Potential threats of microplastics and pathogenic bacteria to the immune system of the mussels Mytilus galloprovincialis.

As one of the main components of marine pollution, microplastics (MPs) inevitably enter the mussel aquaculture environment. At the same time, pathogenic bacteria, especially pathogens such as Vibrio, can cause illness outbreaks, leading to large-scale death of mussels. The potential harm of MPs and pathogenic bacteria to bivalve remains unclear. This study designed two experiments (1) mussels (Mytilus galloprovincialis) were exposed to 100 particles/L or 1,000 particles/L polymethyl methacrylate (PMMA, 17.01 ± 6.74 µm) MPs and 1 × 107 CFU/mL Vibrio parahaemolyticus at the same time (14 days), and (2) mussels were exposed to 100 particles/L or 1,000 particles/L MPs for a long time (30 days) and then exposed to 1 × 107 CFU/mL V. parahaemolyticus to explore the effects of these two stresses on the mussel immune system. The results showed that after the combined exposure of V. parahaemolyticus and MPs, the lysosomal membrane stability of hemocytes decreased, lysozyme activity was inhibited, and hemocytes were induced to produce more lectins and defensins to fight pathogenic invasion. Long-term exposure to MPs caused a large amount of energy consumption in mussels, inhibited most of the functions of humoral immunity, increased the risk of mussel infection with pathogenic bacteria, and negatively affected mussel condition factor, the number of hemocytes, and the number of byssuses. Mussels may allocate more energy to deal with MPs and pathogenic bacterial infections rather than for growth. Above all, MPs exposure can affect mussel immune function or reduce its stress resistance, which in turn has an impact on mollusk farming.

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.

Differential Expression of Long Non-Coding RNA (lncRNA) in Mediterranean Mussel (Mytilus galloprovincialis) Hemocytes under Immune Stimuli.

The Mediterranean mussel is one of the most economically relevant bivalve mollusk species in Europe and China. The absence of massive mortalities and their resistance to pathogens affecting other cultured bivalves has been under study in recent years. The transcriptome response of this species to different immune stimuli has been extensively studied, and even the complexity of its genome, which has recently been sequenced, has been suggested as one of the factors contributing to this resistance. However, studies concerning the non-coding RNA profiles remain practically unexplored-especially those corresponding to the lncRNAs. To the best of our knowledge, this is the second characterization and study of lncRNAs in this bivalve species. In this work, we identified the potential repertoire of lncRNAs expressed in mussel hemocytes, and using RNA-Seq we analyzed the lncRNA profile of mussel hemocytes stimulated in vitro with three different immune stimuli: LPS, poly I:C, and ß-glucans. Compared to unstimulated hemocytes, LPS induced the highest modulation of lncRNAs, whereas poly I:C and ß-glucans induced a similar discrete response. Based on the potential cis-regulatory activity of the lncRNAs, we identified the neighboring protein-coding genes of the regulated lncRNAs to estimate-at least partially-the processes in which they are implicated. After applying correlation analyses, it seems that-especially for LPS-the lncRNAs could participate in the regulation of gene expression, and substantially contribute to the immune response.

The Evolution and Diversity of Interleukin-17 Highlight an Expansion in Marine Invertebrates and Its Conserved Role in Mucosal Immunity.

The interleukin-17 (IL-17) family consists of proinflammatory cytokines conserved during evolution. A comparative genomics approach was applied to examine IL-17 throughout evolution from poriferans to higher vertebrates. Cnidaria was highlighted as the most ancient diverged phylum, and several evolutionary patterns were revealed. Large expansions of the IL-17 repertoire were observed in marine molluscs and echinoderm species. We further studied this expansion in filter-fed Mytilus galloprovincialis, which is a bivalve with a highly effective innate immune system supported by a variable pangenome. We recovered 379 unique IL-17 sequences and 96 receptors from individual genomes that were classified into 23 and 6 isoforms after phylogenetic analyses. Mussel IL-17 isoforms were conserved among individuals and shared between closely related Mytilidae species. Certain isoforms were specifically implicated in the response to a waterborne infection with Vibrio splendidus in mussel gills. The involvement of IL-17 in mucosal immune responses could be conserved in higher vertebrates from these ancestral lineages.

The Endocannabinoid System in the Mediterranean Mussel Mytilus galloprovincialis: Possible Mediators of the Immune Activity?

