Hemoglobin wonders: a fascinating gas transporter dive into molluscs.

Hemoglobin (Hb) has been identified in at least 14 molluscan taxa so far. Research spanning over 130 years on molluscan Hbs focuses on their genes, protein structures, functions, and evolution. Molluscan Hbs are categorized into single-, two-, and multiple-domain chains, including red blood cell, gill, and extracellular Hbs, based on the number of globin domains and their respective locations. These Hbs exhibit variation in assembly, ranging from monomeric and dimeric to higher-order multimeric forms. Typically, molluscan Hbs display moderately high oxygen affinity, weak cooperativity, and varying pH sensitivity. Hb's potential role in antimicrobial pathways could augment the immune defense of bivalves, which may be a complement to their lack of adaptive immunity. The role of Hb as a respiratory protein in bivalves likely originated from the substitution of hemocyanin. Molluscan Hbs demonstrate adaptive evolution in response to environmental changes via various strategies (e.g. increasing Hb types, multimerization, and amino acid residue substitutions at key sites), enhancing or altering functional properties for habitat adaptation. Concurrently, an increase in Hb assembly diversity, coupled with a downward trend in oxygen affinity, is observed during molluscan differentiation and evolution. Hb in Protobranchia, Heteroconchia, and Pteriomorphia bivalves originated from separate ancestors, with Protobranchia inheriting a relative ancient molluscan Hb gene. In bivalves, extracellular Hbs share a common origin, while gill Hbs likely emerged from convergent evolution. In summary, research on molluscan Hbs offers valuable insights into the origins, biological variations, and adaptive evolution of animal Hbs.

A chromosome-level genome for the nudibranch gastropod Berghia stephanieae helps parse clade-specific gene expression in novel and conserved phenotypes.

BACKGROUND: How novel phenotypes originate from conserved genes, processes, and tissues remains a major question in biology. Research that sets out to answer this question often focuses on the conserved genes and processes involved, an approach that explicitly excludes the impact of genetic elements that may be classified as clade-specific, even though many of these genes are known to be important for many novel, or clade-restricted, phenotypes. This is especially true for understudied phyla such as mollusks, where limited genomic and functional biology resources for members of this phylum have long hindered assessments of genetic homology and function. To address this gap, we constructed a chromosome-level genome for the gastropod Berghia stephanieae (Valdés, 2005) to investigate the expression of clade-specific genes across both novel and conserved tissue types in this species. RESULTS: The final assembled and filtered Berghia genome is comparable to other high-quality mollusk genomes in terms of size (1.05 Gb) and number of predicted genes (24,960 genes) and is highly contiguous. The proportion of upregulated, clade-specific genes varied across tissues, but with no clear trend between the proportion of clade-specific genes and the novelty of the tissue. However, more complex tissue like the brain had the highest total number of upregulated, clade-specific genes, though the ratio of upregulated clade-specific genes to the total number of upregulated genes was low. CONCLUSIONS: Our results, when combined with previous research on the impact of novel genes on phenotypic evolution, highlight the fact that the complexity of the novel tissue or behavior, the type of novelty, and the developmental timing of evolutionary modifications will all influence how novel and conserved genes interact to generate diversity.

Molluscan Genomes Reveal Extensive Differences in Photopigment Evolution Across the Phylum.

In animals, opsins and cryptochromes are major protein families that transduce light signals when bound to light-absorbing chromophores. Opsins are involved in various light-dependent processes, like vision, and have been co-opted for light-independent sensory modalities. Cryptochromes are important photoreceptors in animals, generally regulating circadian rhythm, they belong to a larger protein family with photolyases, which repair UV-induced DNA damage. Mollusks are great animals to explore questions about light sensing as eyes have evolved multiple times across, and within, taxonomic classes. We used molluscan genome assemblies from 80 species to predict protein sequences and examine gene family evolution using phylogenetic approaches. We found extensive opsin family expansion and contraction, particularly in bivalve xenopsins and gastropod Go-opsins, while other opsins, like retinochrome, rarely duplicate. Bivalve and gastropod lineages exhibit fluctuations in opsin repertoire, with cephalopods having the fewest number of opsins and loss of at least 2 major opsin types. Interestingly, opsin expansions are not limited to eyed species, and the highest opsin content was seen in eyeless bivalves. The dynamic nature of opsin evolution is quite contrary to the general lack of diversification in mollusk cryptochromes, though some taxa, including cephalopods and terrestrial gastropods, have reduced repertoires of both protein families. We also found complete loss of opsins and cryptochromes in multiple, but not all, deep-sea species. These results help set the stage for connecting genomic changes, including opsin family expansion and contraction, with differences in environmental, and biological features across Mollusca.

