Transcriptomic analysis of turbot (Scophthalmus maximus) treated with zymosan a reveals that lncRNAs and inflammation-related genes mediate the protection conferred against Aeromonas salmonicida.

Aeromonas salmonicida is one of the most harmful pathogens in finfish aquaculture worldwide. Immunostimulants such as ß-glucans are used to enhance the immunity of cultured fish. However, their effects on fish physiology are not completely understood. In the present work, we evaluated the effect of a single intraperitoneal (ip) injection of zymosan A on fish survival against A. salmonicida infection. A single administration of this compound protected fish against A. salmonicida challenge and reduce the bacterial load in the head kidney one week after its administration. Transcriptome analyses of head kidney samples revealed several molecular mechanisms involved in the protection conferred by zymosan A and their regulation by long noncoding RNAs. The transcriptome profile of turbot exposed only to zymosan A was practically unaltered one week after ip injection. However, the administration of this immunostimulant induced significant transcriptomic changes once the fish were in contact with the bacteria and increased the survival of the infected turbot. Our results suggest that the restraint of the infection-induced inflammatory response, the management of apoptotic cell death, cell plasticity and cellular processes involving cytoskeleton dynamics support the protective effects of zymosan A. All this information provides insights on the cellular and molecular mechanisms involved in the protective effects of this widely used immunostimulant.

RNA-Seq analysis of juvenile gilthead sea bream (Sparus aurata) provides some clues regarding their resistance to the nodavirus RGNNV genotype.

Gilthead sea bream (Sparus aurata) is considered an asymptomatic carrier for the nodavirus genotype affecting European sea bass (Dicentrarchus labrax), RGNNV. Only larvae and juveniles of sea bream have been found to be susceptible to the RGNNV/SJNNV reassortant. Nevertheless, the molecular bases of the high resistance of sea bream against RGNNV are not known, and the overall transcriptome response to the virus remains unexplored. In this work, we conducted the first RNA-Seq analysis of sea bream infected with RGNNV to elucidate the immune mechanisms involved in their resistance. Since we recently published the transcriptome response of sea bass infected with RGNNV, we wanted to take the same tissues (brain and head kidney) at the same time points (24 and 72 h postinfection) to conduct comparative analyses. Sea bream responded to RGNNV challenge with a powerful immune arsenal characterized by the high expression of a multitude of type I interferon-related genes, immune receptors and antigen presentation-related genes in both tissues. Moreover, complement-, coagulation- and angiogenesis-related genes were highly enriched in the head kidney at the earlier sampling point. Interestingly, despite the strong immune response found in the brain, inflammation seems to have been restrained, resulting in a neuroprotective scenario. While the response in sea bass was characterized by the activation of the stress axis, which could lead to immunosuppression and neuronal damage, genes involved in these processes were not modulated in sea bream. An efficient antiviral response accompanied by low inflammation and the absence of stimulation of the stress response seem to play a role in the success of sea bream in resisting RGNNV infection.

Transcriptome and Genome Analyses Applied to Aquaculture Research.

The Special Issue "Transcriptome and Genome Analyses Applied to Aquaculture Research" had great success among the researchers specialized in different fields of aquaculture [...].

Editorial of Special Issue "The 2nd Edition: Vaccines for Aquaculture".

The Special Issue "Vaccines for Aquaculture" of the journal Vaccines had a great success among fish immunologists, with 17 published manuscripts [...].

The Immune System of Marine Organisms as Source for Drugs against Infectious Diseases.

The high proliferation of microorganisms in aquatic environments has allowed their coevolution for billions of years with other living beings that also inhabit these niches. Among the different existing types of interaction, the eternal competition for supremacy between the susceptible species and their pathogens has selected, as part of the effector division of the immune system of the former ones, a vast and varied arsenal of efficient antimicrobial molecules, which is highly amplified by the broad biodiversity radiated, above any others, at the marine habitats. At present, the great recent scientific and technological advances already allow the massive discovery and exploitation of these defense compounds for therapeutic purposes against infectious diseases of our interest. Among them, antimicrobial peptides and antimicrobial metabolites stand out because of the wide dimensions of their structural diversities, mechanisms of action, and target pathogen ranges. This revision work contextualizes the research in this field and serves as a presentation and scope identification of the Special Issue from Marine Drugs journal "The Immune System of Marine Organisms as Source for Drugs against Infectious Diseases".