Anandamide (AEA) is one of the best characterized members of the endocannabinoid family and its involvement in many pathophysiological processes has been well documented in vertebrates and invertebrates. Here, we report the biochemical and functional characterization of key elements of the endocannabinoid system in hemocytes isolated from the Mediterranean mussel Mytilus galloprovincialis. We also show the effects of exogenous AEA, as well as of capsaicin, on the cell ability to migrate and to activate the respiratory burst, upon in vitro stimulation of phagocytosis. Interestingly, our findings show that both AEA and capsaicin suppress the hemocyte response and that the use of selective antagonists of CB2 and TRPV1 receptors revert their inhibitory effects. Overall, present data support previous evidence on the presence of endocannabinoid signaling in mollusks and advance our knowledge about the evolutionary origins of this endogenous system and its role in the innate response of mollusks.

Immunological Responses of Marine Bivalves to Contaminant Exposure: Contribution of the -Omics Approach.

The increasing number of data studies on the biological impact of anthropogenic chemicals in the marine environment, together with the great development of invertebrate immunology, has identified marine bivalves as a key invertebrate group for studies on immunological responses to pollutant exposure. Available data on the effects of contaminants on bivalve immunity, evaluated with different functional and molecular endpoints, underline that individual functional parameters (cellular or humoral) and the expression of selected immune-related genes can distinctly react to different chemicals depending on the conditions of exposure. Therefore, the measurement of a suite of immune biomarkers in hemocytes and hemolymph is needed for the correct evaluation of the overall impact of contaminant exposure on the organism's immunocompetence. Recent advances in -omics technologies are revealing the complexity of the molecular players in the immune response of different bivalve species. Although different -omics represent extremely powerful tools in understanding the impact of pollutants on a key physiological function such as immune defense, the -omics approach has only been utilized in this area of investigation in the last few years. In this work, available information obtained from the application of -omics to evaluate the effects of pollutants on bivalve immunity is summarized. The data shows that the overall knowledge on this subject is still quite limited and that to understand the environmental relevance of any change in immune homeostasis induced by exposure to contaminants, a combination of both functional assays and cutting-edge technology (transcriptomics, proteomics, and metabolomics) is required. In addition, the utilization of metagenomics may explain how the complex interplay between the immune system of bivalves and its associated bacterial communities can be modulated by pollutants, and how this may in turn affect homeostatic processes of the host, host-pathogen interactions, and the increased susceptibility to disease. Integrating different approaches will contribute to knowledge on the mechanism responsible for immune dysfunction induced by pollutants in ecologically and economically relevant bivalve species and further explain their sensitivity to multiple stressors, thus resulting in health or disease.

Immune-responsive gene 1 (IRG1) and dimethyl itaconate are involved in the mussel immune response.

Immune-responsive gene 1 (irg1) is a gene that is well-conserved among different taxa and is highly expressed in the mussel Mytilus galloprovincialis at the constitutive level. The expression of this gene increases after a bacterial infection, primarily in haemocytes. irg1 catalyses the production of itaconic acid from cis-aconitic acid in the Krebs cycle. Recently, itaconate has been revealed as an immune metabolite involved in macrophage polarization. In this work, we studied the effects of exogenous dimethyl itaconate (DI) on mussels in vitro and in vivo at relevant previously described endogenous concentrations and in mussels infected with Vibrio splendidus. DI did not have adverse effects on the haemocytes viability, apoptotic cells, proliferation and phagocytic activity; however, haemocyte size, velocity and accumulated distance were decreased. The antibacterial activity of DI in vitro and in vivo was observed with high concentrations of DI, that is, 30 and 50 mM, respectively. Furthermore, DI inhibited total ROS, increased mitochondrial ROS and modulated antioxidant genes, such as SOD and CAT, related to Nrf2 activation. In this research, we have demonstrated some important pathways in haemocytes in which itaconate can be involved after its production in a bacterial infection.

Shift in Immune Parameters After Repeated Exposure to Nanoplastics in the Marine Bivalve Mytilus.