A combined phylogenetic strategy illuminates the evolution of Goniodorididae nudibranchs (Mollusca, Gastropoda, Heterobranchia).

Goniodorididae is a family of small dorid nudibranchs distributed worldwide that feed on entoprocts, ascidians, and bryozoans. The evolutionary relationships between its taxa have been uncertain due to the limited taxa available for phylogenetic analyses; some genera being paraphyletic. The family includes a remarkable number of synonymized genera in which the species richness is unequally distributed, while some genera have dozens of species others are monospecific. Some clades are very uniform morphologically while others are considered highly variable. To increase backbone phylogenetic resolution a target enrichment approach of ultra-conserved elements was aimed at representative Goniodorididae species for the first time. Additionally, we increase species representation by including mitochondrial markers cytochrome c oxidase subunit I and ribosomal RNA 16S as well as nuclear Histone 3 and ribosomal RNA 18S from 109 Goniodorididae species, out of approximately 160 currently valid species. Maximum likelihood and Bayesian inference analyses were performed to infer the phylogeny of the family. As a result, two subfamilies and eleven genera were elucidated. The synonymized genera Bermudella, Cargoa, and Ceratodoris are here resurrected and a new genus, Naisdoris gen. nov., is described. The clades included taxa with shared prey preference, showing that trophic behavior could have driven species evolution and morphological uniqueness within the family Goniodorididae.

Phylogenomic reconstruction of Solenogastres (Mollusca, Aplacophora) informs hypotheses on body size evolution.

Body size is a fundamental characteristic of animals that impacts every aspect of their biology from anatomical complexity to ecology. In Mollusca, Solenogastres has been considered important to understanding the group's early evolution as most morphology-based phylogenetic reconstructions placed it as an early branching molluscan lineage. Under this scenario, molluscs were thought to have evolved from a small, turbellarian-like ancestor and small (i.e., macrofaunal) body size was inferred to be plesiomorphic for Solenogastres. More recently, phylogenomic studies have shown that aplacophorans (Solenogastres + Caudofoveata) form a clade with chitons (Polyplacophora), which is sister to all other molluscs, suggesting a relatively large-bodied (i.e., megafaunal) ancestor for Mollusca. Meanwhile, recent investigations into aplacophoran phylogeny have called the assumption that the last common ancestor of Solenogastres was small-bodied into question, but sampling of meiofaunal species was limited, biasing these studies towards large-bodied taxa and leaving fundamental questions about solenogaster body size evolution unanswered. Here, we supplemented available data with transcriptomes from eight diverse meiofaunal species of Solenogastres and conducted phylogenomic analyses on datasets of up to 949 genes. Maximum likelihood analyses support the meiofaunal family Meiomeniidae as the sister group to all other solenogasters, congruent with earlier ideas of a small-bodied ancestor of Solenogastres. In contrast, Bayesian Inference analyses support the large-bodied family Amphimeniidae as the sister group to all other solenogasters. Investigation of phylogenetic signal by comparing site-wise likelihood scores for the two competing hypotheses support the Meiomeniidae-first topology. In light of these results, we performed ancestral character state reconstruction to explore the implications of both hypotheses on understanding of Solenogaster evolution and review previous hypotheses about body size evolution and its potential consequences for solenogaster biology. Both hypotheses imply that body size evolution has been highly dynamic over the course of solenogaster evolution and that their relatively static body plan has successfully allowed for evolutionary transitions between meio-, macro- and megafaunal size ranges.

