Histone derived antimicrobial peptides identified from Mytilus coruscus serum by peptidomics.

Histones and their N-terminal or C-terminal derived peptides have been studied in vertebrates and presented as potential antimicrobial agents playing important roles in the innate immune defenses. Although histones and their derived peptides had been reported as components of innate immunity in invertebrates, the knowledge about the histone derived antimicrobial peptides (HDAPs) in invertebrates are still limited. Using a peptidomic technique, a set of peptide fragments derived from the histones was identified in this study from the serum of microbes challenged Mytilus coruscus. Among the 85 identified histone-derived-peptides with high confidence, 5 HDAPs were chemically synthesized and the antimicrobial activities were verified, showing strong growth inhibition against Gram-positive bacteria, Gram-negative bacteria, and fungus. The gene expression level of the precursor histones matched by representative HDAPs were further tested using q-PCR, and the results showed a significant upregulation of the histone gene expression levels in hemocytes, gill, and mantle of the mussel after immune stress. In addition, three identified HDAPs were selected for preparation of specific antibodies, and the corresponding histones and their derived C-terminal fragments were detected by Western blotting in the blood cell and serum of immune challenged mussel, respectively, indicating the existence of HDAPs in M. coruscus. Our findings revealed the immune function of histones in Mytilus, and confirmed the existence of HDAPs in the mussel. The identified Mytilus HDAPs represent a new source of immune effector with antimicrobial function in the innate immune system, and thus provide promising candidates for the treatment of microbial infections in aquaculture and medicine.

Bacterial c-di-GMP signaling gene affects mussel larval metamorphosis through outer membrane vesicles and lipopolysaccharides.

Biofilms serve as crucial cues for settlement and metamorphosis in marine invertebrates. Within bacterial systems, c-di-GMP functions as a pivotal signaling molecule regulating both biofilm formation and dispersion. However, the molecular mechanism of how c-di-GMP modulates biofilm-induced larval metamorphosis remains elusive. Our study reveals that the deletion of a c-di-GMP related gene in Pseudoalteromonas marina led to an increase in the level of bacterial c-di-GMP by knockout technique, and the mutant strain had an enhanced ability to produce more outer membrane vesicles (OMVs) and lipopolysaccharides (LPS). The mutant biofilms had higher induction activity for larval metamorphosis in mussels Mytilus coruscus, and OMVs play a major role in the induction activity. We further explored the function of LPS in OMVs. Extracted LPS induced high larval metamorphosis rate, and LPS content were subject to c-di-GMP and LPS-biosynthesis gene. Thus, we postulate that the impact of c-di-GMP on biofilm-induced metamorphosis is mediated through OMVs and LPS.

Identification and expression analysis of two steamer-like retrotransposons in the Chilean blue mussel (Mytilus chilensis).

BACKGROUND: Disseminated neoplasia (DN) is a proliferative cell disorder of the circulatory system of bivalve mollusks. The disease is transmitted between individuals and can also be induced by external chemical agents such as bromodeoxyuridine. In Mya arenaria, we have cloned and characterized an LTR-retrotransposon named Steamer. Steamer mRNA levels and gene copy number correlates with DN and can be used as a marker of the disease. So far, the only mollusk where a retrotransposon expression relates to DN is Mya arenaria. On the other hand, it has been reported that the Chilean blue mussel Mytilus chilensis can also suffers DN. Our aim was to identify retrotransposons in Mytilus chilensis and to study their expression levels in the context of disseminated neoplasia. RESULTS: Here we show that 7.1% of individuals collected in August 2018, from two farming areas, presents morphological characteristics described in DN. Using Steamer sequence to interrogate the transcriptome of M. chilensis we found two putative retrotransposons, named Steamer-like elements (MchSLEs). MchSLEs are present in the genome of M. chilensis and MchSLE1 is indeed an LTR-retrotransposon. Neither expression, nor copy number of the reported MchSLEs correlate with DN status but both are expressed at different levels among individual animals. We also report that in cultured M. chilensis haemocytes MchSLEs1 expression can be induced by bromodeoxyuridine. CONCLUSIONS: We conclude that SLEs present in Mytilus chilensis are differentially expressed among individuals and do not correlate with disseminated neoplasia. Treatment of haemocytes with a stressor like bromodeoxyuridine induces expression of MchSLE1 suggesting that in Mytilus chilensis environmental stressors can induce activation of LTR-retrotransposon.