Zebrafish as a Vertebrate Model for Studying Nodavirus Infections.

Nervous necrosis virus (NNV) is a neurotropic pathogenic virus affecting a multitude of marine and freshwater fish species that has a high economic impact on aquaculture farms worldwide. Therefore, the development of new tools and strategies aimed at reducing the mortality caused by this virus is a pivotal need. Although zebrafish is not considered a natural host for NNV, the numerous experimental advantages of this species make zebrafish an attractive model for studying different aspects of the disease caused by NNV, viral encephalopathy and retinopathy (VER). In this work, we established the best way and age to infect zebrafish larvae with NNV, obtaining significant mortalities in 3-day-postfertilization larvae when the virus was inoculated directly into the brain or by intramuscular microinjection. As occurs in naturally susceptible fish species, we confirmed that after intramuscular injection the virus was able to migrate to the central nervous system (CNS). As expected, due to the severe damage that this virus causes to the CNS, alterations in the swimming behavior of the zebrafish larvae were also observed. Taking advantage of the existence of transgenic fluorescent zebrafish lines, we were able to track the migration of different innate immune cells, mainly neutrophils, to the site of infection with NNV via the brain. However, we did not observe colocalization between the viral particles and neutrophils. RNA-Seq analysis of NNV-infected and uninfected larvae at 1, 3 and 5 days postinfection (dpi) revealed a powerful modulation of the antiviral immune response, especially at 5 dpi. We found that this response was dominated by, though not restricted to, the type I interferon system, the major defence mechanism in the innate immune response against viral pathogens. Therefore, as zebrafish larvae are able to develop the main characteristic of NNV infection and respond with an efficient immune arsenal, we confirmed the suitability of zebrafish larvae for modelling VER disease and studying different aspects of NNV pathogenesis, immune response and screening of antiviral drugs.

Acute Inflammation Induces Neuroendocrine and Opioid Receptor Genes Responses in the Seabass Dicentrarchus labrax Brain.

In fish, as observed in mammals, any stressful event affects the immune system to a larger or shorter extent. The neuroendocrine-immune axis is a bi-directional network of mobile compounds and their receptors that are shared between both systems (neuroendocrine and immune) and that regulate their respective responses. However, how and to what extent immunity modulates the neuroendocrine system is not yet fully elucidated. This study was carried out to understand better central gene expression response patterns in a high-valued farmed fish species to an acute peripheral inflammation, focusing on genes related to the hypothalamus-pituitary-interrenal axis and the opioid system. European seabass, Dicentrarchus labrax, were intra-peritoneally injected with either Freund's Incomplete Adjuvant to induce a local inflammatory response or Hanks Balances Salt Solution to serve as the control. An undisturbed group was also included to take into account the effects due to handling procedures. To evaluate the outcomes of an acute immune response, fish were sampled at 4, 24, 48, and 72 h post-injection. The brain was sampled and dissected for isolation of different regions: telencephalon, optic tectum, hypothalamus, and pituitary gland. The expression of several genes related to the neuroendocrine response was measured by real-time PCR. Data were statistically analyzed by ANOVA and discriminant analyses to obtain these genes' responsiveness for the different brain regions. Serotonergic receptors were upregulated in the telencephalon, whereas the optic tectum inhibited these transcription genes. The hypothalamus showed a somewhat delayed response in which serotonin and glucocorticoid receptors were concerned. Still, the hypothalamic corticotropin-releasing hormone played an important role in differentiating fish undergoing an inflammatory response from those not under such conditions. Opioid receptors gene expression increased in both the hypothalamus and the telencephalon, while in the optic tectum, most were downregulated. However, no changes in the pituitary gland were observed. The different brain regions under immune stimulation demonstrated clear, distinct responses regarding gene transcription rates as well as the time period needed for the effect to occur. Further, more integrative studies are required to associate functions to the evaluated genes more safely and better understand the triggering mechanisms.

Surgical face masks as a source of emergent pollutants in aquatic systems: Analysis of their degradation product effects in Danio rerio through RNA-Seq.