Transcriptional landscape of small non-coding RNAs reveals diversity of categories and functions in molluscs.

Small non-coding RNAs (sncRNAs) are non-coding RNA molecules that play various roles in metazoans. Among the sncRNAs, microRNAs (miRNAs) guide post-translational gene regulation during cellular development, proliferation, apoptosis, and differentiation, while PIWI-interacting RNAs (piRNAs) suppress transposon activity to safeguard the genome from detrimental insertion mutagenesis. While an increasing number of piRNAs are being identified in the soma and germlines of various organisms, they are scarcely reported in molluscs. To unravel the small RNA (sRNA) expression patterns and genomic function in molluscs, we generated a comprehensive sRNA dataset by sRNA sequencing (sRNA-seq) of eight mollusc species. Abundant miRNAs were identified and characterized in all investigated molluscs, and ubiquitous piRNAs were discovered in both somatic and gonadal tissues in six of the investigated molluscs, which are more closely associated with transposon silencing. Tens of piRNA clusters were also identified based on the genomic mapping results, which varied among different tissues and species. Our dataset serves as important reference data for future genomic and genetic studies on sRNAs in these molluscs and related species, especially in elucidating the ancestral state of piRNAs in bilaterians.

Flipping Shells! Unwinding LR Asymmetry in Mirror-Image Molluscs.

In seeking to understand the establishment of left-right (LR) asymmetry, a limiting factor is that most animals are ordinarily invariant in their asymmetry, except when manipulated or mutated. It is therefore surprising that the wider scientific field does not appear to fully appreciate the remarkable fact that normal development in molluscs, especially snails, can flip between two chiral types without pathology. Here, I describe recent progress in understanding the evolution, development, and genetics of chiral variation in snails, and place it in context with other animals. I argue that the natural variation of snails is a crucial resource towards understanding the invariance in other animal groups and, ultimately, will be key in revealing the common factors that define cellular and organismal LR asymmetry.

Hiker, a new family of DNA transposons encoding transposases with DD35E motifs, displays a distinct phylogenetic relationship with most known DNA transposon families of IS630-Tc1-mariner (ITm).

DNA transposons play a crucial role in determining the size and structure of eukaryotic genomes. In this study, a new family of IS630-Tc1-mariner (ITm) DNA transposons, named Hiker (HK), was identified. HK is characterized by a DD35E catalytic domain and is distinct from all previously known families of the ITm group. Phylogenetic analyses showed that DD35E/Hiker forms a monophyletic clade with DD34E/Gambol, indicating that they may represent a separate superfamily of ITm. A total of 178 Hiker species were identified, with 170 found mainly in Actinopterygii, one in Chondrichthyes, six in Anura and one in Mollusca. Gambol (GM), on the other hand, are found in invertebrates, with 18 in Arthropoda and one in Platyhelminthes. Hiker transposons have a total length ranging from 2.14 to 3.67 kb and contain a single open reading frame that encodes a protein of approximately 370 amino acids (range 311-413 aa). They are flanked by short terminal inverted repeats (TIRs) of 16-30 base pairs and two base pair (TA) target-site duplications. In contrast, most transposons of the Gambol family have a total length of 1.35-5.96 kb, encode a transposase protein of approximately 350 amino acids (range 306-374 aa), and are flanked by TIRs that range from 32 to 1097 bp in length. Both Hiker and Gambol transposases have several conserved motifs, including helix-turn-helix (HTH) motifs and a DDE domain. Our study observed multiple amplification waves and repeated horizontal transfer (HT) events of HK transposons in vertebrate genomes, indicating their role in diversifying and shaping the genomes of Actinopterygii, Chondrichthyes, and Anura. Conversely, GM transposons showed few Horizontal transfer events. According to cell-based transposition assays, most HK transposons are likely inactive due to the truncated DNA binding domains of their transposases. We present an updated classification of the ITm group based on these findings, which will enhance the understanding of both the evolution of ITm transposons and that of their hosts.

