A multi-tissue de novo transcriptome assembly and relative gene expression of the vulnerable freshwater salmonid Thymallus ligericus.

Freshwater ecosystems are among the most endangered ecosystems worldwide. While numerous taxa are on the verge of extinction as a result of global changes and direct or indirect anthropogenic activity, genomic and transcriptomic resources represent a key tool for comprehending species' adaptability and serve as the foundation for conservation initiatives. The Loire grayling, Thymallus ligericus, is a freshwater European salmonid endemic to the upper Loire River basin. The species is comprised of fragmented populations that are dispersed over a small area and it has been identified as a vulnerable species. Here, we provide a multi-tissue de novo transcriptome assembly of T. ligericus. The completeness and integrity of the transcriptome were assessed before and after redundancy removal with lineage-specific libraries from Eukaryota, Metazoa, Vertebrata, and Actinopterygii. Relative gene expression was assessed for each of the analyzed tissues, using the de novo assembled transcriptome and a genome-based analysis using the available T. thymallus genome as a reference. The final assembly, with a contig N50 of 1221 and Benchmarking Universal Single-Copy Orthologs (BUSCO) scores above 94%, is made accessible along with structural and functional annotations and relative gene expression of the five tissues (NCBI SRA and FigShare databases). This is the first transcriptomic resource for this species, which provides a foundation for future research on this and other salmonid species that are increasingly exposed to environmental stressors.

An historical "wreck": A transcriptome assembly of the naval shipworm, Teredo navalis Linnaeus, 1978.

Historically famous for their negative impact on human-built marine wood structures, mollusc shipworms play a central ecological role in marine ecosystems. Their association with bacterial symbionts, providing cellulolytic and nitrogen-fixing activities, underscores their exceptional wood-eating and wood-boring behaviours, improving energy transfer and the recycling of essential nutrients locked in the wood cellulose. Importantly, from a molecular standpoint, a minute of omic resources are available from this lineage of Bivalvia. Here, we produced and assembled a transcriptome from the globally distributed naval shipworm, Teredo navalis (family Teredinidae). The transcriptome was obtained by sequencing the total RNA from five equidistant segments of the whole body of a T. navalis specimen. The quality of the produced assembly was accessed with several statistics, revealing a highly contiguous (1194 N50) and complete (over 90% BUSCO scores for Eukaryote and Metazoan databases) transcriptome, with nearly 38,000 predicted ORF, more than half being functionally annotated. Our findings pave the way to investigate the unique evolutionary biology of these highly modified bivalves and lay the foundation for an adequate gene annotation of a full genome sequence of the species.

A whole-body transcriptome assembly of the annelid worm Hediste diversicolor.

The Annelida phylum is composed of a myriad of species exhibiting key phenotypic adaptations. They occupy key ecological niches in a variety of marine, freshwater and terrestrial ecosystems. Importantly, the increment of omic resources is rapidly modifying the taxonomic landscape and knowledge of species belonging to this phylum. Here, we comprehensively characterised and annotated a transcriptome of the common ragworm, Hediste diversicolor (OF Müller). This species belongs to the family Nereididae and inhabits estuarine and lagoon areas on the Atlantic coasts of Europe and North America. Ecologically, H. diversicolor plays an important role in benthic food webs. Given its commercial value, H. diversicolor is a promising candidate for aquaculture development and production in farming facilities, under a circular economy framework. We used Illumina next-generation sequencing technology, to produce a total of 105 million (M) paired-end (PE) raw reads and generate the first whole-body transcriptome assembly of H. diversicolor species. This high-quality transcriptome contains 69,335 transcripts with an N50 transcript length of 2313 bp and achieved a BUSCO gene completeness of 97.7% and 96% in Eukaryota and Metazoa lineage-specific profile libraries. Our findings offer a valuable resource for multiple biological applications using this species.

Population Genomics Reveals the Underlying Structure of the Small Pelagic European Sardine and Suggests Low Connectivity within Macaronesia.