Surgical face masks are the most popularised and effective personal equipment for protecting public health during the COVID-19 pandemic. They are composed of plastic polymer fibres with a large amount of inorganic and organic compounds that can be released into aquatic environments through degradation processes. This source of microplastics and inorganic and organic substances could potentially impact aquatic organisms. In this study, the toxicogenomic effects of face masks at different stages of degradation in water were analysed in zebrafish larvae (Danio rerio) through RNA-Seq. Larvae were exposed for 10 days to three treatments: 1) face mask fragments in an initial stage of degradation (poorly degraded masks -PDM- products) with the corresponding water; 2) face mask fragments in an advanced stage of degradation (highly degraded masks -HDM- products) with the corresponding water; and 3) water derived from HDM (W-HDM). Transcriptome analyses revealed that the three treatments provoked the down-regulation of genes related to reproduction, especially the HDM products, suggesting that degradation products derived from face masks could act as endocrine disruptors. The affected genes are involved in different steps of reproduction, including gametogenesis, sperm-egg recognition and binding or fertilisation. Immune-related genes and metabolic processes were also differentially affected by the treatments.

An environmentally relevant concentration of antibiotics impairs the immune system of zebrafish (Danio rerio) and increases susceptibility to virus infection.

In this work, we analysed the transcriptome and metatranscriptome profiles of zebrafish exposed to an environmental concentration of the two antibiotics most frequently detected in European inland surface water, sulfamethoxazole (SMX) and clarithromycin (CLA). We found that those animals exposed to antibiotics (SMX+CLA) for two weeks showed a higher bacterial load in both the intestine and kidney; however, significant differences in the relative abundance of certain bacterial classes were found only in the intestine, which also showed an altered fungal profile. RNA-Seq analysis revealed that the complement/coagulation system is likely the most altered immune mechanism, although not the only one, in the intestine of fish exposed to antibiotics, with numerous genes inhibited compared to the control fish. On the other hand, the effect of SMX+CLA in the kidney was more modest, and an evident impact on the immune system was not observed. However, infection of both groups with spring viremia of carp virus (SVCV) revealed a completely different response to the virus and an inability of the fish exposed to antibiotics to respond with an increase in the transcription of complement-related genes, a process that was highly activated in the kidney of the untreated zebrafish after SVCV challenge. Together with the higher susceptibility to SVCV of zebrafish treated with SMX+CLA, this suggests that complement system impairment is one of the most important mechanisms involved in antibiotic-mediated immunosuppression. We also observed that zebrafish larvae exposed to SMX+CLA for 7 days showed a lower number of macrophages and neutrophils.

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.

Characterization of the turbot (Scophthalmus maximus) interleukin-18: Identification of splicing variants, phylogeny, synteny and expression analysis.

Interleukin-18 (IL-18) is a pro-inflammatory cytokine that belongs to the interleukin-1 (IL-1) family of cytokines. As occurs with IL-1ß, it is synthetized as an inactive precursor peptide that is mainly processed by the cysteine protease caspase-1 in the inflammasome complex. In mammals, and in collaboration with IL-12, it has been described as an important cytokine controlling the Th1-mediated immune responses through the induction of IFN-γ. Although its function in mammals is well stablished, the activity of this cytokine in teleost remains to be elucidated. This could be due, among other things, to the absence of this gene in the fish model species zebrafish, but also to its complex regulation. As it was observed for rainbow trout and human, il18 splicing variants were also found in turbot, which could represent a regulatory mechanism of its bioactivity. In the case of turbot, three splicing variants were observed (SV1-3), and one of them showed an insertion of 10 amino acids in the middle of the potential caspase-1 cleavage position, reflecting that this is probably a form resistant to the processing by the inflammasome. Phylogenetic and three-dimensional analyses of turbot Il18 revealed that it is relatively well-conserved in vertebrates, although only a partial conservation of the gene synteny was observed between fish and mammals. As it was expected, turbot il18 splicing variants were mainly expressed in immune tissues under healthy conditions, and their expression was induced by a bacterial challenge, although certain inhibitions were observed after viral and parasitic infections. In the case of the viral challenge, il18 downregulations did not seem to be due to the effect of type I IFNs.

An integrative toxicogenomic analysis of plastic additives.