Role of maternal spiralian-specific homeobox gene SPILE-E in the specification of blastomeres along the animal-vegetal axis during the early cleavage stages of mollusks.

Spiralians, one of the major clades of bilaterians, share a unique development known as spiralian development, characterized by the formation of tiers of cells called quartets, which exhibit different developmental potentials along the animal-vegetal axis. Recently, spiralian-specific TALE-type homeobox genes (SPILE) have been identified, some of which show zygotic and staggered expression patterns along the animal-vegetal axis and function in quartet specification in mollusks. However, it is unclear which maternal molecular components control the zygotic expression of these transcription factors. In this study, we focused on SPILE-E, a maternal transcription factor, and investigated its expression and function in mollusks. We found that the maternal and ubiquitous expression of SPILE-E in the cleavage stages is conserved in molluskan species, including limpets, mussels, and chitons. We knocked down SPILE-E in limpets and revealed that the expression of transcription factors specifically expressed in the first quartet (1q2 ; foxj1b) and second quartet (2q; SPILE-B) was abolished, whereas the macromere-quartet marker (SPILE-C) was ectopically expressed in 1q2 in SPILE-E morphants. Moreover, we showed that the expression of SPILE-A, which upregulates SPILE-B but represses SPILE-C expression, decreased in SPILE-E morphants. Consistent with changes in the expression pattern of the above transcription factors, SPILE-E-morphant larvae exhibited patchy or complete loss of expression of marker genes of ciliated cells and shell fields, possibly reflecting incomplete specification of 1q2 and 2q. Our results provide a molecular framework for quartet specification and highlight the importance of maternal lineage-specific transcription factors in the development and evolution of spiralians.

A Phylogenomic Backbone for Gastropod Molluscs.

Gastropods have survived several mass extinctions during their evolutionary history resulting in extraordinary diversity in morphology, ecology, and developmental modes, which complicate the reconstruction of a robust phylogeny. Currently, gastropods are divided into six subclasses: Caenogastropoda, Heterobranchia, Neomphaliones, Neritimorpha, Patellogastropoda, and Vetigastropoda. Phylogenetic relationships among these taxa historically lack consensus, despite numerous efforts using morphological and molecular information. We generated sequence data for transcriptomes derived from 12 taxa belonging to clades with little or no prior representation in previous studies in order to infer the deeper cladogenetic events within Gastropoda and, for the first time, infer the position of the deep-sea Neomphaliones using a phylogenomic approach. We explored the impact of missing data, homoplasy, and compositional heterogeneity on the inferred phylogenetic hypotheses. We recovered a highly supported backbone for gastropod relationships that is congruent with morphological and mitogenomic evidence, in which Patellogastropoda, true limpets, are the sister lineage to all other gastropods (Orthogastropoda) which are divided into two main clades 1) Vetigastropoda $s.l.$ (including Pleurotomariida $+$ Neomphaliones) and 2) Neritimorpha $+$ (Caenogastropoda $+$ Heterobranchia). As such, our results support the recognition of five subclasses (or infraclasses) in Gastropoda: Patellogastropoda, Vetigastropoda, Neritimorpha, Caenogastropoda, and Heterobranchia. [Compositional heterogeneity; fast-evolving; long-branch attraction; missing data; Mollusca; phylogenetics; systematic error.].

MolluscDB: an integrated functional and evolutionary genomics database for the hyper-diverse animal phylum Mollusca.