The European sardine (Sardina pilchardus, Walbaum 1792) is indisputably a commercially important species. Previous studies using uneven sampling or a limited number of makers have presented sometimes conflicting evidence of the genetic structure of S. pilchardus populations. Here, we show that whole genome data from 108 individuals from 16 sampling areas across 5000 km of the species' distribution range (from the Eastern Mediterranean to the archipelago of Azores) support at least three genetic clusters. One includes individuals from Azores and Madeira, with evidence of substructure separating these two archipelagos in the Atlantic. Another cluster broadly corresponds to the center of the distribution, including the sampling sites around Iberia, separated by the Almeria-Oran front from the third cluster that includes all of the Mediterranean samples, except those from the Alboran Sea. Individuals from the Canary Islands appear to belong to the Mediterranean cluster. This suggests at least two important geographical barriers to gene flow, even though these do not seem complete, with many individuals from around Iberia and the Mediterranean showing some patterns compatible with admixture with other genetic clusters. Genomic regions corresponding to the top outliers of genetic differentiation are located in areas of low recombination indicative that genetic architecture also has a role in shaping population structure. These regions include genes related to otolith formation, a calcium carbonate structure in the inner ear previously used to distinguish S. pilchardus populations. Our results provide a baseline for further characterization of physical and genetic barriers that divide European sardine populations, and information for transnational stock management of this highly exploited species towards sustainable fisheries.

Unravelling the main immune repertoire of Paracentrotus lividus following Vibrio anguillarum bath challenge.

Paracentrotus lividus is the most abundant echinoid species in the North East Atlantic Ocean and Mediterranean Sea. Although there is abundant genomic information of the species, there is no deep characterisation of the genes involved in the immune response. Here, a reference transcriptome of male and female coelomocytes was produced. The generated P. lividus transcriptome assembly has 203,511 transcripts, N50 transcript length of 1079 bp, and more than 90% estimated gene completeness in Eukaryota and Metazoa BUSCO databases, respectively. Differential gene expression analyses showed 54 and 55 up-regulated genes in P. lividus female and male coelomocyte tissues, respectively. These results suggest a similar immune gene repertoire between sexes. To examine the immune response, P. lividus was challenged with Vibrio anguillarum, one of the candidate pathogens for bald disease. Immune parameters were evaluated at cell and humoral levels, as well as the expression analysis of immune related genes at an early response stage. No differences were found at cellular and humoral levels with the exception of the increase of nitric oxide in perivisceral fluid of challenged animals. At the gene expression level, a total of 2721 genes were upregulated in challenged animals, 13.6 times higher expression than control group. Our analysis revealed that four major KEGG pathways were enriched in challenged animals: Autophagy (KEGG:04140), Endocytosis (KEGG:04144), Phagosome (KEGG:04145) and Protein processing in endoplasmic reticulum (KEGG:04141). Several toll-like receptors (TLR), scavenger receptors cysteine-rich (SRCR) or nucleotide-binding oligomerisation domain like receptors (NLR) were identified as major family genes for pathogen recognition and immune defence. This study provides a valuable transcriptomic resource and unfolds the molecular basis of immune response to V. anguillarum exposure. Overall, our findings contribute to the conservation effort of the P. lividus populations, as well as its sustainable exploitation in an aquaculture context.

Dataset of the complete mitogenome of the deep-sea sailfin roughshark, Oxynotus paradoxus Frade, 1929.

Chondrichthyans comprise a diverse group of vertebrate species with extraordinary ecological relevance. Yet, multiple members of this evolutionary lineage are associated with significant extinction risk. The sailfin roughshark Oxynotus paradoxus is a deep-water benthic shark currently listed as vulnerable due to population declines in parts of its range. Here we provide the first complete mitochondrial genome of O. paradoxus, comprising also the first record for the genus and family Oxynotidae. These data can facilitate future monitoring of the genetic diversity in this and related species. Genomic DNA was extracted from O. paradoxus collected in the eastern North Atlantic off western Portugal (37.59°N, 9.51°W) and sent for Illumina Paired-End (2 × 150 bp) library construction and whole genome sequencing on a Novaseq6000 platform. Trimmomatic (version 0.38) was used to remove adapters and MitoZ (version 3.4) to assemble and annotate the mitogenome. This mitogenome with 17 100 bp has a total of 38 genes, 13 of which are protein-coding genes, 23 transfer RNA genes, and 2 ribosomal RNA genes. Eight transfer RNAs and 1 protein-coding gene (NADH dehydrogenase subunit 6, NAD6) are in the complementary strand. In the provided phylogenetic inference, with all available and verified Squalomorphii mitogenomes, the four orders are well separated, and as expected, O. paradoxus is placed in the Squaliformes order. This data reinforces the need for more genomic resources for the Oxynotidae family.