Bivalves are widespread in coastal environments subjected to a wide range of environmental fluctuations: however, the rapidly occurring changes due to several anthropogenic factors can represent a significant threat to bivalve immunity. The mussel Mytilus spp. has extremely powerful immune defenses toward different potential pathogens and contaminant stressors. In particular, the mussel immune system represents a significant target for different types of nanoparticles (NPs), including amino-modified nanopolystyrene (PS-NH2) as a model of nanoplastics. In this work, the effects of repeated exposure to PS-NH2 on immune responses of Mytilus galloprovincialis were investigated after a first exposure (10 µg/L; 24 h), followed by a resting period (72-h depuration) and a second exposure (10 µg/L; 24 h). Functional parameters were measured in hemocytes, serum, and whole hemolymph samples. In hemocytes, transcription of selected genes involved in proliferation/apoptosis and immune response was evaluated by qPCR. First exposure to PS-NH2 significantly affected hemocyte mitochondrial and lysosomal parameters, serum lysozyme activity, and transcription of proliferation/apoptosis markers; significant upregulation of extrapallial protein precursor (EPp) and downregulation of lysozyme and mytilin B were observed. The results of functional hemocyte parameters indicate the occurrence of stress conditions that did not however result in changes in the overall bactericidal activity. After the second exposure, a shift in hemocyte subpopulations, together with reestablishment of basal functional parameters and of proliferation/apoptotic markers, was observed. Moreover, hemolymph bactericidal activity, as well as transcription of five out of six immune-related genes, all codifying for secreted proteins, was significantly increased. The results indicate an overall shift in immune parameters that may act as compensatory mechanisms to maintain immune homeostasis after a second encounter with PS-NH2.

Vibrio-bivalve interactions in health and disease.

In the marine environment, bivalve mollusks constitute habitats for bacteria of the Vibrionaceae family. Vibrios belong to the microbiota of healthy oysters and mussels, which have the ability to concentrate bacteria in their tissues and body fluids, including the hemolymph. Remarkably, these important aquaculture species respond differently to infectious diseases. While oysters are the subject of recurrent mass mortalities at different life stages, mussels appear rather resistant to infections. Thus, Vibrio species are associated with the main diseases affecting the worldwide oyster production. Here, we review the current knowledge on Vibrio-bivalve interaction in oysters (Crassostrea sp.) and mussels (Mytilus sp.). We discuss the transient versus stable associations of vibrios with their bivalve hosts as well as technical issues limiting the monitoring of these bacteria in bivalve health and disease. Based on the current knowledge of oyster/mussel immunity and their interactions with Vibrio species pathogenic for oyster, we discuss how differences in immune effectors could contribute to the higher resistance of mussels to infections. Finally, we review the multiple strategies evolved by pathogenic vibrios to circumvent the potent immune defences of bivalves and how key virulence mechanisms could have been positively or negatively selected in the marine environment through interactions with predators.

Molecular features of interleukin-1 receptor-associated kinase-b and a in Mytilus coruscus, regulating their function by infection of aquatic pathogens and the expression of their serine/threonine protein kinase functional domains.

Interleukin-1 receptor-associated kinases (IRAKs) play important roles in the innate immune system of TLR (Toll-like receptor) signaling pathway. In this paper, interleukin-1 receptor-associated kinase-b (designated as McIRAK-b) and interleukin-1 receptor-associated kinase-a (named as McIRAK-a) were obtained based on the transcriptome data, the full length of McIRAK-b was 1815 bp and McIRAK-a was 3168bp, encoding 532 and 978 amino acids, respectively. BLASTp analysis and phylogenetic relationship strongly suggested that the deduced amino acid sequence of McIRAK-b had high homology with IRAK-4 and McIRAK-a was similar to IRAK-1 of other mollusks, especially at their function domains. The expressions of McIRAK-b and McIRAK-a were detected in six tissues including adductor muscle, hemocyte, gills, gonad and hepatopancreas, and the highest expressions appeared both in gills. The expressions of McIRAK-b and McIRAK-a in gills were observed with time-dependent manners after bacterial infections. After being challenged with Vibrio alginolyticus, McIRAK-b expressed significantly and got the peak at 8 h (9.47 times compared with the control group), but the peak appeared at 4 h by being infected with Vibrio parahaemolyticus (12.02 times compared with the control group). The highest point of McIRAK-a mRNA appeared at 12 h (5.16 times) after being challenged with V.alginolyticus and 8 h (4.21 times) for V.parahaemolyticus challenge. The results suggested that IRAK-b and IRAK-a might be important in immune signaling pathway of mussels. The kinase functional domain sequences (S_TKc) of McIRAK-b and McIRAK-a expressed in BL21(DE3) and purified by Ni-NAT Superflow resin conforming to the expected molecular weight with many active sites for their conferring protein-protein interaction functions. This study may provide some further understandings of the regulatory mechanisms in the bivalve innate immune system for IRAKs family.

