Behavioural Fever Promotes an Inflammatory Reflex Circuit in Ectotherms.

BACKGROUND: The communication between the brain and the immune system is a cornerstone in animal physiology. This interaction is mediated by immune factors acting in both health and pathogenesis, but it is unclear how these systems molecularly and mechanistically communicate under changing environmental conditions. Behavioural fever is a well-conserved immune response that promotes dramatic changes in gene expression patterns during ectotherms' thermoregulatory adaptation, including those orchestrating inflammation. However, the molecular regulators activating the inflammatory reflex in ectotherms remain unidentified. METHODS: We revisited behavioural fever by providing groups of fish a thermal gradient environment during infection. Our novel experimental setup created temperature ranges in which fish freely moved between different thermal gradients: (1) wide thermoregulatory range; T° = 6.4 °C; and (2) restricted thermoregulatory range; T° = 1.4 °C. The fish behaviour was investigated during 5-days post-viral infection. Blood, spleen, and brain samples were collected to determine plasmatic pro- and anti-inflammatory cytokine levels. To characterize genes' functioning during behavioural fever, we performed a transcriptomic profiling of the fish spleen. We also measured the activity of neurotransmitters such as norepinephrine and acetylcholine in brain and peripheral tissues. RESULTS: We describe the first set of the neural components that control inflammatory modulation during behavioural fever. We identified a neuro-immune crosstalk as a potential mechanism promoting the fine regulation of inflammation. The development of behavioural fever upon viral infection triggers a robust inflammatory response in vivo, establishing an activation threshold after infection in several organs, including the brain. Thus, temperature shifts strongly impact on neural tissue, specifically on the inflammatory reflex network activation. At the molecular level, behavioural fever causes a significant increase in cholinergic neurotransmitters and their receptors' activity and key anti-inflammatory factors such as cytokine Il10 and Tgfß in target tissues. CONCLUSION: These results reveal a cholinergic neuronal-based mechanism underlying anti-inflammatory responses under induced fever. We performed the first molecular characterization of the behavioural fever response and inflammatory reflex activation in mobile ectotherms, identifying the role of key regulators of these processes. These findings provide genetic entry points for functional studies of the neural-immune adaptation to infection and its protective relevance in ectotherm organisms.

Viral Infection Drives the Regulation of Feeding Behavior Related Genes in Salmo salar.

The feeding behavior in fish is a complex activity that relies on the ability of the brain to integrate multiple signals to produce appropriate responses in terms of food intake, energy expenditure, and metabolic activity. Upon stress cues including viral infection or mediators such as the proinflammatory cytokines, prostaglandins, and cortisol, both Pomc and Npy/Agrp neurons from the hypothalamus are stimulated, thus triggering a response that controls both energy storage and expenditure. However, how appetite modulators or neuro-immune cues link pathogenesis and energy homeostasis in fish remains poorly understood. Here, we provide the first evidence of a molecular linkage between inflammation and food intake in Salmon salar. We show that in vivo viral challenge with infectious pancreatic necrosis virus (IPNV) impacts food consumption by activating anorexic genes such as mc4r, crf, and pomcb and 5-HT in the brain of S. salar. At the molecular level, viral infection induces an overall reduction in lipid content in the liver, favoring the production of AA and EPA associated with the increment of elovl2 gene. In addition, infection upregulates leptin signaling and inhibits insulin signaling. These changes are accompanied by a robust inflammatory response represented by the increment of Il-1b, Il-6, Tnfa, and Pge2 as well as an increased cortisol level in vivo. Thus, we propose a model in which hypothalamic neurons respond to inflammatory cytokines and stress-related molecules and interact with appetite induction/inhibition. These findings provide evidence of crosstalk between pathogenesis-driven inflammation and hypothalamic-pituitary-adrenocortical axes in stress-induced food intake behavior in fish.

Early transcriptomic responses associated with the membrane-initiated action of cortisol in the skeletal muscle of rainbow trout (Oncorhynchus mykiss).