An Ancestral Major Histocompatibility Complex Organization in Cartilaginous Fish: Reconstructing MHC Origin and Evolution.

Cartilaginous fish (sharks, rays, and chimeras) comprise the oldest living jawed vertebrates with a mammalian-like adaptive immune system based on immunoglobulins (Ig), T-cell receptors (TCRs), and the major histocompatibility complex (MHC). Here, we show that the cartilaginous fish "adaptive MHC" is highly regimented and compact, containing (i) a classical MHC class Ia (MHC-Ia) region containing antigen processing (antigen peptide transporters and immunoproteasome) and presenting (MHC-Ia) genes, (ii) an MHC class II (MHC-II) region (with alpha and beta genes) with linkage to beta-2-microglobulin (ß2m) and bromodomain-containing 2, (iii) nonclassical MHC class Ib (MHC-Ib) regions with 450 million-year-old lineages, and (iv) a complement C4 associated with the MHC-Ia region. No MHC-Ib genes were found outside of the elasmobranch MHC. Our data suggest that both MHC-I and MHC-II genes arose after the second round of whole-genome duplication (2R) on a human chromosome (huchr) 6 precursor. Further analysis of MHC paralogous regions across early branching taxa from all jawed vertebrate lineages revealed that Ig/TCR genes likely arose on a precursor of the huchr9/12/14 MHC paralog. The ß2m gene is linked to the Ig/TCR genes in some vertebrates suggesting that it was present at 1R, perhaps as the donor of C1 domain to the primordial MHC gene. In sum, extant cartilaginous fish exhibit a conserved and prototypical MHC genomic organization with features found in various vertebrates, reflecting the ancestral arrangement for the jawed vertebrates.

Mitochondrial replication's role in vertebrate mtDNA strand asymmetry.

Mitogenomes are defined as compact and structurally stable over aeons. This perception results from a vertebrate-centric vision, where few types of mtDNA rearrangements are described. Here, we bring a new light to the involvement of mitochondrial replication in the strand asymmetry of the vertebrate mtDNA. Using several species of deep-sea hatchetfish (Sternoptychidae) displaying distinct mtDNA structural arrangements, we unravel the inversion of the coding direction of protein-coding genes (PCGs). This unexpected change is coupled with a strand asymmetry nucleotide composition reversal and is shown to be directly related to the strand location of the Control Region (CR). An analysis of the fourfold redundant sites of the PCGs (greater than 6000 vertebrates), revealed the rarity of this phenomenon, found in nine fish species (five deep-sea hatchetfish). Curiously, in Antarctic notothenioid fishes (Trematominae), where a single PCG inversion (the only other record in fish) is coupled with the inversion of the CR, the standard asymmetry is disrupted for the remaining PCGs but not yet reversed, suggesting a transitory state. Our results hint that a relaxation of the classic vertebrate mitochondrial structural stasis promotes disruption of the natural balance of asymmetry of the mtDNA. These findings support the long-lasting hypothesis that replication is the main molecular mechanism promoting the strand-specific compositional bias of this unique and indispensable molecule.

Extensive gene loss parallels kidney aglomerulism in Syngnathidae.

The eccentric seahorses, seadragons, pipehorses and pipefishes (Syngnathidae) have an aglomerular kidney1. Here, we show that nephron genes2 conserved in Bilateria are secondarily eroded/deleted in Syngnathidae genomes. A transcriptome enrichment analysis suggests the predominance of excretion processes in the Syngnathidae kidney. In a lineage where crypsis and idleness are tightly associated, we propose that aglomerulism evolved as an energy-saving strategy.

Examination of gammarid transcriptomes reveals a widespread occurrence of key metabolic genes from epibiont bdelloid rotifers in freshwater species.