Transgressive gene expression and expression plasticity under thermal stress in a stable hybrid zone.

Interspecific hybridization can lead to myriad outcomes, including transgressive phenotypes in which the hybrids are more fit than either parent species. Such hybrids may display important traits in the context of climate change, able to respond to novel environmental conditions not previously experienced by the parent populations. While this has been evaluated in an agricultural context, the role of transgressive hybrids under changing conditions in the wild remains largely unexplored; this is especially true regarding transgressive gene expression. Using the blue mussel species complex (genus Mytilus) as a model system, we investigated the effects of hybridization on temperature induced gene expression plasticity by comparing expression profiles in parental species and their hybrids following a 2-week thermal challenge. Hybrid expression plasticity was most often like one parent or the other (50%). However, a large fraction of genes (26%) showed transgressive expression plasticity (i.e. the change in gene expression was either greater or lesser than that of both parent species), while only 2% were intermediately plastic in hybrids. Despite their close phylogenetic relationship, there was limited overlap in the differentially expressed genes responding to temperature, indicating interspecific differences in the responses to high temperature in which responses from hybrids are distinct from both parent species. We also identified differentially expressed long non-coding RNAs (lncRNAs), which we suggest may contribute to species-specific differences in thermal tolerance. Our findings provide important insight into the impact of hybridization on gene expression under warming. We propose transgressive hybrids may play an important role in population persistence under future warming conditions.

The Mytilus chilensis Steamer-like Element-1 Retrotransposon Antisense mRNA Harbors an Internal Ribosome Entry Site That Is Modulated by hnRNPK.

LTR-retrotransposons are transposable elements characterized by the presence of long terminal repeats (LTRs) directly flanking an internal coding region. They share genome organization and replication strategies with retroviruses. Steamer-like Element-1 (MchSLE-1) is an LTR-retrotransposon identified in the genome of the Chilean blue mussel Mytilus chilensis. MchSLE-1 is transcribed; however, whether its RNA is also translated and the mechanism underlying such translation remain to be elucidated. Here, we characterize the MchSLE-1 translation mechanism. We found that the MchSLE-1 5' and 3'LTRs command transcription of sense and antisense RNAs, respectively. Using luciferase reporters commanded by the untranslated regions (UTRs) of MchSLE-1, we found that in vitro 5'UTR sense is unable to initiate translation, whereas the antisense 5'UTR initiates translation even when the eIF4E-eIF4G interaction was disrupted, suggesting the presence of an internal ribosomal entry site (IRES). The antisense 5'UTR IRES activity was tested using bicistronic reporters. The antisense 5'UTR has IRES activity only when the mRNA is transcribed in the nucleus, suggesting that nuclear RNA-binding proteins are required to modulate its activity. Indeed, heterogeneous nuclear ribonucleoprotein K (hnRNPK) was identified as an IRES trans-acting factor (ITAF) of the MchSLE-1 IRES. To our knowledge, this is the first report describing an IRES in an antisense mRNA derived from a mussel LTR-retrotransposon.

Expansion and diversity of caspases in Mytilus coruscus contribute to larval metamorphosis and environmental adaptation.