Cortisol is a critical neuroendocrine regulator of the stress response in fish. Cortisol practically affects all tissues by interacting with an intracellular receptor and modulating target gene expression. However, cortisol also interacts with components of the plasma membrane in a nongenomic process that activates rapid signaling. Until now, the implication of this novel cortisol signaling for the global transcriptional response has not been explored. In the present work, we evaluated the effects of the membrane-initiated actions of cortisol on the in vivo transcriptome of rainbow trout (Oncorhynchus mykiss) skeletal muscle. RNA-Seq analyses were performed to examine the transcriptomic changes in rainbow trout stimulated by physiological concentrations of cortisol and cortisol coupled with bovine serum albumin (cortisol-BSA), a membrane-impermeable analog of cortisol. A total of 660 million paired-ends reads were generated. Reads mapped onto the reference genome revealed that 1,737; 897; and 1,012 transcripts were differentially expressed after 1, 3, and 9 h of cortisol-BSA treatment, respectively. Gene Ontology analysis showed that this novel action of cortisol modulates several biological processes, such as mRNA processing, ubiquitin-dependent protein catabolic processes, and transcription regulation. In addition, a KEGG analysis revealed that focal adhesion was the main signaling pathway that was upregulated at all the times tested. Taking these results together, we propose that the membrane-initiated cortisol action contributes significantly in the regulation of stress-mediated gene expression.

Membrane-initiated cortisol action modulates early pyruvate dehydrogenase kinase 2 (pdk2) expression in fish skeletal muscle.

Cortisol is the main glucocorticoid hormone in teleosts involved in the regulation of metabolic adjustments under both normal and stressful physiological conditions. In the skeletal muscle, cortisol modulates the energetic metabolism promoting the mobilization of glucose and other energetic substrates to overcome the stress stimulus. The effects of cortisol-mediated stress response are attributed to canonical/genomic mechanisms which involve the interaction of the hormone with its intracellular glucocorticoid receptor and, consequently, modulation of target genes. However, cortisol also can interact with membrane components, activating rapid signaling pathways with unknown contribution during the early stress response. In the present work, we evaluated the impact of membrane-initiated cortisol action over the expression of the critical modulator of energetic metabolism, pyruvate dehydrogenase kinase 2 (pdk2), in fish skeletal muscle. Juvenile rainbow trout were intraperitoneally administered with stress-related doses of cortisol and cortisol-BSA, and the expression of pdk2 was assayed by using RT-qPCR. Our results reveal that pdk2 mRNA levels increased in the skeletal muscle at one hour in both cortisol- and cortisol-BSA-treated fish. Moreover, in vitro studies revealed a biphasic response over the pdk2 regulation in myotubes mediated first through membrane-cortisol signaling pathways followed by the classic cortisol action. Finally, pdk2 up-regulation owing to cortisol and cortisol-BSA is reverted in RU486 treated myotubes, suggesting that GR signaling participates in both cortisol signaling pathways. This work suggests that non-classical cortisol pathways contribute to regulate the early metabolic response to stress in fish skeletal muscle.

The expression of TRPV channels, prostaglandin E2 and pro-inflammatory cytokines during behavioural fever in fish.

A fever, or increased body temperature, is a symptom of inflammation, which is a complex defence reaction of the organism to pathogenic infections. After pathogens enter the body, immune cells secrete a number of agents, the functions of which stimulate the body to develop a functional immune and fever response. In mammals it is known that PGE2 is the principal mediator of fever. The extent to which PGE2 and other pro-inflammatory cytokines such as TNF-α, IL-6, or IL-1ß could be involved in the induction of behavioural fever in fish remains to be clarified. Several members of the transient receptor potential (TRP) family of ion channels have been implicated as transducers of thermal stimuli, including TRPV1 and TRPV2, which are activated by heat. Here we show that members of the TRP family, TRPV1 and TRPV4, may participate in the coordination of temperature sensing during the behavioural fever. To examine the behavioral fever mechanism in Salmo salar an infection with IPNV, infectious pancreatic necrosis virus, was carried out by an immersion challenge with 10 × 105 PFU/mL-1 of IPNV. Behavioural fever impacted upon the expression levels of both TRPV1 and TRPV4 mRNAs after the viral challenge and revealed a juxtaposed regulation of TRPV channels. Our results suggest that an increase in the mRNA abundance of TRPV1 is tightly correlated with a significant elevation in the expression of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α and PGE2) in the Pre-Optic Area (POA) and cytokine release in plasma. Together, these data indicate that the reduction of TRPV4 expression during behavioural fever may contribute to the onset of behavioural fever influencing movement toward higher water temperatures. Our data also suggest an effect of TRPV channels in the regulation of behavioural fever through activation of EP3 receptors in the central nervous system by PGE2 induced by plasma-borne cytokines. These results highlight for first time in mobile ectotherms the key role of pro-inflammatory cytokines and TRPV channels in behavioural fever that likely involves a complex integration of prostaglandin induction, cytokine recognition and temperature sensing.

Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs.

This study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene expression in S. aurata with an emphasis upon immunity and the immune response.

Comparative immunity of Salmo salar and Oncorhynchus kisutch during infestation with the sea louse Caligus rogercresseyi: An enrichment transcriptome analysis.

Caligus rogercresseyi, an ectoparasite affecting the Chilean salmon industry, can cause immunosuppression and physiological stress in farmed fish. Interestingly, coho salmon (Oncorhynchus kisutch) are notably resistant to infestation, whereas Atlantic salmon (Salmo salar) are phenotypically more susceptible to sea lice. However, comparative studies on immune responses to C. rogercresseyi have not been conducted. In this study, Illumina sequencing was conducted to evaluate head kidney and skin samples taken 7 and 14 days post-infestation, yielding a total of 1492 and 1522 contigs annotated to immune-related genes for Atlantic and coho salmon, respectively. Both species evidenced an upregulation of inflammatory genes. Atlantic salmon had highly upregulated TLR22 and MHCII at 14 days post-infestation, while coho salmon had highly upregulated stat5 and il1r transcripts. Fourteen transcripts related to TH1, TH2, TLR, and macrophage responses were corroborated via RT-qPCR. Statistical analyses indicated an upregulation of mmp13, cox2, il10, ccr3, tlr22a2, and tlr21 in Atlantic salmon and of ifnγ, cd83, T-bet, tlr13, and tlr19 in coho salmon. These results suggest strong differences between the Atlantic and coho salmon immune responses, where coho salmon, the more resistant species, presented a primary TH1 response. Additionally, putative roles of TLRs in salmonids against sea lice were evidenced. This study is the first comparative transcriptome analysis that reveals species-specific immune responses in salmons infected with C. rogercresseyi.

Density-dependent effects of Caligus rogercresseyi infestation on the immune responses of Salmo salar.

Sea lice infestations are a particular concern in the salmonid aquaculture industry due to damaging effects on fish growth, disease/infection susceptibility, and survival. Despite the impacts of sea lice parasitism, few studies have determined corresponding physiological thresholds, or the quantity of sea lice that can trigger measurable effects in the host immune response. The present study evaluated the mRNA expressions of immune-related genes in Salmo salar (Atlantic salmon) under infestation challenges with contrasting loads of the sea louse Caligus rogercresseyi. Specifically, two groups of S. salar were infected with either 35 (i.e. low parasitic load) or 100 (i.e. high parasitic load) copepodids per fish. At 14 days post-infestation, the mRNA levels of immune-related genes (e.g. related to oxidative stress, pro- and inflammatory responses, and the adaptive TH1/TH2 pathways) were assessed through RT-qPCR. Significant differences were found in relation to parasitic load, suggesting density-dependent effects that activated the S. salar immune system. Higher parasitic load promoted strong inflammatory and oxidative stress responses that were correlated with the TH1 immune response. This study highlights the molecular signatures for distinct parasitic loads, providing new perspectives towards fully understanding parasite-host interactions.

Pesticides Drive Stochastic Changes in the Chemoreception and Neurotransmission System of Marine Ectoparasites.