In developed countries, contact with plastics is constant. Plastics contain a vast number of additives such as plasticisers, stabilisers, antioxidants, flame retardants, etc., that can impact human health. Most of them have been studied separately; however, an integrative approach to identify genes, biological processes, molecular functions, and diseases linked to exposure to these compounds has not been addressed until now. The genes most commonly affected by plastic additives are related to apoptosis, cell death, proliferation and differentiation, immunity and insulin-related processes, and are mainly associated with cancer, mental disorders, diabetes mellitus type II and obesity. The most commonly affected molecular functions included steroid hormone receptor activity implicated in cancer, mental disorders, immune signalling and gonadotropin-releasing hormones. These processes and functions affected by plastic additives are related to the diseases of the developed world, most of which are linked to the endocrine system, such as cancer, diabetes, infertility and obesity. The strong interconnection among the top 50 genes modulated by plastic additives shows that the pathways affected are strongly interrelated. Therefore, studying the effects of plastic additives through a single-compound approach cannot be sufficient and a holistic approach is more appropriate for evaluating the potential effects of plastics in human health.

Interactions between the Parasite Philasterides dicentrarchi and the Immune System of the Turbot Scophthalmus maximus. A Transcriptomic Analysis.

The present study analyses the interactions between Philasterides dicentrarchi (a ciliate parasite that causes high mortalities in cultured flatfish) and the peritoneal cells of the turbot Scophthalmus maximus during an experimental infection. The transcriptomic response was evaluated in the parasites and in the fish peritoneal cells, at 1, 2 and 4 h post-infection (hpi) in turbot injected intraperitoneally (ip) with 107 ciliates and at 12 and 48 hpi in turbot injected ip with 105 ciliates. Numerous genes were differentially expressed (DE) in P. dicentrarchi, relative to their expression in control ciliates (0 hpi): 407 (369 were up-regulated) at 1 hpi, 769 (415 were up-regulated) at 2 hpi and 507 (119 were up-regulated) at 4 hpi. Gene ontology (GO) analysis of the DE genes showed that the most representative categories of biological processes affected at 1, 2 and 4 hpi were biosynthetic processes, catabolic processes, biogenesis, proteolysis and transmembrane transport. Twelve genes of the ABC transporter family and eight genes of the leishmanolysin family were DE at 1, 2 and 4 hpi. Most of these genes were strongly up-regulated (UR), suggesting that they are involved in P. dicentrarchi infection. A third group of UR genes included several genes related to ribosome biogenesis, DNA transcription and RNA translation. However, expression of tubulins and tubulin associated proteins, such as kinesins or dyneins, which play key roles in ciliate division and movement, was down-regulated (DR). Similarly, genes that coded for lysosomal proteins or that participate in the cell cycle mitotic control, glycolysis, the Krebs cycle and/or in the electron transport chain were also DR. The transcriptomic analysis also revealed that in contrast to many parasites, which passively evade the host immune system, P. dicentrarchi strongly stimulated turbot peritoneal cells. Many genes related to inflammation were DE in peritoneal cells at 1, 2 and 4 hpi. However, the response was much lower at 12 hpi and almost disappeared completely at 48 hpi in fish that were able to kill P. dicentrarchi during the first few hpi. The genes that were DE at 1, 2 and 4 hpi were mainly related to the apoptotic process, the immune response, the Fc-epsilon receptor signalling pathway, the innate immune response, cell adhesion, cell surface receptors, the NF-kappaB signalling pathway and the MAPK cascade. Expression of toll-like receptors 2, 5 and 13 and of several components of NF-κB, MAPK and JAK/STAT signalling pathways was UR in the turbot peritoneal cells. Genes expressing chemokines and chemokine receptors, genes involved in prostaglandin and leukotriene synthesis, prostaglandins, leukotriene receptors, proinflammatory cytokines and genes involved in apoptosis were strongly UR during the first four hours of infection. However, expression of anti-inflammatory cytokines such as Il-10 and lipoxygenases with anti-inflammatory activity (i.e., arachidonate 15-lipoxygenase) were only UR at 12 and/or 48 hpi, indicating an anti-inflammatory state in these groups of fish. In conclusion, the present study shows the regulation of several genes in P. dicentrarchi during the early stages of infection, some of which probably play important roles in this process. The infection induced a potent acute inflammatory response, and many inflammatory genes were regulated in peritoneal cells, showing that the turbot uses all the protective mechanisms it has available to prevent the entry of the parasite.

