are depositing the enamel matrix, and (ii) AMTN structure was different, a change which mostly resulted from an intraexonic splicing in the large exon 8 of an ancestral mammal. The present study was performed to know whether the differences in AMTN structure and expression in rodents compared to non-mammalian tetrapods dated back to an early ancestral mammal or were acquired later in mammalian evolution. We sequenced, assembled and screened the jaw transcriptome of a neonate opossum Monodelphis domestica, a marsupial. We found two AMTN transcripts. Variant 1, representing 70.8% of AMTN transcripts, displayed the structure known in rodents, whereas variant 2 (29.2%) exhibited the nonmammalian tetrapod structure. Then, we studied AMTN expression during amelogenesis in a neonate specimen. We obtained similar data as those reported in rodents. These findings indicate that more than 180 million years ago, before the divergence of marsupials and placentals, changes occurred in AMTN function and structure. The spatiotemporal expression was delayed to the maturation stage of amelogenesis and the intraexonic splicing gave rise to isoform 1, encoded by variant 1 and lacking the RGD motif. The ancestral isoform 2, housing the RGD, was initially conserved, as demonstrated here in a marsupial, then secondarily lost in the placental lineages. These findings bring new elements towards our understanding of the non-prismatic to prismatic enamel transition that occurred at the onset of mammals.

A comparative thermal tolerance study was undertaken on two sister species of Euphausiids (Antarctic krills) Euphausia superba and Euphausia crystallorophias. Both are essential components of the Southern Ocean ecosystem, but occupy distinct environmental geographical locations with slightly different temperature regimes. They therefore provide a useful model system for the investigation of adaptations to thermal tolerance.Initial CTmax studies showed that E. superba was slightly more thermotolerant than E. crystallorophias. Five Hsp70 mRNAs were characterized from the RNAseq data of both species and subsequent expression kinetics studies revealed notable differences in induction of each of the 5 orthologues between the two species, with E. crystallorophias reacting more rapidly than E. superba. Furthermore, analyses conducted to estimate the evolutionary rates and selection strengths acting on each gene tended to support the hypothesis that diversifying selection has contributed to the diversification of this gene family, and led to the selective relaxation on the inducible C form with its possible loss of function in the two krill species.The sensitivity of the epipelagic species E. crystallorophias to temperature variations and/or its adaptation to cold is enhanced when compared with its sister species, E. superba. These results indicate that ice krill could be the first of the two species to be impacted by the warming of coastal waters of the Austral ocean in the coming years due to climate change.

Sepia officinalis egg protection is ensured by a complex capsule produced by the female accessory genital glands and the ink bag. Our study is focused on the proteins constituting the main egg case. De novo transcriptomes from female genital glands provided essential databases for protein identification. A proteomic approach in SDS-PAGE coupled with MS unveiled a new egg case protein family: SepECPs, for Sepia officinalis Egg Case Proteins. N-glycosylation was demonstrated by PAS staining SDS-PAGE gels. These glycoproteins are mainly produced in the main nidamental glands. SepECPs share high sequence homology, especially in the signal peptide and the three cysteine-rich domains. SepECPs have a high number of cysteines, with conserved motifs involved in 3D-structure. SDS-PAGE showed that SepECPs could form dimers; this result was confirmed by TEM observations, which also revealed a protein network. This network is similar to the capsule network, and it associates these structural proteins with polysaccharides, melanin and bacteria to form a tight mesh. Its hardness and elasticity provide physical protection to the embryo. In addition, SepECPs also have bacteriostatic antimicrobial activity on GRAM- bacteria. By observing the SepECP / Vibrio aestuarianus complex in SEM, we demonstrated the ability of these proteins to agglomerate bacteria and thus inhibit their growth. These original proteins identified from the outer egg case ensure the survival of the species by providing physical and chemical protection to the embryos released in the environment without any maternal protection.

The applied exploitation of microalgae cultures has to date almost exclusively involved the use of wild type strains, deposited over decades in dedicated culture collections. Concomitantly, the concept of improving algae with selection programs for particular specific purposes is slowly emerging. Studying since a decade an economically and ecologically important haptophyte Tisochrysis lutea (Tiso), we took advantage of the availability of wild type (Tiso-Wt) and selected (Tiso-S2M2) strains to conduct a molecular variations study. This endeavour presented substantial challenges: the genome assembly was not yet available, the life cycle unknown and genetic diversity of Tiso-Wt poorly documented. This study brings the first molecular data in order to set up a selection strategy for that microalgae. Following high-throughput Illumina sequencing, transcriptomes of Tiso-Wt and Tiso-S2M2 were de novo assembled and annotated. Genetic diversity between both strains was analyzed and revealed a clear conservation, while a comparison of transcriptomes allowed identification of polymorphisms resulting from the selection program. Of 34,374 transcripts, 291 were differentially expressed and 165 contained positional polymorphisms (SNP, Indel). We focused on lipid over-accumulation of the Tiso-S2M2 strain and 8 candidate genes were identified by combining analysis of positional polymorphism, differential expression levels, selection signature and by study of putative gene function. Moreover, genetic analysis also suggests the existence of a sexual cycle and genetic recombination in Tisochrysis lutea.