Scientific efforts to elucidate the mechanisms of chemical communication between organisms in marine environments are increasing. This study applied novel molecular technology to outline the effects of two xenobiotic drugs, deltamethrin (DM) and azamethiphos (AZA), on the neurotransmission system of the copepod ectoparasite Caligus rogercresseyi. Transcriptome sequencing and bioinformatics analyses were conducted to evaluate treatment effects on the glutamatergic synaptic pathway of the parasite, which is closely related to chemoreception and neurotransmission. After drug treatment with DM or AZA, stochastic mRNA expression patterns of glutamatergic synapse pathway components were observed. Both DM and AZA promoted a down-regulation of the glutamate-ammonia ligase, and DM activated a metabotropic glutamate receptor that is a suggested inhibitor of neurotransmission. Furthermore, the delousing drugs drove complex rearrangements in the distribution of mapped reads for specific metabotropic glutamate receptor domains. This study introduces a novel methodological approach that produces high-quality results from transcriptomic data. Using this approach, DM and AZA were found to alter the expression of numerous mRNAs tightly linked to the glutamatergic signaling pathway. These data suggest possible new targets for xenobiotic drugs that play key roles in the delousing effects of antiparasitics in sea lice.

Evidence for the Induction of Key Components of the NOTCH Signaling Pathway via Deltamethrin and Azamethiphos Treatment in the Sea Louse Caligus rogercresseyi.

The extensive use of organophosphates and pyrethroids in the aquaculture industry has negatively impacted parasite sensitivity to the delousing effects of these antiparasitics, especially among sea lice species. The NOTCH signaling pathway is a positive regulator of ABC transporter subfamily C expression and plays a key role in the generation and modulation of pesticide resistance. However, little is known about the molecular mechanisms behind pesticide resistance, partly due to the lack of genomic and molecular information on the processes involved in the resistance mechanism of sea lice. Next-generation sequencing technologies provide an opportunity for rapid and cost-effective generation of genome-scale data. The present study, through RNA-seq analysis, determined that the sea louse Caligus rogercresseyi (C. rogercresseyi) specifically responds to the delousing drugs azamethiphos and deltamethrin at the transcriptomic level by differentially activating mRNA of the NOTCH signaling pathway and of ABC genes. These results suggest that frequent antiparasitic application may increase the activity of inhibitory mRNA components, thereby promoting inhibitory NOTCH output and conditions for increased resistance to delousing drugs. Moreover, data analysis underscored that key functions of NOTCH/ABC components were regulated during distinct phases of the drug response, thus indicating resistance modifications in C. rogercresseyi resulting from the frequent use of organophosphates and pyrethroids.

Fish can show emotional fever: stress-induced hyperthermia in zebrafish.

Whether fishes are sentient beings remains an unresolved and controversial question. Among characteristics thought to reflect a low level of sentience in fishes is an inability to show stress-induced hyperthermia (SIH), a transient rise in body temperature shown in response to a variety of stressors. This is a real fever response, so is often referred to as 'emotional fever'. It has been suggested that the capacity for emotional fever evolved only in amniotes (mammals, birds and reptiles), in association with the evolution of consciousness in these groups. According to this view, lack of emotional fever in fishes reflects a lack of consciousness. We report here on a study in which six zebrafish groups with access to a temperature gradient were either left as undisturbed controls or subjected to a short period of confinement. The results were striking: compared to controls, stressed zebrafish spent significantly more time at higher temperatures, achieving an estimated rise in body temperature of about 2-4°C. Thus, zebrafish clearly have the capacity to show emotional fever. While the link between emotion and consciousness is still debated, this finding removes a key argument for lack of consciousness in fishes.

High-throughput transcriptome analysis of ISAV-infected Atlantic salmon Salmo salar unravels divergent immune responses associated to head-kidney, liver and gills tissues.