BACKGROUND: Apoptosis is involved (directly and indirectly) in several physiological processes including tissue remodeling during the development, the turnover of immune cells, and a defense against harmful stimuli. The disordered apoptotic process participates in the pathogenesis of various diseases, such as neoplasms, and chronic inflammatory or systemic autoimmune diseases, which are associated with its inadequate regulation. Caspases are vital components of the apoptotic pathway that are involved in developmental and immune processes. However, genome-wide identification and functional analysis of caspase have not been conducted in Mytilus coruscus, which is an economically important bivalve. RESULTS: Here, 47 caspase genes were identified from the genomes of M. coruscus, and the expansion of caspase-2/9 and caspase-3/6/7 genes were observed. Tandem duplication acts as an essential driver of gene expansion. The expanded caspase genes were highly diverse in terms of sequence, domain structure, and spatiotemporal expression profiles, suggesting their functional differentiation. The high expression of the expanded caspase genes at the pediveliger larvae stage and the result of apoptosis location in the velum suggest that the apoptosis mediated by them plays a critical role in the metamorphosis of M. coruscus larvae. In gill, caspase genes respond differently to the challenge of different strains, and most caspase-2/9 and caspase-3/6/7 genes were induced by copper stress, whereas caspase-8/10 genes were suppressed. Additionally, most caspase genes were upregulated in the mantle under ocean acidification which could weaken the biomineralization capacity of the mantle tissue. CONCLUSIONS: These results provide a comprehensive overview of the evolution and function of the caspase family and enhanced the understanding of the biological function of caspases in M. coruscus larval development and response to biotic and abiotic challenges.

Transcriptomic Analysis Provides Insights into Candidate Genes and Molecular Pathways Involved in Growth of Mytilus coruscus Larvae.

Growth is a fundamental aspect of aquaculture breeding programs, pivotal for successful cultivation. Understanding the mechanisms that govern growth and development differences across various stages can significantly boost seedling production of economically valuable species, thereby enhancing aquaculture efficiency and advancing the aquaculture industry. Mytilus coruscus, a commercially vital marine bivalve, underscores this importance. To decipher the intricate molecular mechanisms dictating growth and developmental disparities in marine shellfish, we conducted transcriptome sequencing and meticulously analyzed gene expression variations and molecular pathways linked to growth traits in M. coruscus. This study delved into the molecular and gene expression variations across five larval development stages, with a specific focus on scrutinizing the differential expression patterns of growth-associated genes using RNA sequencing and quantitative real-time PCR analysis. A substantial number of genes-36,044 differentially expressed genes (DEGs)-exhibited significant differential expression between consecutive developmental stages. These DEGs were then categorized into multiple pathways (Q value < 0.05), including crucial pathways such as the spliceosome, vascular smooth muscle contraction, DNA replication, and apoptosis, among others. In addition, we identified two pivotal signaling pathways-the Hedgehog (Hh) signaling pathway and the TGF-beta (TGF-ß) signaling pathway-associated with the growth and development of M. coruscus larvae. Ten key growth-related genes were pinpointed, each playing crucial roles in molecular function and the regulation of growth traits in M. coruscus. These genes and pathways associated with growth provide deep insights into the molecular basis of physiological adaptation, metabolic processes, and growth variability in marine bivalves.

First characterization of the nuclear receptor superfamily in the Mediterranean mussel Mytilus galloprovincialis: developmental expression dynamics and potential susceptibility to environmental chemicals.

Endocrine-disrupting chemicals (EDCs) represent a global threat to human health and the environment. In vertebrates, lipophilic EDCs primarily act by mimicking endogenous hormones, thus interfering with the transcriptional activity of nuclear receptors (NRs). The demonstration of the direct translation of these mechanisms into perturbation of NR-mediated physiological functions in invertebrates, however, has rarely proven successful, as the modes of action of EDCs in vertebrates and invertebrates seem to be distinct. In the present work, we investigated the members of the NR superfamily in a bivalve mollusk, the Mediterranean mussel Mytilus galloprovincialis. In addition to annotating the M. galloprovincialis NR complement, we assessed the potential developmental functions and susceptibility to EDC challenge during early development by gene expression analyses. Our results indicate that a majority of mussel NRs are dynamically expressed during early development, including receptors characterized by a potential susceptibility to EDCs. This study thus indicates that NRs are major regulators of early mussel development and that NR-mediated endocrine disruption in the mussel could be occurring at a larger scale and at earlier stages of the life cycle than previously anticipated. Altogether, these findings will have significant repercussions for our understanding of the stability of natural mussel populations. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

The Mediterranean mussel Mytilus galloprovincialis: a novel model for developmental studies in mollusks.