Mollusca represents the second largest animal phylum but remains poorly explored from a genomic perspective. While the recent increase in genomic resources holds great promise for a deep understanding of molluscan biology and evolution, access and utilization of these resources still pose a challenge. Here, we present the first comprehensive molluscan genomics database, MolluscDB (http://mgbase.qnlm.ac), which compiles and integrates current molluscan genomic/transcriptomic resources and provides convenient tools for multi-level integrative and comparative genomic analyses. MolluscDB enables a systematic view of genomic information from various aspects, such as genome assembly statistics, genome phylogenies, fossil records, gene information, expression profiles, gene families, transcription factors, transposable elements and mitogenome organization information. Moreover, MolluscDB offers valuable customized datasets or resources, such as gene coexpression networks across various developmental stages and adult tissues/organs, core gene repertoires inferred for major molluscan lineages, and macrosynteny analysis for chromosomal evolution. MolluscDB presents an integrative and comprehensive genomics platform that will allow the molluscan community to cope with ever-growing genomic resources and will expedite new scientific discoveries for understanding molluscan biology and evolution.

Is adaptation limited by mutation? A timescale-dependent effect of genetic diversity on the adaptive substitution rate in animals.

Whether adaptation is limited by the beneficial mutation supply is a long-standing question of evolutionary genetics, which is more generally related to the determination of the adaptive substitution rate and its relationship with species effective population size (Ne) and genetic diversity. Empirical evidence reported so far is equivocal, with some but not all studies supporting a higher adaptive substitution rate in large-Ne than in small-Ne species. We gathered coding sequence polymorphism data and estimated the adaptive amino-acid substitution rate ωa, in 50 species from ten distant groups of animals with markedly different population mutation rate θ. We reveal the existence of a complex, timescale dependent relationship between species adaptive substitution rate and genetic diversity. We find a positive relationship between ωa and θ among closely related species, indicating that adaptation is indeed limited by the mutation supply, but this was only true in relatively low-θ taxa. In contrast, we uncover no significant correlation between ωa and θ at a larger taxonomic scale, suggesting that the proportion of beneficial mutations scales negatively with species' long-term Ne.

Mineralize It or Not: Comparative Proteomics and Elemental Analysis Reveal Ancestral Compositions of Iron Mineralized Molluscan Radulae.

The radula is a unique foraging organ to Mollusca, which is important for their evolution and taxonomic classification. Many radulae are mineralized with metals. Although the remarkable mechanical properties of mineralized radulae are well-studied, the formation of mineralization from nonmineralized radulae is poorly understood. Taking advantage of the recently sequenced octopus and chiton genomes, we were able to identify more species-specific radular proteins by proteomics. Comparing these proteomes with the known limpet radula proteome enabled us to gain insight into the molecular components of nonmineralized and mineralized radula, highlighting that iron mineralization in the chiton radula is possibly due to the evolution of ferritins and peroxiredoxins. Through an in vitro binding assay, ferritin is shown to be important to iron accumulation into the nonmineralized radula. Moreover, radular proteomes reflect their adaption to dietary habits to some extent. The octopus radula has many scaffold modification proteins to suit flexibility while the chiton radula has abundant sugar metabolism proteins (e.g., glycosyl hydrolases) to adapt to algae feeding. This study provides a foundation for the understanding of molluscan radula formation and evolution and may inspire the synthesis of iron nanomaterials.

The development of CRISPR for a mollusc establishes the formin Lsdia1 as the long-sought gene for snail dextral/sinistral coiling.

The establishment of left-right body asymmetry is a key biological process that is tightly regulated genetically. In the first application of CRISPR/Cas9 to a mollusc, we show decisively that the actin-related diaphanous gene Lsdia1 is the single maternal gene that determines the shell coiling direction of the freshwater snail Lymnaea stagnalis Biallelic frameshift mutations of the gene produced sinistrally coiled offspring generation after generation, in the otherwise totally dextral genetic background. This is the gene sought for over a century. We also show that the gene sets the chirality at the one-cell stage, the earliest observed symmetry-breaking event linked directly to body handedness in the animal kingdom. The early intracellular chirality is superseded by the inter-cellular chirality during the 3rd cleavage, leading to asymmetric nodal and Pitx expression, and then to organismal body handedness. Thus, our findings have important implications for chiromorphogenesis in invertebrates as well as vertebrates, including humans, and for the evolution of snail chirality. This article has an associated 'The people behind the papers' interview.

Convergent evolution of barnacles and molluscs sheds lights in origin and diversification of calcareous shell and sessile lifestyle.