BACKGROUND: The Ice krill, Euphausia crystallorophias is one of the species at the base of the Southern Ocean food chain. Given their significant contribution to the biomass of the Southern Ocean, it is vitally important to gain a better understanding of their physiology and, in particular, anticipate their responses to climate change effects in the warming seas around Antarctica. METHODOLOGY/PRINCIPAL FINDINGS: Illumina sequencing was used to produce a transcriptome of the ice krill. Analysis of the assembled contigs via two different methods, produced 36 new pre-pro-peptides, coding for 61 neuropeptides or peptide hormones belonging to the following families: Allatostatins (A, B et C), Bursicon (α and β), Crustacean Hyperglycemic Hormones (CHH and MIH/VIHs), Crustacean Cardioactive Peptide (CCAP), Corazonin, Diuretic Hormones (DH), the Eclosion Hormone (EH), Neuroparsin, Neuropeptide F (NPF), small Neuropeptide F (sNPF), Pigment Dispersing Hormone (PDH), Red Pigment Concentrating Hormone (RPCH) and finally Tachykinin. LC/MS/MS proteomics was also carried out on eyestalk extracts, which are the major site of neuropeptide synthesis in decapod crustaceans. Results confirmed the presence of six neuropeptides and six precursor-related peptides previously identified in the transcriptome analyses. CONCLUSIONS: This study represents the first comprehensive analysis of neuropeptide hormones in a Eucarida non-decapod Malacostraca, several of which are described for the first time in a non-decapod crustacean. Additionally, there is a potential expansion of PDH and Neuropeptide F family members, which may reflect certain life history traits such as circadian rhythms associated with diurnal migrations and also the confirmation via mass spectrometry of several novel pre-pro-peptides, of unknown function. Knowledge of these essential hormones provides a vital framework for understanding the physiological response of this key Southern Ocean species to climate change and provides a valuable resource for studies into the molecular phylogeny of these organisms and the evolution of neuropeptide hormones.

Microbiome research has grown substantially over the past decade in terms of the range of biomes sampled, identified taxa, and the volume of data derived from the samples. In particular, experimental approaches such as metagenomics, metabarcoding, metatranscriptomics and metaproteomics have provided profound insights into the vast, hitherto unknown, microbial biodiversity. The ELIXIR Marine Metagenomics Community, initiated amongst researchers focusing on marine microbiomes, has concentrated on promoting standards around microbiome-derived sequence analysis, as well as understanding the gaps in methods and reference databases, and solutions to computational overheads of performing such analyses. Nevertheless, the methods used and the challenges faced are not confined to marine studies, but are broadly applicable to all other biomes. Thus, expanding this Community to a more inclusive ELIXIR Microbiome Community will enable it to encompass a broad range of biomes and link expertise across ‘omics technologies. Furthermore, engaging with a large number of researchers will improve the efficiency and sustainability of bioinformatics infrastructure and resources for microbiome research (standards, data, tools, workflows, training), which will enable a deeper understanding of the function and taxonomic composition of the different microbial communities.

Threats to global biodiversity are increasingly recognised by scientists and the public as a critical challenge. Molecular sequencing technologies offer means to catalogue, explore, and monitor the richness and biogeography of life on Earth. However, exploiting their full potential requires tools that connect biodiversity infrastructures and resources. As a research infrastructure developing services and technical solutions that help integrate and coordinate life science resources across Europe, ELIXIR is a key player. To identify opportunities, highlight priorities, and aid strategic thinking, here we survey approaches by which molecular technologies help inform understanding of biodiversity. We detail example use cases to highlight how DNA sequencing is: resolving taxonomic issues; Increasing knowledge of marine biodiversity; helping understand how agriculture and biodiversity are critically linked; and playing an essential role in ecological studies. Together with examples of national biodiversity programmes, the use cases show where progress is being made but also highlight common challenges and opportunities for future enhancement of underlying technologies and services that connect molecular and wider biodiversity domains. Based on emerging themes, we propose key recommendations to guide future funding for biodiversity research: biodiversity and bioinformatic infrastructures need to collaborate closely and strategically; taxonomic efforts need to be aligned and harmonised across domains; metadata needs to be standardised and common data management approaches widely adopted; current approaches need to be scaled up dramatically to address the anticipated explosion of molecular data; bioinformatics support for biodiversity research needs to be enabled and sustained; training for end users of biodiversity research infrastructures needs to be prioritised; and community initiatives need to be proactive and focused on enabling solutions. For sequencing data to deliver their full potential they must be connected to knowledge: together, molecular sequence data collection initiatives and biodiversity research infrastructures can advance global efforts to prevent further decline of Earth’s biodiversity.

Threats to global biodiversity are increasingly recognised by scientists and the public as a critical challenge. Molecular sequencing technologies offer means to catalogue, explore, and monitor the richness and biogeography of life on Earth. However, exploiting their full potential requires tools that connect biodiversity infrastructures and resources. As a research infrastructure developing services and technical solutions that help integrate and coordinate life science resources across Europe, ELIXIR is a key player. To identify opportunities, highlight priorities, and aid strategic thinking, here we survey approaches by which molecular technologies help inform understanding of biodiversity. We detail example use cases to highlight how DNA sequencing is: resolving taxonomic issues; Increasing knowledge of marine biodiversity; helping understand how agriculture and biodiversity are critically linked; and playing an essential role in ecological studies. Together with examples of national biodiversity programmes, the use cases show where progress is being made but also highlight common challenges and opportunities for future enhancement of underlying technologies and services that connect molecular and wider biodiversity domains. Based on emerging themes, we propose key recommendations to guide future funding for biodiversity research: biodiversity and bioinformatic infrastructures need to collaborate closely and strategically; taxonomic efforts need to be aligned and harmonised across domains; metadata needs to be standardised and common data management approaches widely adopted; current approaches need to be scaled up dramatically to address the anticipated explosion of molecular data; bioinformatics support for biodiversity research needs to be enabled and sustained; training for end users of biodiversity research infrastructures needs to be prioritised; and community initiatives need to be proactive and focused on enabling solutions. For sequencing data to deliver their full potential they must be connected to knowledge: together, molecular sequence data collection initiatives and biodiversity research infrastructures can advance global efforts to prevent further decline of Earth’s biodiversity.