A model organism in developmental biology is defined by its experimental amenability and by resources created for the model system by the scientific community. For the most powerful invertebrate models, the combination of both has already yielded a thorough understanding of developmental processes. However, the number of developmental model systems is still limited, and their phylogenetic distribution heavily biased. Members of one of the largest animal lineages, the Spiralia, for example, have long been neglected. In order to remedy this shortcoming, we have produced a detailed developmental transcriptome for the bivalve mollusk Mytilus galloprovincialis, and have expanded the list of experimental protocols available for this species. Our high-quality transcriptome allowed us to identify transcriptomic signatures of developmental progression and to perform a first comparison with another bivalve mollusk: the Pacific oyster Crassostrea gigas. To allow co-labelling studies, we optimized and combined protocols for immunohistochemistry and hybridization chain reaction to create high-resolution co-expression maps of developmental genes. The resources and protocols described here represent an enormous boost for the establishment of Mytilus galloprovincialis as an alternative model system in developmental biology.

Contamination acts as a genotype-dependent barrier to gene flow, causing genetic erosion and fine-grained population subdivision in Mussels from the Strait of Istanbul.

This study provides evidence of fine-grained genetic structuring in Mediterranean mussels (Mytilus galloprovincialis) from the Strait of Istanbul, caused by barriers to gene flow via contaminant-mediated selection. In this study, mitochondrial D-loop sequences were analyzed in mussels from 8 localities, all less than 30 kilometers apart, with differing contaminant loads. The results were: 1) Intra-population genetic differentiation (ΦST) between sites with high and low contaminant loads was high (up to 0.459), even at distances of only a few kilometers. 2) Genetic diversity was negatively correlated with the contaminant load ("genetic erosion"). 3) There was evidence of selection, based on haplotype frequencies and neutrality tests (Tajima's D), with purifying selection at the most contaminated site and balancing selection at the least contaminated. 4) Genetic distance was not correlated with geographic distance (no isolation-by-distance), but was correlated with contaminant load at each site. 5) Population dendrograms and Bayesian estimators of migration indicated that gene flow between sites was affected by contamination. For the dendrograms of the sampling sites, the clades clustered according to contaminant load more than geographic distance. Overall, these results suggest that 1) contamination may serve as a genotype-dependent dispersal barrier (i.e., contamination may not affect total number of migrants, just the relative proportions of the haplotypes in the established immigrants), leading strong population differentiation over short distances, and 2) genetic erosion may occur by a combination of selection and altered patterns of haplotype-specific gene flow. These effects may be more pronounced in the Strait of Istanbul than in other locations because of the riverine nature and strong, uni-directional current of the strait.

Patterns of admixture and introgression in a mosaic Mytilus galloprovincialis and Mytilus edulis hybrid zone in SW England.

The study of hybrid zones offers important insights into speciation. Earlier studies on hybrid populations of the marine mussel species Mytilus edulis and Mytilus galloprovincialis in SW England provided evidence of admixture but were constrained by the limited number of molecular markers available. We use 57 ancestry-informative SNPs, most of which have been mapped genetically, to provide evidence of distinctive differences between admixed populations in SW England and asymmetrical introgression from M. edulis to M. galloprovincialis. We combine the genetic study with analysis of phenotypic traits of potential ecological and adaptive significance. We demonstrate that hybrid individuals have brown mantle edges unlike the white or purple in the parental species, suggesting allelic or non-allelic genomic interactions. We report differences in gonad development stage between the species consistent with a prezygotic barrier between the species. By incorporating results from publications dating back to 1980, we confirm the long-term stability of the hybrid zone despite higher viability of M. galloprovincialis. This stability coincides with a dramatic change in temperature of UK coastal waters and suggests that these hybrid populations might be resisting the effects of global warming. However, a single SNP locus associated with the Notch transmembrane signalling protein shows a markedly different pattern of variation to the others and might be associated with adaptation of M. galloprovincialis to colder northern temperatures.

SNP genotyping revealed a hybrid zone between Mytilus chilensis and M. platensis in southern South America (the Strait of Magellan, Isla Grande de Tierra del Fuego and the Falkland Islands).