Previous data revealed the unexpected presence of genes encoding for long-chain polyunsaturated fatty acid (LC-PUFA) biosynthetic enzymes in transcriptomes from freshwater gammarids but not in marine species, even though closely related species were compared. This study aimed to clarify the origin and occurrence of selected LC-PUFA biosynthesis gene markers across all published gammarid transcriptomes. Through systematic searches, we confirmed the widespread occurrence of sequences from seven elongases and desaturases involved in LC-PUFA biosynthesis, in transcriptomes from freshwater gammarids but not marine species, and clarified that such occurrence is independent from the gammarid species and geographical origin. The phylogenetic analysis established that the retrieved elongase and desaturase sequences were closely related to bdelloid rotifers, confirming that multiple transcriptomes from freshwater gammarids contain contaminating rotifers' genetic material. Using the Adineta steineri genome, we investigated the genomic location and exon-intron organization of the elongase and desaturase genes, establishing they are all genome-anchored and, importantly, identifying instances of horizontal gene transfer. Finally, we provide compelling evidence demonstrating Bdelloidea desaturases and elongases enable these organisms to perform all the reactions for de novo biosynthesis of PUFA and, from them, LC-PUFA, an advantageous trait when considering the low abundance of these essential nutrients in freshwater environments.

A PacBio Hi-Fi Genome Assembly of the Painter's Mussel Unio pictorum (Linnaeus, 1758).

The highly diverse group of freshwater mussels from order Unionida is found in the world's freshwater systems due to several fascinating evolutionary adaptations, including "parental care," and most notably, an obligatory parasitic phase in their early life cycle, called glochidia, which infests and uses fish for nutrition and dispersal. Freshwater mussels play essential ecological roles in freshwater habitats, including water filtration, sediment bioturbation, and nutrient cycling. However, these species are also highly threatened, being one of the faunal groups with the highest recorded extinction rate in the wild. Genomics methods have an incredible potential to promote biodiversity conservation, allowing the characterization of population health, identification of adaptive genetic elements, delineation of conservation units, and providing a framework for predictive assessments of the impact of anthropogenic threats and climate change. Unfortunately, only six freshwater mussel species have had their whole genomes sequenced to date, and only two of these are European species. Here, we present the first genome assembly of the Painter's Mussel, Unio pictorum (Linnaeus, 1758), the type species representative of the order and the most widespread species of the genus in Europe. We used long-read PacBio Hi-Fi sequencing reads to produce a highly contiguous assembly that will pave the way for the study of European freshwater mussels in the Genome Era.

PacBio Hi-Fi genome assembly of the Iberian dolphin freshwater mussel Unio delphinus Spengler, 1793.

Mussels of order Unionida are a group of strictly freshwater bivalves with nearly 1,000 described species widely dispersed across world freshwater ecosystems. They are highly threatened showing the highest record of extinction events within faunal taxa. Conservation is particularly concerning in species occurring in the Mediterranean biodiversity hotspot that are exposed to multiple anthropogenic threats, possibly acting in synergy. That is the case of the dolphin freshwater mussel Unio delphinus Spengler, 1793, endemic to the western Iberian Peninsula with recently strong population declines. To date, only four genome assemblies are available for the order Unionida and only one European species. We present the first genome assembly of Unio delphinus. We used the PacBio HiFi to generate a highly contiguous genome assembly. The assembly is 2.5 Gb long, possessing 1254 contigs with a contig N50 length of 10 Mbp. This is the most contiguous freshwater mussel genome assembly to date and is an essential resource for investigating the species' biology and evolutionary history that ultimately will help to support conservation strategies.

Corrigendum: Uncovering a 500 million year old history and evidence of pseudogenization for TLR15.

[This corrects the article DOI: 10.3389/fimmu.2022.1020601.].

The Crown Pearl V2: an improved genome assembly of the European freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758).

Contiguous assemblies are fundamental to deciphering the composition of extant genomes. In molluscs, this is considerably challenging owing to the large size of their genomes, heterozygosity, and widespread repetitive content. Consequently, long-read sequencing technologies are fundamental for high contiguity and quality. The first genome assembly of Margaritifera margaritifera (Linnaeus, 1758) (Mollusca: Bivalvia: Unionida), a culturally relevant, widespread, and highly threatened species of freshwater mussels, was recently generated. However, the resulting genome is highly fragmented since the assembly relied on short-read approaches. Here, an improved reference genome assembly was generated using a combination of PacBio CLR long reads and Illumina paired-end short reads. This genome assembly is 2.4 Gb long, organized into 1,700 scaffolds with a contig N50 length of 3.4 Mbp. The ab initio gene prediction resulted in 48,314 protein-coding genes. Our new assembly is a substantial improvement and an essential resource for studying this species' unique biological and evolutionary features, helping promote its conservation.