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus causing high mortality in farmed Atlantic salmon (Salmo salar). The collective data from the Atlantic salmon-ISAV interactions, performed "in vitro" using various salmon cell lines and "in vivo" fish infected with different ISAV isolates, have shown a strong regulation of immune related transcripts during the infection. Despite this strong defence response, the majority of fish succumb to infections with ISAV. The deficient protection of the host against ISAV is in part due to virulence factors of the virus, which allow evade the host-defence machinery. As such, the viral replication is uninhibited and viral loads quickly spread to several tissues causing massive cellular damage before the host can develop an effective cell-mediated and humoral outcome. To interrogate the correlation of the viral replication with the host defence response, we used fish that have been infected by cohabitation with ISAV-injected salmons. Whole gene expression patterns were measured with RNA-seq using RNA extracted from Head-kidney, Liver and Gills. The results show divergent mRNA abundance of functional modules related to interferon pathway, adaptive/innate immune response and cellular proliferation/differentiation. Furthermore, gene regulation in distinct tissues during the infection process was independently controlled within the each tissue and the observed mRNA expression suggests high modulation of the ISAV-segment transcription. Importantly this is the first time that strong correlations between functional modules containing significant immune process with protein-protein affinities and viral-segment transcription have been made between different tissues of ISAV-infected fish.

Sublethal neurotoxicity of saxitoxin in early zebrafish development: Impact on sensorimotor function and neurotransmission systems.

Saxitoxin (STX) represents a marine toxin of significant concern due to its deleterious implications for aquatic ecosystems and public food safety. As a potent paralytic agent, the role of STX in obstructing voltage-gated sodium channels (VGSCs) is well-characterized. Yet, the mechanistic details underlying its low-dose toxicity remain largely enigmatic. In the current study, zebrafish embryos and larvae were subjected to subchronic exposure of graded STX concentrations (0, 1, 10, and 100 µg/L) until the 7th day post-fertilization. A tactile stimulus-based assay was employed to evaluate potential behavioral perturbations resulting from STX exposure. Both behavioral and transcription level analyses unveiled a compromised tactile response, which was found to be associated with a notable upregulation in the mRNA of two distinct VGSC isoforms, specifically the scn8aa/ab and scn1Laa/ab transcripts, even at the minimal STX dose. Notably, exposure to this lowest STX concentration also resulted in alterations in the transcriptional patterns of pivotal genes for cholinergic and GABAergic pathways, including ache and gabra1. Furthermore, STX induced a marked decrease in the levels of the neurotransmitter GABA. Our findings underscore that prolonged low-dose STX exposure during early development can significantly compromise the tactile response behavior in zebrafish. This study reveals that chronic low-dose STX exposure of developing zebrafish alters neurotransmission pathways that converge on altered tactile behavior.

Behavioural fever is a synergic signal amplifying the innate immune response.

Behavioural fever, defined as an acute change in thermal preference driven by pathogen recognition, has been reported in a variety of invertebrates and ectothermic vertebrates. It has been suggested, but so far not confirmed, that such changes in thermal regime favour the immune response and thus promote survival. Here, we show that zebrafish display behavioural fever that acts to promote extensive and highly specific temperature-dependent changes in the brain transcriptome. The observed coupling of the immune response to fever acts at the gene-environment level to promote a robust, highly specific time-dependent anti-viral response that, under viral infection, increases survival. Fish that are not offered a choice of temperatures and that therefore cannot express behavioural fever show decreased survival under viral challenge. This phenomenon provides an underlying explanation for the varied functional responses observed during systemic fever. Given the effects of behavioural fever on survival and the fact that it exists across considerable phylogenetic space, such immunity-environment interactions are likely to be under strong positive selection.

Effect of Fish Stock Density on Hormone Genes Expression from Brain and Gastrointestinal Tract of Salmo salar.