The calcareous shell and sessile lifestyle are the representative phenotypes of many molluscs, which happen to be present in barnacles, a group of unique crustaceans. The origin of these phenotypes is unclear, but it may be embodied in the convergent genetics of such distant groups (interphylum). Herein, we perform comprehensive comparative genomics analysis in barnacles and molluscs, and reveal a genome-wide strong convergent molecular evolution between them, including coexpansion of biomineralization and organic matrix genes for shell formation, and origination of lineage-specific orphan genes for settlement. Notably, the expanded biomineralization gene encoding alkaline phosphatase evolves a novel, highly conserved motif that may trigger the origin of barnacle shell formation. Unlike molluscs, barnacles adopt novel organic matrices and cement proteins for shell formation and settlement, respectively, and their calcareous shells have potentially originated from the cuticle system of crustaceans. Therefore, our study corroborates the idea that selection pressures driving convergent evolution may strongly act in organisms inhabiting similar environments regardless of phylogenetic distance. The convergence signatures shed light on the origin of the shell and sessile lifestyle of barnacles and molluscs. In addition, notable non-convergence signatures are also present and may contribute to morphological and functional specificities.

Duplication and subsequent functional diversification of aquaporin family in Pacific abalone Haliotis discus hannai.

Aquaporins (AQPs) are a group of proteins that evolved to mediate specific permeation of water and other small solutes, playing important roles in osmoregulation and nutrition, especially for aquatic animals. Genome-wide characterization of the AQP family in a typical mollusc, Pacific abalone, suggested that tandem duplication and retroduplication led to the dramatic expansion and diversification of AQP genes. Structural analysis indicated that tandem duplicated AQPs showed abnormal characteristics. The conserved amino acids in the key site of the Ar/R region were replaced by the others. These substitutions altered the pore diameter and properties of the inner surface and could accommodate the pass through of other molecules except water. Functional analysis indicated that abnormal Ar/R region of the tandemly adjacent members led to the different permeability, suggesting the neofunctionalization of tandemly duplicated genes. Mutation analysis indicated that at the key site of Ar/R region, just a single amino acid substitute could alter the permeability of HdAQPs, further explaining the mechanism of neofunctionalization between the tandem duplicated HdAQPs. Our observations provided strong evidence that duplication and subsequent neofunctionalization have led to structural and functional diversity of AQPs in Pacific abalone, providing insights into the evolution of AQPs in molluscs.

Dynamic evolutionary history of spiralian-specific TALE homeobox genes in mollusks.

Homeobox genes play essential roles in the early development of many animals. Although the repertoire of most homeobox genes, including three amino acid loop extension (TALE)-type homeobox genes, is conserved in animals, spiralian-TALE (SPILE) genes are a notable exception. In this study, SPILE genes were extracted from the genomic data of 22 mollusk species and classified into four clades (-A/C, -B, -D, and -E) to determine which SPILE genes exhibit dynamic repertoire changes. While SPILE-D and -E duplications were rarely observed, SPILE-B duplication was observed in the bivalve lineage and SPILE-A/C duplication was observed in multiple clades. Conversely, most or all SPILE genes were lost in cephalopods and in some gastropod lineages. SPILE gene expression patterns were also analyzed in multiple mollusk species using publicly available RNA-seq data. The majority of SPILE genes examined, particularly those in the A/C- and B-clades, were specifically expressed during early development, suggesting that most SPILE genes exert specific roles in early development. This comprehensive cataloging and characterization revealed a dynamic evolutionary history, including SPILE-A/C and -B gene duplications and the loss of SPILE genes in several lineages. Furthermore, this study provides a useful resource for studying the molecular mechanism of spiralian early development and the evolution of young and lineage-specific transcription factors.

Identification of peptidoglycan recognition proteins in hemocytes and kidney of common periwinkle Littorinalittorea.