Decay of Skin-Specific Gene Modules in Pangolins.

The mammalian skin exhibits a rich spectrum of evolutionary adaptations. The pilosebaceous unit, composed of the hair shaft, follicle, and the sebaceous gland, is the most striking synapomorphy. The evolutionary diversification of mammals across different ecological niches was paralleled by the appearance of an ample variety of skin modifications. Pangolins, order Pholidota, exhibit keratin-derived scales, one of the most iconic skin appendages. This formidable armor is intended to serve as a deterrent against predators. Surprisingly, while pangolins have hair on their abdomens, the occurrence of sebaceous and sweat glands is contentious. Here, we explore various molecular modules of skin physiology in four pangolin genomes, including that of sebum production. We show that genes driving wax monoester formation, Awat1/2, show patterns of inactivation in the stem pangolin branch, while the triacylglycerol synthesis gene Dgat2l6 seems independently eroded in the African and Asian clades. In contrast, Elovl3 implicated in the formation of specific neutral lipids required for skin barrier function is intact and expressed in the pangolin skin. An extended comparative analysis shows that genes involved in skin pathogen defense and structural integrity of keratinocyte layers also show inactivating mutations: associated with both ancestral and independent pseudogenization events. Finally, we deduce that the suggested absence of sweat glands is not paralleled by the inactivation of the ATP-binding cassette transporter Abcc11, as previously described in Cetacea. Our findings reveal the sophisticated and complex history of gene retention and loss as key mechanisms in the evolution of the highly modified mammalian skin phenotypes.

A novel assembly pipeline and functional annotations for targeted sequencing: A case study on the globally threatened Margaritiferidae (Bivalvia: Unionida).

The proliferation of genomic sequencing approaches has significantly impacted the field of phylogenetics. Target capture approaches provide a cost-effective, fast and easily applied strategy for phylogenetic inference of non-model organisms. However, several existing target capture processing pipelines are incapable of incorporating whole genome sequencing (WGS). Here, we develop a new pipeline for capture and de novo assembly of the targeted regions using whole genome re-sequencing reads. This new pipeline captured targeted loci accurately, and given its unbiased nature, can be used with any target capture probe set. Moreover, due to its low computational demand, this new pipeline may be ideal for users with limited resources and when high-coverage sequencing outputs are required. We demonstrate the utility of our approach by incorporating WGS data into the first comprehensive phylogenomic reconstruction of the freshwater mussel family Margaritiferidae. We also provide a catalogue of well-curated functional annotations of these previously uncharacterized freshwater mussel-specific target regions, representing a complementary tool for scrutinizing phylogenetic inferences while expanding future applications of the probe set.

The chromosome-level genome and key genes associated with mud-dwelling behavior and adaptations of hypoxia and noxious environments in loach (Misgurnus anguillicaudatus).

BACKGROUND: The loach (Misgurnus anguillicaudatus), the most widely distributed species of the family Cobitidae, displays a mud-dwelling behavior and intestinal air-breathing, inhabiting the muddy bottom of extensive freshwater habitats. However, lack of high-quality reference genome seriously limits the interpretation of the genetic basis of specialized adaptations of the loach to the adverse environments including but not limited to the extreme water temperature, hypoxic and noxious mud environment. RESULTS: This study generated a 1.10-Gb high-quality, chromosome-anchored genome assembly, with a contig N50 of 3.83 Mb. Multiple comparative genomic analyses found that proto-oncogene c-Fos (fos), a regulator of bone development, is positively selected in loach. Knockout of fos (ID: Mis0086400.1) led to severe osteopetrosis and movement difficulties, combined with the comparison results of bone mineral density, supporting the hypothesis that fos is associated with loach mud-dwelling behavior. Based on genomic and transcriptomic analysis, we identified two key elements involved in the intestinal air-breathing of loach: a novel gene (ID: mis0158000.1) and heat shock protein beta-1 (hspb1). The flavin-containing monooxygenase 5 (fmo5) genes, central to xenobiotic metabolism, undergone expansion in loach and were identified as differentially expressed genes in a drug stress trial. A fmo5-/- (ID: Mis0185930.1) loach displayed liver and intestine injury, indicating the importance of this gene to the adaptation of the loach to the noxious mud. CONCLUSIONS: Our work provides valuable insights into the genetic basis of biological adaptation to adverse environments.