Potential Involvement of lncRNAs in the Modulation of the Transcriptome Response to Nodavirus Challenge in European Sea Bass (Dicentrarchus labrax L.).

Long noncoding RNAs (lncRNAs) are being increasingly recognised as key modulators of various biological mechanisms, including the immune response. Although investigations in teleosts are still lagging behind those conducted in mammals, current research indicates that lncRNAs play a pivotal role in the response of fish to a variety of pathogens. During the last several years, interest in lncRNAs has increased considerably, and a small but notable number of publications have reported the modulation of the lncRNA profile in some fish species after pathogen challenge. This study was the first to identify lncRNAs in the commercial species European sea bass. A total of 12,158 potential lncRNAs were detected in the head kidney and brain. We found that some lncRNAs were not common for both tissues, and these lncRNAs were located near coding genes that are primarily involved in tissue-specific processes, reflecting a degree of cellular specialisation in the synthesis of lncRNAs. Moreover, lncRNA modulation was analysed in both tissues at 24 and 72 h after infection with nodavirus. Enrichment analysis of the neighbouring coding genes of the modulated lncRNAs revealed many terms related to the immune response and viral infectivity but also related to the stress response. An integrated analysis of the lncRNAs and coding genes showed a strong correlation between the expression of the lncRNAs and their flanking coding genes. Our study represents the first systematic identification of lncRNAs in European sea bass and provides evidence regarding the involvement of these lncRNAs in the response to nodavirus.

RNA-Seq analysis of European sea bass (Dicentrarchus labrax L.) infected with nodavirus reveals powerful modulation of the stress response.

Nodavirus, or nervous necrosis virus (NNV), is the causative agent of viral encephalopathy and retinopathy (VER), a severe disease affecting numerous fish species worldwide. European sea bass, a cultured species of great economic importance, is highly susceptible to the disease. To better understand the response of this organism to NNV, we conducted RNA-Seq analysis of the brain and head kidney from experimentally infected and uninfected sea bass juveniles at 24 and 72 hours post-infection (hpi). Contrary to what was expected, we observed modest modulation of immune-related genes in the brain, the target organ of this virus, and some of these genes were even downregulated. However, genes involved in the stress response showed extremely high modulation. Accordingly, the genes encoding the enzymes implicated in the synthesis of cortisol were almost the only overexpressed genes in the head kidney at 24 hpi. This stress response was attenuated after 72 h in both tissues, and a progressive immune response against the virus was mounted. Moreover, experiments were conducted to determine how stress activation could impact NNV replication. Our results show the complex interplay between viral activity, the stress reaction and the immune response.

Zebrafish pten Genes Play Relevant but Distinct Roles in Antiviral Immunity.

The PTEN (phosphatase and TENsin homolog on chromosome 10) gene encodes a bifunctional phosphatase that acts as a tumor suppressor. However, PTEN has been implicated in different immune processes, including autophagy, inflammation, regulation of natural killer (NK) cell cytolytic activity and type I interferon responses. Unlike mammals, zebrafish possess two pten genes (ptena and ptenb). This study explores the involvement of both zebrafish pten genes in antiviral defense. Although ptena-/- and ptenb-/- larvae were more susceptible to Spring viremia of carp virus (SVCV), the viral replication rate was lower in the mutant larvae than in the wild-type larvae. We observed that both mutant lines showed alterations in the transcription of numerous genes, including those related to the type I interferon (IFN) system, cytolytic activity, autophagy and inflammation, and some of these genes were regulated in opposite ways depending on which pten gene was mutated. Even though the lower replication rate of SVCV could be associated with impaired autophagy in the mutant lines, the higher mortality observed in the ptena-/- and ptenb-/- larvae does not seem to be associated with an uncontrolled inflammatory response.

Conserved function of zebrafish (Danio rerio) Gdf15 as a sepsis tolerance mediator.