Hybrid zones among mussel species have been extensively studied in the northern hemisphere. In South America, it has only recently become possible to study the natural hybrid zones, due to the clarification of the taxonomy of native mussels of the Mytilus genus. Analysing 54 SNP markers, we show the genetic species composition and admixture in the hybrid zone between M. chilensis and M. platensis in the southern end of South America. Bayesian, non-Bayesian clustering and re-assignment algorithms showed that the natural hybrid zone between M. chilensis and M. platensis in the Strait of Magellan, Isla Grande de Tierra del Fuego and the Falkland Islands shows clinal architecture. The hybrid zone can be divided into three different areas: the first one is on the Atlantic coast where only pure M. platensis and hybrid were found. In the second one, inside the Strait of Magellan, pure individuals of both species and mussels with variable degrees of hybridisation coexist. In the last area at the Strait in front of Punta Arenas City, fjords on the Isla Grande de Tierra del Fuego, and at the Beagle Channel, only M. chilensis and a low number of hybrids were found. According to the proportion of hybrids, bays with protected conditions away from strong currents would give better conditions for hybridisation. We do not find evidence of any other mussel species such as M. edulis, M. galloprovincialis, M. planulatus or M. trossulus in the zone.

Transcriptome analysis reveals tissue-specific responses of Mytilus unguiculatus to Vibrio alginolyticus infection.

Mytilus unguiculatus is an important economic bivalve species with wide consumption and aquaculture value. Disease is one of the primary limiting factors in mussel aquaculture, thus understanding the response of different tissues of M. unguiculatus to pathogens is crucial for disease prevention and control. In this study, we investigated the physiological and transcriptomic responses of the gills, adductor muscle, and mantle of M. unguiculatus infected with Vibrio alginolyticus. The results showed that V. alginolyticus infection caused inflammation and tissue structure changes in the gill, adductor muscle and mantle of M. unguiculatus. Meanwhile, the activities of superoxide dismutase and catalase in the three tissues increased, while the total antioxidant capacity decreased, suggesting that M. unguiculatus have an activated defense mechanism against infection-induced oxidative stress, despite a compromised total antioxidant capacity. Transcriptomic studies reveal that infected M. unguiculatus exhibits upregulation of endocytosis, lysosome activity, cellular apoptosis, and immune-related signaling pathways, indicating that M. unguiculatus responds to pathogen invasion by upregulating efferocytosis. Compared with the gill and adductor muscle, the mantle had a higher level of mytimycin, mytilin and myticin, and the three tissues also increased the expression of mytimycin to cope with the invasion of pathogens. In addition, the analysis of genes related to taste transduction pathways and muscle contraction and relaxation found that after infection with V. alginolyticus, M. unguiculatus may reduce appetite by inhibiting taste transduction in the gill, while improving muscle contraction of the adductor muscle and keeping the shell closed, to resist further invasion of pathogens and reduce the risk of pathogen transmission in the population.

Multi-omics reveals largely distinct transcript- and protein-level responses to the environment in an intertidal mussel.

Organismal responses to stressful environments are influenced by numerous transcript- and protein-level mechanisms, and the relationships between expression changes at these levels are not always straightforward. Here, we used paired transcriptomic and proteomic datasets from two previous studies from gill of the California mussel, Mytilus californianus, to explore how simultaneous transcript and protein abundance patterns may diverge under different environmental scenarios. Field-acclimatized mussels were sampled from two disparate intertidal sites; individuals from one site were subjected to three further treatments (common garden, low-intertidal or high-intertidal outplant) that vary in temperature and feeding time. Assessing 1519 genes shared between the two datasets revealed that both transcript and protein expression patterns differentiated the treatments at a global level, despite numerous underlying discrepancies. There were far more instances of differential expression between treatments in transcript only (1451) or protein only (226) than of the two levels shifting expression concordantly (68 instances). Upregulated expression of cilium-associated transcripts (likely related to feeding) was associated with relatively benign field treatments. In the most stressful treatment, transcripts, but not proteins, for several molecular chaperones (including heat shock proteins and endoplasmic reticulum chaperones) were more abundant, consistent with a threshold model for induction of translation of constitutively available mRNAs. Overall, these results suggest that the relative importance of transcript- and protein-level regulation (translation and/or turnover) differs among cellular functions and across specific microhabitats or environmental contexts. Furthermore, the degree of concordance between transcript and protein expression can vary across benign versus acutely stressful environmental conditions.

