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Esta tese doutoral demostra a importancia das branquias para o sistema inmune do mexillón, levando a cabo o recoñecemento específico dos patóxenos incorporados durante o filtraxe da auga e a activación das defensas inmunes mediante a produción de citoquinas pro-inflamatorias. Estudaronse dúas familias xénicas de importancia clave nestos dous procesos, os TLRs (implicados no recoñemento de patóxenos) e as IL-17s (implicadas na sinalización inmune), amosando en mexillón as maiores expansións de todos os metazoos e unha gran diversidade xenómica relacionada coa diversificación funcional dos repertorios

Recent evidence has demonstrated the unique properties of the innate immune system, known as innate immune memory, immune priming, or trained immunity. These properties have been described as the ability of the innate immune system to learn from previous microbial experiences, which improves survival after subsequent infection. In this review, we present the state of knowledge on trained immunity in invertebrates and provide a comprehensive overview of these capabilities in cultured marine molluscs, which are currently threatened by recurrent diseases. Studies have shown that exposure to environmental microbiota, pathogens, or derived elements, can provide a stronger response and protection against future infections. These studies highlight common and distinct features of protection, mechanisms, specificity, and duration that vary with immune markers, and methods of stimulation. While the cellular and molecular basis of these responses is only partially understood, effects on phagocytosis, haemocyte populations, apoptosis, oxidative stress, and immune gene expression have been suggested. Finally, we propose a framework for future research to go beyond the current evidence and address potential limitations in the implementation of trained immunity‐based strategies to control disease. Immune training may provide a unique opportunity to promote the sustainable development of marine mollusc aquaculture.

Additional data derived from "Bivalves present the largest and most diversified repertoire of Toll-like receptors in the animal kingdom, suggesting broad-spectrum pathogen recognition in marine waters". -TLR sequences indetified in the annotation of 85 metazoan genomes -TLR sequences de novo indentified in the genomes of three mussel species -Snakemake pipeline and script to calculate saturation curves from the salmon output of the mapping of different mussel SRA transcriptomic samples to the mussel genome -Pipeline used to calculate the modulated transcripts for different mussel transcriptomes

Illumina sequencing of mussel hemocytes transcriptome after viral VHSV stimulation: three control and three infected individuals 24 hours after the stimulation

Toll-like receptors (TLRs) are the most widespread class of membrane-bound innate immune receptors, responsible of specific pathogen recognition and production of immune effectors through the activation of intracellular signaling cascades. The repertoire of TLRs was analyzed in 85 metazoans, enriched on molluscan species, an underrepresented phylum in previous studies. Following an ancient evolutionary origin, suggested by the presence of TLR genes in Anthozoa (Cnidaria), these receptors underwent multiple independent gene family expansions, the most significant of which occurred in bivalve molluscs. Marine mussels (Mytilus spp.) had the largest TLR repertoire in the animal kingdom, with evidence of several lineage-specific expanded TLR subfamilies with different degrees of orthology conservation within bivalves. Phylogenetic analyses revealed that bivalve TLR repertoires were more diversified than their counterparts in deuterostomes or ecdysozoans. The complex evolutionary history of TLRs, characterized by lineage-specific expansions and losses, along with episodic positive selection acting on the extracellular recognition domains, suggests that functional diversification might be a leading evolutionary force. We analyzed a comprehensive transcriptomic data set from Mytilus galloprovincialis and built transcriptomic correlation clusters with the TLRs expressed in gills and in hemocytes. The implication of specific TLRs in different immune pathways was evidenced, as well as their specific modulation in response to different biotic and abiotic stimuli. We propose that, in a similar fashion to the remarkable functional specialization of vertebrate TLRs, the expansion of the TLR gene family in bivalves attends to a functional specification motivated by the biological particularities of these organisms and their living environment

Presence/absence variation (PAV) is a well-known phenomenon in prokaryotes that was described for the first time in bivalves in 2020 in Mytilus galloprovincialis. The objective of the present study was to further our understanding of the PAV phenomenon in mussel biology. The distribution of PAV was studied in a mussel chromosome-level genome assembly, revealing a widespread distribution but with hotspots of dispensability. Special attention was given to the effect of PAV in gene expression, since dispensable genes were found to be inherently subject to distortions due to their sparse distribution among individuals. Furthermore, the high expression and strong tissue specificity of some dispensable genes, such as myticins, strongly supported their biological relevance. The significant differences in the repertoire of dispensable genes associated with two geographically distinct populations suggest that PAV is involved in local adaptation. Overall, the PAV phenomenon would provide a key selective advantage at the population level

C-type lectins belong to a widely conserved family of lectins characterized in Metazoa. They show important functional diversity and immune implications, mainly as pathogen recognition receptors. In this work, C-type lectin-like proteins (CTLs) of a set of metazoan species were analyzed, revealing an important expansion in bivalve mollusks, which contrasted with the reduced repertoires of other mollusks, such as cephalopods. Orthology relationships demonstrated that these expanded repertoires consisted of CTL subfamilies conserved within Mollusca or Bivalvia and of lineage-specific subfamilies with orthology only between closely related species. Transcriptomic analyses revealed the importance of the bivalve subfamilies in mucosal immunity, as they were mainly expressed in the digestive gland and gills and modulated with specific stimuli. CTL domain-containing proteins that had additional domains (CTLDcps) were also studied, revealing interesting gene families with different conservation degrees of the CTL domain across orthologs from different taxa. Unique bivalve CTLDcps with specific domain architectures were revealed, corresponding to uncharacterized bivalve proteins with putative immune function according to their transcriptomic modulation, which could constitute interesting targets for functional characterization.