Establishing typical values for hemocyte mortality in individual California mussels, Mytilus californianus.

Hemocytes are immune cells in the hemolymph of invertebrates that play multiple roles in response to stressors; hemocyte mortality can thus serve as an indicator of overall animal health. However, previous research has often analyzed hemolymph samples pooled from several individuals, which precludes tracking individual responses to stressors over time. The ability to track individuals is important, however, because large inter-individual variation in response to stressors can confound the interpretation of pooled samples. Here, we describe protocols for analysis of inter- and intra-individual variability in hemocyte mortality across repeated hemolymph samples of California mussels, Mytilus californianus, free from typical abiotic stressors. To assess individual variability in hemocyte mortality with serial sampling, we created four groups of 15 mussels each that were repeatedly sampled four times: at baseline (time zero) and three subsequent times separated by either 24, 48, 72, or 168 h. Hemocyte mortality was assessed by fluorescence-activated cell sorting (FACS) of cells stained with propidium iodide. Our study demonstrates that hemolymph can be repeatedly sampled from individual mussels without mortality; however, there is substantial inter- and intra-individual variability in hemocyte mortality through time that is partially dependent on the sampling interval. Across repeated samples, individual mussels' hemocyte mortality had, on average, a range of ~6% and a standard deviation of ~3%, which was minimized with sampling periods ≥72 h apart. Due to this intra-individual variability, obtaining ≥2 samples from a specimen will more accurately establish an individual's baseline. Pooled-sample means were similar to individual-sample means; however, pooled samples masked the individual variation in each group. Overall, these data lay the foundation for future work exploring individual mussels' temporal responses to various stressors on a cellular level.

Stimulation of Mytilus galloprovincialis Hemocytes With Different Immune Challenges Induces Differential Transcriptomic, miRNomic, and Functional Responses.

Mediterranean mussels (Mytilus galloprovincialis) are marine bivalve molluscs with high resilience to biotic and abiotic stress. This resilience is one of the reasons why this species is such an interesting model for studying processes such as the immune response. In this work, we stimulated mussel hemocytes with poly I:C, ß-glucans, and LPS and then sequenced hemocyte mRNAs (transcriptome) and microRNAs (miRNome) to investigate the molecular basis of the innate immune responses against these pathogen-associated molecular patterns (PAMPs). An immune transcriptome comprising 219,765 transcripts and an overview of the mussel miRNome based on 5,175,567 non-redundant miRNA reads were obtained. The expression analyses showed opposite results in the transcriptome and miRNome; LPS was the stimulus that triggered the highest transcriptomic response, with 648 differentially expressed genes (DEGs), while poly I:C was the stimulus that triggered the highest miRNA response, with 240 DE miRNAs. Our results reveal a powerful immune response to LPS as well as activation of certain immunometabolism- and ageing/senescence-related processes in response to all the immune challenges. Poly I:C exhibited powerful stimulating properties in mussels, since it triggered the highest miRNomic response and modulated important genes related to energy demand; these effects could be related to the stronger activation of these hemocytes (increased phagocytosis, increased NO synthesis, and increased velocity and accumulated distance). The transcriptome results suggest that after LPS stimulation, pathogen recognition, homeostasis and cell survival processes were activated, and phagocytosis was induced by LPS. ß-glucans elicited a response related to cholesterol metabolism, which is important during the immune response, and it was the only stimulus that induced the synthesis of ROS. These results suggest a specific and distinct response of hemocytes to each stimulus from a transcriptomic, miRNomic, and functional point of view.

Transcriptomic Response of Mussel Gills After a Vibrio splendidus Infection Demonstrates Their Role in the Immune Response.

Mussels (Mytilus galloprovincialis) are filter feeder bivalves that are constantly in contact with a wide range of microorganisms, some of which are potentially pathogenic. How mussels recognize and respond to pathogens has not been fully elucidated to date; therefore, we investigated the immune mechanisms that these animals employ in response to a bacterial bath infection from the surrounding water, mimicking the response that mussels mount under natural conditions. After the bath infection, mussels were able to remove the bacteria from their bodies and from the water tank. Accordingly, antibacterial activity was detected in gill extracts, demonstrating that this tissue plays a central role in removing and clearing potential pathogens. A transcriptomic study performed after a bath infection with Vibrio splendidus identified a total of 1,156 differentially expressed genes. The expression levels of genes contributing to a number of biological processes, such as immune response activation pathways and their regulation with cytokines, cell recognition, adhesion and apoptosis, were significantly modulated after infection, suggesting that the gills play important roles in pathogen recognition, as well as being activators and regulators of the mussel innate immune response. In addition to RNA-seq analysis, long non-coding RNAs and their neighboring genes were also analyzed and exhibited modulation after the bacterial challenge. The response of gills against bath infection was compared with the findings of a previous transcriptomic study on hemocytes responding to systemic infection, demonstrating the different and specific functions of gills. The results of this study indicate that recognition processes occur in the gill, thereby activating the effector agents of the immune response to overcome bacterial infection.