Genetic features of bivalve transmissible neoplasia in blue mussels from the Kola Bay (Barents Sea) suggest a recent trans-Arctic migration of the cancer lineages.

Ecology and biogeography of bivalve transmissible neoplasia (BTN) are underexplored due to its recent discovery and a challenging diagnostics. Blue mussels harbour two evolutionary lineages of BTN, MtrBTN1 and MtrBTN2, both derived from Mytilus trossulus. MtrBTN1 has been found only in M. trossulus from North Pacific. MtrBTN2 parasitizes different Mytilus spp. worldwide. BTN in M. trossulus in the Atlantic sector has never been studied. We looked for BTN in mussels from the Barents Sea using flow cytometry of cells, qPCR with primers specific to cancer-associated alleles and sequencing of mtDNA and nuclear loci. Both MtrBTN1 and MtrBTN2 were present in our material, though their prevalence was low (~0.4%). All cancers parasitized M. trossulus except one, MtrBTN1, which was found in a hybrid between M. trossulus and M. edulis. The mtDNA haplotypes found in both lineages were nearly identical to those known from the Northwest Pacific but not from elsewhere. Our results suggest that these two lineages may have arrived in the Barents Sea in recent decades with the maritime transport along the Northern Sea Route. A young evolutionary age of MtrBTN1 seems to indicate that it is an emerging disease in the process of niche expansion. Comparing the new and the published sequence data on tumour suppressor p53, we proved that the prevalence of BTN in mussels can reach epizootic levels. The finding of diverse recombinants between paternally and maternally inherited mtDNAs in somatic tissues of M. trossulus was an unexpected result of our study.

Genetic Diversity, Population Structure, and Environmental Adaptation Signatures of Chinese Coastal Hard-Shell Mussel Mytilus coruscus Revealed by Whole-Genome Sequencing.

The hard-shell mussel (Mytilus coruscus) is widespread in the temperate coastal areas of the northwest Pacific and holds a significant position in the shellfish aquaculture market in China. However, the natural resources of this species have been declining, and population genetic studies of M. coruscus are also lacking. In this study, we conducted whole-genome resequencing (WGR) of M. coruscus from eight different latitudes along the Chinese coast and identified a total of 25,859,986 single nucleotide polymorphism (SNP) markers. Our findings indicated that the genetic diversity of M. coruscus from the Zhoushan region was lower compared with populations from other regions. Furthermore, we observed that the evolutionary tree clustered into two primary branches, and the Zhangzhou (ZZ) population was in a separate branch. The ZZ population was partly isolated from populations in other regions, but the distribution of branches was not geographically homogeneous, and a nested pattern emerged, consistent with the population differentiation index (FST) results. To investigate the selection characteristics, we utilized the northern M. coruscus populations (Dalian and Qingdao) and the central populations (Zhoushan and Xiangshan) as reference populations and the southern ZZ population as the target population. Our selection scan analysis identified several genes associated with thermal responses, including Hsp70 and CYP450. These genes may play important roles in the adaptation of M. coruscus to different living environments. Overall, our study provides a comprehensive understanding of the genomic diversity of coastal M. coruscus in China and is a valuable resource for future studies on genetic breeding and the evolutionary adaptation of this species.

Screening of genes encoding proteins that interact with Nrf2: Probing a cDNA library from Mytilus coruscus using a yeast two-hybrid system.