As filter-feeding bivalves, mussels have been traditionally studied as possible vectors of different bacterial or viral pathogens. The absence of a known viral pathogen in these bivalves makes it particularly interesting to study the interaction of the mussel innate immune system with a virus of interest. In the present work, mussels were challenged with viral haemorrhagic septicaemia virus (VHSV), which is a pathogen in several fish species. The viral load was eliminated after 24 h and mussels evidenced antiviral activity towards VHSV, demonstrating that the virus was recognized and eliminated by the immune system of the host and confirming that mussels are not VHSV vectors in the marine environment. The transcriptome activating the antiviral response was studied, revealing the involvement of cytoplasmic viral sensors with the subsequent activation of the JAK-STAT pathway and several downstream antiviral effectors. The inflammatory response was inhibited with the profound downregulation of MyD88, shifting the immune balance towards antiviral functions. High modulation of retrotransposon activity was observed, revealing a mechanism that facilitates the antiviral response and that had not been previously observed in these species. The expression of several inhibitors of apoptosis and apoptosis-promoting genes was modulated, although clear inhibition of apoptosis in bivalves after severe viral infection and subsequent disease was not observed in this study. Finally, the modulated expression of several long noncoding RNAs that were correlated with genes involved in the immune response was detected

Mussels (Mytilus galloprovincialis) were exposed during 24 hours to a waterborne infection with 10E8 CFU/ml Vibrio splendidus (reference strain LGP32) in the tank water. Five biological replicates were used for each infected and control conditions

This study presents the results of SARS-CoV-2 surveillance in sewage water of 11 municipalities and marine bioindicators in Galicia (NW of Spain) from May 2020 to May 2021. An integrated pipeline was developed including sampling, pre-treatment and biomarker quantification, RNA detection, SARS-CoV-2 sequencing, mechanistic mathematical modeling and forecasting. The viral load in the inlet stream to the wastewater treatment plants (WWTP) was used to detect new outbreaks of COVID-19, and the data of viral load in the wastewater in combination with data provided by the health system was used to predict the evolution of the pandemic in the municipalities under study within a time horizon of 7 days. Moreover, the study shows that the viral load was eliminated from the treated sewage water in the WWTP, mainly in the biological reactors and the disinfection system. As a result, we detected a minor impact of the virus in the marine environment through the analysis of seawater, marine sediments and, wild and aquacultured mussels in the final discharge point of the WWTP.

Transcriptomic studies performed under different infection models have revealed the implication of conserved innate immune gene families in the defensive response of mussels. Many of those families are however greatly expanded in comparison with phylogenetically close species. We have deepened in the evolutionary and selection processes that gave rise to these gene families, particularly the pro-inflammatory interleukin IL-17 and the immune Toll-like receptors (TLRs ). Comparative genomics was applied to examine the evolution of these genes from poriferans to higher vertebrates. Common evolutionary patterns were revealed. Cnidaria was highlighted as the most ancient, diverged phylum and expansions of different magnitude were found as well in other marine bivalves and echinoderm species. Concerning mussels, the different clusters obtained in phylogenetic analyses were conserved by the three analyzed species (Mytilus galloprovincialis, M. edulis and M. coruscus). Selection analyses were performed on those clusters/isoforms and the inter-genomic variation was studied to reveal the degree of conservation of these genes or their subjection to presence/absence variation. Former transcriptomic studies had revealed the implication of TLRs in recognizing bacterial infections in hemocytes and gills. and subsequent activation of IL-17 as immune mediators leading to the canonical pathway of NF-kB inflammation. For the current study, a massive expression dataset was built using all the transcriptomic data available for mussel (M. gal/oprovincialis). Clusters of correlated genes were obtained from this dataset. Different expression trends were obtained for different members of the studied gene families. The expression analysis revealed different correlated transcripts that could interact with specific forms of our target genes in response to different stimuli. These results would point towards a clear functional specificity that would be allowed by the great underlying variabilit

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

In recent years, the immune response of mussels (Mytilus galloprovincialis) has been studied at the transcriptomic level against several bacterial infections. As a result, different immune mechanisms have been revealed, including both conserved essential innate pathways and particularities of the mussel immune response according to its nature and environment. However, there is often a lack of functional verification because mussels are a non-model species and because transcriptomic and proteomic information is not always well correlated. In the current study, a high-throughput quantitative proteomics study coupled to LC-MS/MS analysis using isobaric tandem mass tags (TMTs) for protein labeling was employed to study the mussel gill immune response to a Vibrio splendidus bath (waterborne) infection at a functional protein level. A total of 4,242 proteins were identified and quantified, of which 226 were differentially expressed (DEPs) after infection, giving to the study a depth that was lacking in previous proteomic studies of the bivalve immune response. Modulated proteins evidenced an important cytoskeletal disruption caused by bacterial infection. A conserved network of associated proteins was modulated, regulating oxidative stress and NF-kB inflammatory responses and leading to innate immunity effectors. Proteomic results were submitted to an integrated analysis with those obtained in a previous transcriptomic approach with the same infection. Half of all the quantified proteins had a concordant transcriptomic expression trend, but this concordance increased when focusing on the DEPs. The correlation was higher within the immune-related DEPs, and the activation of the conserved NF-kB pro-inflammatory pathway was the main response in both approaches. The results of both techniques could be integrated to obtain a more complete vision of the 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

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

Bivalves are susceptible to numerous diseases that compromise their culture, producing economic losses all over the world. Mussels that cohabitate in the same areas as oysters and clams have experienced massive mortalities due to viral and bacterial infections. As an example, the ostreid herpesvirus 1 (OsHV-1) has caused massive mortalities in oysters (Crassostrea gigas) in different parts of the world. However, until now, high mortalities have never been reported in the field for Mediterranean mussel (Mytilus galloprovincialis). This resistance could be due to the high expression of antimicrobial peptides that has been observed in mussel