Nanoplastics: From tissue accumulation to cell translocation into Mytilus galloprovincialis hemocytes. resilience of immune cells exposed to nanoplastics and nanoplastics plus Vibrio splendidus combination.

Plastic litter is an issue of global concern. In this work Mytilus galloprovincialis was used to study the distribution and effects of polystyrene nanoplastics (PS NPs) of different sizes (50 nm, 100 nm and 1 µm) on immune cells. Internalization and translocation of NPs to hemolymph were carried out by in vivo experiments, while endocytic routes and effects of PS NPs on hemocytes were studied in vitro. The smallest PS NPs tested were detected in the digestive gland and muscle. A fast and size-dependent translocation of PS NPs to the hemolymph was recorded after 3 h of exposure. The internalization rate of 50 nm PS NPs was lower when caveolae and clathrin endocytosis pathways were inhibited. On the other hand, the internalization of larger particles decreased when phagocytosis was inhibited. The hemocytes exposed to NPs had changes in motility, apoptosis, ROS and phagocytic capacity. However, they showed resilience when were infected with bacteria after PS NP exposure being able to recover their phagocytic capacity although the expression of the antimicrobial peptide Myticin C was reduced. Our findings show for the first time the translocation of PS NPs into hemocytes and how their effects trigger the loss of its functional parameters.

Integrated transcriptomic and functional immunological approach for assessing the invasiveness of bivalve alien species.

Biological invasions started when humans moved species beyond their normal geographic limits. Bivalves are the most notoriously invasive species in subtidal aquatic environments. Next-generation sequencing technologies are applied to understand the molecular mechanisms involved in the invasion. The ecological immunology focuses on the role of immunity in invasion, and its magnitude could help to predict the invasiveness of alien species. A remarkable case of invasion has been reported in the Ría de Vigo (Spain) by the black pygmy mussel Xenostrobus securis. In Galicia, the Mediterranean mussel Mytilus galloprovincialis is the predominant cultured bivalve species. Can we predict the invasiveness of alien bivalve species by analyzing their immune response? Can X. securis represent a risk for the autochthonous mussel? We evaluated the suitability of the immune-related hypotheses in our model by using an integrated transcriptomic and functional immunological approach. Our analysis suggests lower immune capabilities in X. securis compared to M. galloprovincialis, probably due to the relocation of energetic resources from the immune response to vital physiological processes to cope with salinity stress. This multidisciplinary approach will help us understand how the immune response can be influenced by the adaptive process and how this immune response can influence the invasion process.

Immune Tolerance in Mytilus galloprovincialis Hemocytes After Repeated Contact With Vibrio splendidus.

Mediterranean mussels (Mytilus galloprovincialis) are sessile filter feeders that live in close contact with numerous marine microorganisms. As is the case in all invertebrates, mussels lack an adaptive immune system, but they respond to pathogens, injuries or environmental stress in a very efficient manner. However, it is not known if they are able to modify their immune response when they reencounter the same pathogen. In this work, we studied the transcriptomic response of mussel hemocytes before and after two consecutive sublethal challenges with Vibrio splendidus. The first exposure significantly regulated genes related to inflammation, migration and response to bacteria. However, after the second exposure, the differentially expressed genes were related to the control and inhibition of ROS production and the resolution of the inflammatory response. Our results also show that the second injection with V. splendidus led to changes at the transcriptional (control of the expression of pro-inflammatory transcripts), cellular (shift in the hemocyte population distribution), and functional levels (inhibition of ROS production). These results suggest that a modified immune response after the second challenge allowed the mussels to tolerate rather than fight the infection, which minimized tissue damage.

Effects of BDE-47 exposure on immune-related parameters of Mytilus galloprovincialis.