Peptidoglycan Recognition Proteins (PGRPs) are a diverse group of proteins involved in innate immunity. In particular, PGRPs have been shown to participate in immune pattern recognition in various mollusks. However, they have not been described in Caenogastropoda, a large molluscan group comprising sea, freshwater and land snails. In this study, four short PGRPs with molecular weights ranging from 21 to 34 kDa and their isoforms were identified and structurally characterized in the kidney and hemocytic transcriptomes of a caenogastropod mollusk Littorina littorea. All of them (LlPGRP1-4) are secretory, possess a signal peptide and a characteristic N-terminal N-acetylmuramoyl-l-alanine amidase (Ami) domain with conserved Zn2+ binding- and amidase catalytic sites. The shortest proteins, LlPGRP1 and LlPGRP2, have no additional conserved motifs on the N-terminus. In longer and most abundantly expressed LlPGRP3 and LlPGRP4 the Ami-domain is combined with an N-terminal SH3-domain and a cysteine-rich motif, respectively. Expression analysis showed that LlPGRPs of the common periwinkle were uninvolved in the immune response to infection with trematode Himasthla elongata though they might act in antibacterial defense.

Structural and evolutionary insights into the multidomain galectin from the red abalone Haliotis rufescens with specificity for sulfated glycans.

Galectins are an evolutionarily ancient family of lectins characterized by their affinity for ß-galactosides and a conserved binding site in the carbohydrate recognition domain (CRD). These lectins are involved in multiple physiological functions, including the recognition of glycans on the surface of viruses and bacteria. This feature supports their role in innate immune responses in marine mollusks. Here, we identified and characterized a galectin, from the mollusk Haliotis rufescens (named HrGal), with four CRDs that belong to the tandem-repeat type. HrGal was purified by affinity chromatography in a galactose-agarose resin and exhibited a molecular mass of 64.11 kDa determined by MALDI-TOF mass spectrometry. The identity of HrGal was verified by sequencing, confirming that it is a 555 amino acid protein with a mass of 63.86 kDa. This protein corresponds to a galectin reported in GenBank with accession number AHX26603. HrGal is stable in the presence of urea, reducing agents, and ions such as Cu2+ and Zn2+. The recombinant galectin (rHrGal) was purified from inclusion bodies in the presence of these ions. A theoretical model obtained with the AlphaFold server exhibits four non-identical CRDs, with a ß sandwich folding and the representative motifs for binding ß-galactosides. This allows us to classify HrGal within the tandem repeat galectin family. On the basis of a phylogenetic analysis, we found that the mollusk sequences form a monophyletic group of tetradomain galectins unrelated to vertebrate galectins. HrGal showed specificity for galactosides and glucosides but only the sulfated sugars heparin and ι-carrageenan inhibited its hemagglutinating activity with a minimum inhibitory concentration of 4 mM and 6.25 X 10-5% respectively. The position of the sulfate groups seemed crucial for binding, both by carrageenans and heparin.

Exploring the Potential of Metatranscriptomics to Describe Microbial Communities and Their Effects in Molluscs.

Metatranscriptomics has emerged as a very useful technology for the study of microbiomes from RNA-seq reads. This method provides additional information compared to the sequencing of ribosomal genes because the gene expression can also be analysed. In this work, we used the metatranscriptomic approach to study the whole microbiome of mussels, including bacteria, viruses, fungi, and protozoans, by mapping the RNA-seq reads to custom assembly databases (including the genomes of microorganisms publicly available). This strategy allowed us not only to describe the diversity of microorganisms but also to relate the host transcriptome and microbiome, finding the genes more affected by the pathogen load. Although some bacteria abundant in the metatranscriptomic analysis were undetectable by 16S rRNA sequencing, a common core of the taxa was detected by both methodologies (62% of the metatranscriptomic detections were also identified by 16S rRNA sequencing, the Oceanospirillales, Flavobacteriales and Vibrionales orders being the most relevant). However, the differences in the microbiome composition were observed among different tissues of Mytilus galloprovincialis, with the fungal kingdom being especially diverse, or among molluscan species. These results confirm the potential of a meta-analysis of transcriptome data to obtain new information on the molluscs' microbiome.