The Nuclear factor Erythroid 2-related factor 2 (Nrf2) is the most important endogenous antioxidant factor in organisms, and it has been demonstrated that it exerts extensive control over the immune response by interacting with crucial innate immunity components directly or indirectly. Although Nrf2 has been widely confirmed to be involved in stress resistance in mammals and some fish, its contribution to mollusks oxidative stress resistance has not frequently been documented. In this investigation, total RNA was taken from the digestive gland of M. coruscus, and a cDNA library was constructed and screened using the GATEWAY recombination technology. The Nrf2 cDNA sequence of M. coruscus was cloned into the pGBKT7 vector to prepare the bait plasmid. Using yeast two-hybrid system, after auxotrophic medium screening, sequencing, and bioinformatics analysis, 13 binding proteins that interacted with Nrf2 were finally identified. They were QM-like protein, 40S ribosomal protein S4 (RPS4), ribosomal protein S2 (RPS2), ribosomal protein L12 (RPL12), EF1-alpha mRNA for elongation factor 1 alpha (eEF1-alpha), ferritin, alpha-amylase, trypsin, vdg3, period clock protein, cyclophilin A isoform 1 (CYP A), serine protease CFSP2, histone variant H2A.Z (H2A.Z). For a better understanding the physiological function of Nrf2 in animals and as a potential target for future research on protein roles in Nrf2 interactions, it is crucial to clarify these protein interactions.

Outer membrane vesicles induce the mussel plantigrade settlement via regulation of c-di-GMP.

Despite the importance of outer membrane vesicles (OMVs) in benthic animal settlement, the underlying molecular mechanism remains elusive. Here, the impact of OMVs and OMVs synthesis-related tolB gene in Mytilus coruscus plantigrade settlement was tested. The OMVs were extracted from Pseudoalteromonas marina through density gradient centrifugation, and a tolB knockout strain, achieved by homologous recombination, was utilized for the investigation. Our results demonstrated that OMVs could significantly enhance M. coruscus plantigrades settlement. Deleting the tolB resulted in downregulation of c-di-GMP, accompanied by a reduction of OMV production, a decline in bacterial motility and increasing biofilm-forming ability. Enzyme treatment resulted in a 61.11% reduction in OMV-inducing activity and a 94.87% reduction in LPS content. Thus, OMVs regulate mussel settlement via LPS, and c-di-GMP is responsible for the OMV-inducing capacity. These findings provide new insights into the interactions between bacteria and mussels.

Validation of the male-specific ORF of the paternally-transmitted mtDNA in Mytilus edulis as a protein-coding gene.

There appears to be an additional set of sex-specific mtDNA-encoded proteins in bivalve species with doubly uniparental mitochondrial inheritance that may be involved in the transmission of the female and male mitogenomes. In the marine mussel Mytilus edulis, the translation of the female-specific open reading frame (F-ORF) was demonstrated but the translation of the male-specific ORF (M-ORF) remains to be shown. Here we validate the male-specific ORF of the paternal mitogenome in M. edulis as a protein-coding gene. The M-ORF protein was detected only in male gonads and localized in sperm mitochondria and acrosome, suggesting that it is involved in a key sperm function in Mytilus edulis.

Aldolase of Mytilus galloprovincialis, Lamarck, 1819: Gene structure, tissue specificity of expression level and activity.

In the present study, the structure of the fructose-1,6-bisphosphataldolase (FBA) gene in Mytilus galloprovincialis (Lamarck, 1819) was analyzed and its tissue specificity of expression level and activity was determined. A 1092 base pairs (bps) complete coding sequence of the FBA gene was assembled from M. galloprovincialis transcriptome. Only one gene encoding FBA (MgFBA) was identified in the M. galloprovincialis genome. The length of MgFBA was 363 amino acids with a molecular mass of 39.7 kDa. According to the amino acid residues, the detected MgFBA gene is a type I aldolase. The FBA gene in M. galloprovincialis had 7 exons; the maximum intron length was about 2.5 kbps. Intraspecific nucleotide diversity (15 mutations) between MgFBAs from the Mediterranean mussels and the Black Sea mussels (present study) was detected. All mutations were synonymous. Tissue specificity in FBA expression level and activity was established. No direct correlation between these functions was found. The highest level of FBA gene expression is found in muscle tissue. According to the phylogenetic analyses, FBA gene in invertebrates could be considered the ancestral gene of muscle type aldolase, which may explain the character of tissue-specific expression.