The persistent pollutants polybrominated diphenyl ethers (PBDEs) have been demonstrated to produce several negative effects on marine organisms. Although Mytilus galloprovincialis was extensively studied as model system, the effects of PBDEs on the innate immune system of mussels remains unclear. In this study, except for the control treatment, specimens of M. galloprovincialis were fed with microalgae treated with increasing concentrations of PBDEs (maximum level 100 ng L-1 of BDE-47 per day). BDE-47 treatment was maintained for 15 days and then the animals were fed with the same control diet, without contaminants, for 15 days. Samples of haemolymph (HL) were obtained at T0, T15 and T30 days of the experiment to evaluate different parameters related to immunity, such as neutral red retention time, and peroxidase, protease, antiprotease, lysozyme and bactericidal activities. BDE-47 exposure for 15 days affected both the stability of haemocytes and humoral parameters. In addition, the obtained results indicated that, at 30 days, after 15 days of culture without contaminant, the immune parameters were still affected, as some of them did not return to the basal levels, and others remained stimulated. Overall the results indicate that BDE-47 exposures at environmentally realistic levels may affect various aspects of immune function in M. galloprovincialis, acting as stressor that can compromise the general welfare.

Tissue distribution and functional characterization of mytimacin-4 in Mytilus galloprovincialis.

Antimicrobial peptides (AMPs) play fundamental roles in the innate immunity of invertebrates. Mytimacin-4 is a kind of AMP gene previously sequenced from Mytilus galloprovincialis based on an identified EST sequence in our lab. In the present study, the tissue distribution and antimicrobial activities of mytimacin-4 were further investigated. A qRT-PCR analysis revealed that mytimacin-4 transcripts were constitutively expressed in all of the tested tissues of M. galloprovincialis, with the highest expression level in the posterior adductor muscle. After challenge by Vibrio anguillarum, the expression level of mytimacin-4 gene was significantly increased at 24 h (P < 0.05) in the mantle and increased at 48 h (P < 0.05) in the posterior adductor muscle. This finding suggested that mytimacin-4 transcripts were inducible upon pathogen infection. A minimal inhibitory concentration (MIC) assay indicated that recombinant mytimacin-4 protein had potent antimicrobial activities against gram-positive and gram-negative bacteria. Among the tested microorganisms, mytimacin-4 protein exhibited strong inhibition activities against Bacillus subtilis and Vibrio parahaemolyticus with MICs of 0.315 µM and 0.62 µM, respectively. This study provides for the first time direct evidence of antimicrobial action of mytimacin-4 in M. galloprovincialis.

Genomics and immunity of the Mediterranean mussel Mytilus galloprovincialis in a changing environment.

The Mediterranean mussel (Mytilus galloprovincialis) is a marine invasive species cultured all over the world. Mussels are an appreciated resource in local aquaculture enterprises because of their robust production and resilience that translates into a reliable economic value. So far, no massive mortalities have been reported in natural or cultured populations of this species. In the last years, the knowledge about its immune system has greatly improved but there are still many questions to be answered. One of them is why mussels, with their high filtering activity, are able to be exposed to a high number of potential pathogens without getting infected and without developing an elevated inflammatory response. The sequencing of the mussel genome has revealed a very complex organization with high heterozygosity, abundance of repetitive sequences and extreme intraspecific sequence diversity among individuals, mainly in immune related genes. Among those genes, antimicrobial peptides are the most expressed gene families in mussels, highly polymorphic and with antimicrobial effect against molluscs pathogens, but also against pathogens of lower vertebrates and humans. The combination of a complex genome with the adaptation of mussel immune system to a changing environment could explain this high variability, not only in immune-related genes, but also in the functional response among individuals sampled in the same location and date.

Two toll-like receptors identified in the mantle of Mytilus coruscus are abundant in haemocytes.

Toll-like receptors (TLRs) are a large family of pattern recognition receptors (PRRs) that play a critical role in innate immunity. TLRs are activated when they recognize microbial associated molecular patterns (MAMPs) of bacteria, viruses, or fungus. In the present study, two TLRs were isolated from the mantle of the hard-shelled mussel (Mytilus coruscus) and designated McTLR2 and McTLR3 based on their sequence similarity and phylogenetic clustering with Crassostrea gigas, CgiTLR2 and CgiTLR3, respectively. Quantitative RT-PCR analysis demonstrated that McTLR2 and McTLR3 were constitutively expressed in many tissues but at low abundance.