A variety of long-term stress conditions may exist in fish cultivation, some of which are so severe that fish can no longer reestablish homeostasis. In teleost fish, the brain and gastrointestinal tract integrate signals that include the perception of stress factors regulating physiological responses, such as social stress by fish population density, where peripheral and central signals, such as peptide hormones, are the main regulators. Therefore, we proposed in this study to analyze the effect of different stock densities (SD) in the gene expression of brain neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP), together with the gastrointestinal peptide hormones leptin (Lep), vasointestinal peptide (VIP), and protachykinin-1 (Prk-1) in Salmo salar post-smolt. The coding sequence of S. salar VIP and Prk-1 precursors were firstly cloned and characterized. Then, the mRNA expression of these genes, together with the NPY, Lep, and CGRP genes, were evaluated in post-smolts kept at 11 Kg/m3, 20 Kg/m3, and 40 Kg/m3. At 14 days of culture, the brain CGRP and liver leptin mRNA levels increased three and tenfold in the post-smolt salmons kept at the highest SD, respectively. The high levels of leptin were kept during all the fish culture experiments. In addition, the highest expression of intestine VIP mRNA was obtained on Day 21 in the group of 40 Kg/m3 returning to baseline on Day 40. In terms of stress biochemical parameters, cortisol levels were increased in the 20 Kg/m3 and 40 Kg/m3 groups on Day 40 and were the highest in the 20 Kg/m3 group on Day 14. This study provides new insight into the gastrointestinal signals that could be affected by chronic stress induced by high stock density in fish farming. Thus, the expression of these peptide hormones could be used as molecular markers to improve production practices in fish aquaculture.

Divergent responses to peptidoglycans derived from different E. coli serotypes influence inflammatory outcome in trout, Oncorhynchus mykiss, macrophages.

BACKGROUND: Pathogen-associated molecular patterns (PAMPs) are structural components of pathogens such as lipopolysaccharide (LPS) and peptidoglycan (PGN) from bacterial cell walls. PAMP-recognition by the host results in an induction of defence-related genes and often the generation of an inflammatory response. We evaluated both the transcriptomic and inflammatory response in trout (O. mykiss) macrophages in primary cell culture stimulated with DAP-PGN (DAP; meso-diaminopimelic acid, PGN; peptidoglycan) from two strains of Escherichia coli (PGN-K12 and PGN-O111:B4) over time. RESULTS: Transcript profiling was assessed using function-targeted cDNA microarray hybridisation (n = 36) and results show differential responses to both PGNs that are both time and treatment dependent. Wild type E. coli (K12) generated an increase in transcript number/diversity over time whereas PGN-O111:B4 stimulation resulted in a more specific and intense response. In line with this, Gene Ontology analysis (GO) highlights a specific transcriptomic remodelling for PGN-O111:B4 whereas results obtained for PGN-K12 show a high similarity to a generalised inflammatory priming response where multiple functional classes are related to ribosome biogenesis or cellular metabolism. Prostaglandin release was induced by both PGNs and macrophages were significantly more sensitive to PGN-O111:B4 as suggested from microarray data. CONCLUSION: Responses at the level of the transcriptome and the inflammatory outcome (prostaglandin synthesis) highlight the different sensitivity of the macrophage to slight differences (serotype) in peptidoglycan structure. Such divergent responses are likely to involve differential receptor sensitivity to ligands or indeed different receptor types. Such changes in biological response will likely reflect upon pathogenicity of certain serotypes and the development of disease.

Interferon Gamma Induces the Increase of Cell-Surface Markers (CD80/86, CD83 and MHC-II) in Splenocytes From Atlantic Salmon.

Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon (Salmo salar). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was detected in IFNγ-stimulated splenocytes. Finally, the expression of IFNγ and cell-surface markers was assessed in Atlantic salmon under field conditions. In vivo results showed that the expression of ifnγ increased simultaneously with the up-regulation of cd80/86, cd83 and mhcii during a natural outbreak of Piscirickettsia salmonis. Overall, the results obtained in this study allow us to propose IFNγ as a candidate molecule to stimulate the phenotypic progression of a small population of immune cells, which will increase antigen presenting cells markers. Thereby, modulatory strategies using IFNγ may generate a robust and coordinated immune response in fish against pathogens that affect aquaculture.

Linking stress coping styles with brain mRNA abundance of selected transcripts for Senegalese sole (Solea senegalensis) juveniles.