GDF15 is frequently detected in patients suffering from various diseases, especially those associated with pro-inflammatory processes and/or metabolic disorders. Accordingly, sepsis, whose major complications are related to metabolic alterations and systemic inflammation, significantly increases the secretion of GDF15. Indeed, this cytokine could be considered a marker of sepsis severity. However, until the last several years, the involvement of GDF15 in these disorders had not been widely characterized. In mice, GDF15 was recently described as a pivotal inducer of sepsis tolerance by mediating metabolic alterations that reduce tissue damage. In this work we describe a zebrafish gdf15 gene. We found that gdf15 follows an expression pattern similar to that observed in mammals, being highly expressed in the liver and kidney and induced after pro-inflammatory stimuli. Moreover, larvae overexpressing gdf15 were more resistant to bacterial and viral challenges without affecting the pathogen load. Consequently, Gdf15 also protected zebrafish larvae against LPS-induced mortality. As in mice, zebrafish Gdf15 seems to induce sepsis tolerance by altering the metabolic parameters of the individuals.

RNA-Seq analysis reveals that spring viraemia of carp virus induces a broad spectrum of PIM kinases in zebrafish kidney that promote viral entry.

PIM kinases are a family of serine/threonine protein kinases that potentiate the progression of the cell cycle and inhibit apoptosis. Because of this, they are considered to be proto-oncogenes, and they represent an interesting target for the development of anticancer drugs. In mammals, three PIM kinases exist (PIM-1, PIM-2 and PIM-3), and different inhibitors have been developed to block their activity. In addition to their involvement in cancer, some publications have reported that the PIM kinases have pro-viral activity, and different mechanisms where PIM kinases favour viral infections have been proposed. Zebrafish possess more than 300 Pim kinase members in their genome, and by using RNA-Seq analysis, we found a high number of Pim kinase genes that were significantly induced after infection with spring viraemia of carp virus (SVCV). Moreover, analysis of the miRNAs modulated by this infection revealed that some of them could be involved in the post-transcriptional regulation of Pim kinase abundance. To elucidate the potential role of the 16 overexpressed Pim kinases in the infectivity of SVCV, we used three different pan-PIM kinase inhibitors (SGI-1776, INCB053914 and AZD1208), and different experiments were conducted both in vitro and in vivo. We observed that the PIM kinase inhibitors had a protective effect against SVCV, indicating that, similar to what is observed in mammals, PIM kinases are beneficial for the virus in zebrafish. Moreover, zebrafish Pim kinases seem to facilitate viral entry into the host cells because when ZF4 cells were pre-incubated with the virus and then were treated with the inhibitors, the protective effect of the inhibitors was abrogated. Although more investigation is necessary, these results show that pan-PIM kinase inhibitors could serve as a useful treatment for preventing the spread of viral diseases.

Zebrafish C-reactive protein isoforms inhibit SVCV replication by blocking autophagy through interactions with cell membrane cholesterol.

In the present work, the mechanisms involved in the recently reported antiviral activity of zebrafish C-reactive protein-like protein (CRP1-7) against the spring viraemia of carp rhabdovirus (SVCV) in fish are explored. The results neither indicate blocking of the attachment or the binding step of the viral replication cycle nor suggest the direct inhibition of G protein fusion activity or the stimulation of the host's interferon system. However, an antiviral state in the host is induced. Further results showed that the antiviral protection conferred by CRP1-7 was mainly due to the inhibition of autophagic processes. Thus, given the high affinity of CRPs for cholesterol and the recently described influence of the cholesterol balance in lipid rafts on autophagy, both methyl-ß-cyclodextrin (a cholesterol-complexing agent) and 25-hydroxycholesterol (a cholesterol molecule with antiviral properties) were used to further describe CRP activity. All the tested compounds exerted antiviral activity by affecting autophagy in a similar manner. Further assays indicate that CRP reduces autophagy activity by initially disturbing the cholesterol ratios in the host cellular membranes, which in turn negatively affects the intracellular regulation of reactive oxygen species (ROS) and increases lysosomal pH as a consequence. Ultimately, here we propose that such pH changes exert an inhibitory direct effect on SVCV replication by disrupting the pH-dependent membrane-fusogenic ability of the viral glycoprotein G, which allows the release of the virus from endosomes into cytoplasm during its entry phase.

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.