The gastric proton pump in gobiid and mudskipper fishes. Evidence of stomach loss?

Stomach loss has occurred independently multiple times during gnathostome evolution with notable frequency within the Teleostei. Significantly, this loss of acid-peptic digestion has been found to correlate with the secondary genomic loss of the gastric proton pump subunits (atp4a, atp4b) and pepsinogens/pepsins (pga, pgc). Gastric glands produce gastric juice containing the acid and pepsin and thus their presence is a hallmark feature of a digestive system capable of acid-peptic digestion. However, in gobiid fishes although oesogaster and gastric glands have been identified histologically, their functional significance has been questioned. In the present study we address whether the gastric proton pump is present and expressed in gastric glands of the goby Neogobius species (Gobiidae) and in members of the family Oxudercidae, a group of amphibious gobiid fishes commonly known as mudskippers (genera: Periophthalmus, Boleophthalmus, Periophthalmodon and Scartelaos). We confirmed the presence of gastric glands and have immunohistochemically localized gastric proton pump expression to these glands in Neogobius fluviatilis and Periophthalmus novemradiatus, Periophthalmus barbarus and Boleophthalmus boddarti. Genome analysis in Neogobius melanostomus, Periophthalmus magnuspinnatus, Scartelaos histophorus, Boleophthalmus pectinirostris, and Periophthalmodon schlosseri revealed the presence of both atp4a and atp4b subunit orthologues in all species in a conserved genomic loci organization. Moreover, it was possible to deduce that the complete open reading frame and the key functional amino acid residues are present. The conserved expression of the gastric proton pump provides clear evidence of the potential for gastric acid secretion indicating that acid digestion is retained in these gobiid fishes and not lost.

The gill transcriptome of threatened European freshwater mussels.

Genomic tools applied to non-model organisms are critical to design successful conservation strategies of particularly threatened groups. Freshwater mussels of the Unionida order are among the most vulnerable taxa and yet almost no genetic resources are available. Here, we present the gill transcriptomes of five European freshwater mussels with high conservation concern: Margaritifera margaritifera, Unio crassus, Unio pictorum, Unio mancus and Unio delphinus. The final assemblies, with N50 values ranging from 1069-1895 bp and total BUSCO scores above 90% (Eukaryote and Metazoan databases), were structurally and functionally annotated, and made available. The transcriptomes here produced represent a valuable resource for future studies on these species' biology and ultimately guide their conservation.

The repertoire of the elongation of very long-chain fatty acids (Elovl) protein family is conserved in tambaqui (Colossoma macropomum): Gene expression profiles offer insights into the sexual differentiation process.

Elongation of very long-chain fatty acids (Elovl) proteins are critical players in the regulation of the length of a fatty acid. At present, eight members of the Elovl family (Elovl1-8), displaying a characteristic fatty acid substrate specificity, have been identified in vertebrates, including teleost fish. In general, Elovl1, Elovl3, Elovl6 and Elovl7 exhibit a substrate preference for saturated and monounsaturated fatty acids, while Elovl2, Elovl4, Elovl5 and Elovl8 use polyunsaturated fatty acids (PUFA) as substrates. PUFA elongases have received considerable attention in aquatic animals due to their involvement in the conversion of C18 PUFAs to long-chain polyunsaturated fatty acids (LC-PUFA). Here, we identified the full repertoire of elovl genes in the tambaqui Colossoma macropomum genome. A detailed phylogenetic and synteny analysis suggests a conservation of these genes among teleosts. Furthermore, based on RNAseq gene expression data, we discovered a gender bias expression of elovl genes during sex differentiation of tambaqui, toward future males. Our findings suggest a role of Elovl enzymes and fatty acid metabolism in tambaqui sexual differentiation.