In fish, proactive and reactive individual stress copying styles (SCS) have been used to resolve variation in molecular expression data. Stress coping styles have been previously described in several stages of Solea senegalensis by validating for the species the use of standard behavioural screening tests. The present study aimed to link behavioural SCS tests with brain transcript abundance in early Senegalese sole juveniles in order to observe the natural variation in a molecular pathway in this species. A total of 50 juveniles were subjected to three individual behavioural (Restraining, New environment and Confinement) and one group (Risk-taking) screening tests. The fish were classified in SCS categories by applying a hierarchical cluster to the variable "Total activity" (the total activity time that the fish was moving in each individual test). Three categories were defined, proactive, intermediate and reactive sole. Six transcripts were chosen and tested, one related to basic metabolism (gapdh-2), three to feeding behaviour (per1, igf-Ia, pparß) and two to the stress response (crh-BP and hsp90aa) in 30 juveniles (10 individuals per SCS category) using rt-qPCR to observe differences in the abundance of those transcripts among SCS. Four transcripts were differentially expressed (DETs) among them. The transcript gapdh-2 showed up-regulation for proactive and intermediate SCS sole while reactive individuals showed down-regulation. Target mRNAs per1, igf-Ia and pparß, showed different levels of up-regulation for proactive and reactive fish while intermediates were highly down-regulated. Surprisingly no differences in stress related transcripts were observed. Correlations were found between variation in coping styles and variation in the abundance of mRNAs involved in important biological functions in Senegalese sole. These results are the first evidence of the relationship between the behavioural individual variation and the fluctuation in brain transcripts abundance in Senegalese sole.

Characterization and expression of NADPH oxidase in LPS-, poly(I:C)- and zymosan-stimulated trout (Oncorhynchus mykiss W.) macrophages.

In vertebrates, the generation of superoxide reactive oxygen species (ROS) via activation of the Nox/Duox family of NADPH oxidases is a prototypical feature of the pathogen-induced defensive responses of activated professional phagocytes. To understand the role of the rainbow trout (Oncorhynchus mykiss) Phox oxidase from a phylogenetic and functional perspective we describe the cloning, sequencing and expression analysis of multiple NADPH components in cultured macrophages. Phylogenetic analyses support the notion of the emergence of Phox-related components before the diversification of basal euteleosts and add to the limited collection of teleost NADPH oxidases. Expression studies using lipopolysaccharide, polyinosine-polycytidylic acid and zymosan to mimic the onset of inflammatory responses in trout macrophages suggest differences in regulation of the NADPH complex throughout the maturation/differentiation period of culture and between different treatments.

Contribution of Non-canonical Cortisol Actions in the Early Modulation of Glucose Metabolism of Gilthead Sea Bream (Sparus aurata).

Teleost fish are exposed to diverse stressors in farming and wildlife conditions during their lifespan. Cortisol is the main glucocorticoid hormone involved in the regulation of their metabolic acclimation under physiological stressful conditions. In this context, increased plasma cortisol is associated with energy substrate mobilization from metabolic tissues, such as liver and skeletal muscle, to rapidly obtain energy and cope with stress. The metabolic actions of cortisol have primarily been attributed to its genomic/classic action mechanism involving the interaction with intracellular receptors, and regulation of stress-responsive genes. However, cortisol can also interact with membrane components to activate rapid signaling pathways. In this work, using the teleost fish gilthead sea bream (Sparus aurata) as a model, we evaluated the effects of membrane-initiated cortisol actions on the early modulation of glucose metabolism. For this purpose, S. aurata juveniles were intraperitoneally administrated with cortisol and with its membrane impermeable analog, cortisol-BSA. After 1 and 6 h of each treatment, plasma cortisol levels were measured, together with glucose, glycogen and lactate in plasma, liver and skeletal muscle. Transcript levels of corticosteroids receptors (gr1, gr2, and mr) and key gluconeogenesis (g6pc and pepck)- and glycolysis (pgam1 and aldo) related genes in the liver were also measured. Cortisol and cortisol-BSA administration increased plasma cortisol levels in S. aurata 1 h after administration. Plasma glucose levels enhanced 6 h after each treatment. Hepatic glycogen content decreased in the liver at 1 h of both cortisol and cortisol-BSA administration, while increased at 6 h due to cortisol but not in response to cortisol-BSA. Expression of gr1, g6pc, pgam1, and aldo were preferentially increased by cortisol-BSA in the liver. Taking all these results in consideration, we suggest that non-canonical cortisol mechanisms contribute to the regulation of the early glucose metabolism responses to stress in S. aurata.