Comparative study of stress and immune-related transcript outcomes triggered by Vibrio anguillarum bacterin and air exposure stress in liver and spleen of gilthead seabream (Sparus aurata), zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss).

The stress and immune-related effects of short-term (1, 6 and 24 h) air exposure stress (1 min), bath vaccination with Vibrio anguillarum bacterin, and both stressors combined were evaluated in liver and spleen of Sparus aurata, Danio rerio and Onchorhynchus mykiss. Expression profiles of immune (interleukin 1 beta: il1ß; tumor necrosis factor alpha: tnfα; interleukin 10: il10; tumor growth factor beta: tgfß1; immunoglobulin M: igm; lysozyme: lys; complement protein c3: c3) and stress-related genes (glucocorticoid receptor: gr; heat shock protein 70: hsp70; and enolase) were analysed by RT-qPCR. Cortisol level was assessed by radioimmunoassay. The gene expression patterns in liver and spleen were found to be differentially regulated in a time- and organ-dependent manner among species. In seabream, a higher il1ß-driven inflammatory response was recorded. In zebrafish, air exposure stress but not bath vaccination alone modulated most of the changes in liver and spleen immune transcripts. Stressed and vaccinated trout showed an intermediate pattern of gene expression, with a lower upregulation of immune-related genes in liver and the absence of changes in the expression of hsp70 and enolase in spleen (as it was observed in seabream but not in zebrafish). Following air exposure, cortisol levels increased in plasma 1 h post-stress (hps) and then decreased at 6 hps in O. mykiss and D. rerio. By contrast, in S.aurata the cortisol level remained higher at 6 hps suggesting a greater degree of responsiveness to this stressor. When fish were exposed to combined air exposure plus bath vaccination cortisol levels were also augmented at 1 and 6 hps in O. mykiss and S.aurata and restored to basal level at 24 hps, whereas in D. rerio the response was higher in response to the combination of both stressors. In addition, V. anguillarum bacterin vaccination triggered cortisol secretion only in D. rerio, suggesting a greater responsiveness of D. rerio hypothalamic-pituitary-interrenal axis. Overall, comparing the tissue transcription responsiveness, liver was found to be more implicated in the response to handling stress compared to spleen. These results also indicate that a species-specific response accounts for the deviations of stress and immune onset in the liver and spleen in these fish species.

Elucidating the Effects of the Lipids Regulators Fibrates and Statins on the Health Status of Finfish Species: A Review.

The most documented fibrates are gemfibrozil, clofibrate and bezafibrate, while for statins, the majority of the published literature focuses on atorvastatin and simvastatin. The present work reviews previously published research concerning the effects of these hypocholesterolaemic pharmaceuticals on fish, with a particular focus on commercially important species, commonly produced by the European aquaculture industry, specifically in recirculated aquaculture systems (RAS). Overall, results suggest that both acute and chronic exposures to lipid-lowering compounds may have adverse effects on fish, disrupting their capacity to excrete exogenous substances, as well as both lipid metabolism and homeostasis, causing severe ontogenetic and endocrinological abnormalities, leading to hampered reproductive success (e.g., gametogenesis, fecundity), and skeletal or muscular malformations, having serious repercussions on fish health and welfare. Nonetheless, the available literature focusing on the effects of statins or fibrates on commonly farmed fish is still limited, and further research is required to understand the implications of this matter on aquaculture production, global food security and, ultimately, human health.

Effects of Fouling Management and Net Coating Strategies on Reared Gilthead Sea Bream Juveniles.

In aquaculture, biofouling management is a difficult and expensive issue. Cuprous oxide has been commonly used to prevent fouling formation. To cheapen net management and reduce the use of copper, the industry has proposed several alternatives. Currently, polyurethane coatings are being explored and commercially implemented. With this alternative, net cleaning is done in situ, reducing the number of nets necessary to raise a batch, thus ideally reducing operational costs. This pilot study compared this new strategy to the use of cuprous oxide. The results show that nets treated with antifouling perform better and bioaccumulation of copper in fish tissues do not pose health risks to fish. Alternatives involving on-site cleaning need to improve efficiency. Although the conditions of this work are not completely comparable to commercial aquaculture conditions, the results might indicate the strengths and constrains of the solutions tested in real life.

Unveiling the effect of dietary essential oils supplementation in Sparus aurata gills and its efficiency against the infestation by Sparicotyle chrysophrii.

A microencapsulated feed additive composed by garlic, carvacrol and thymol essential oils (EOs) was evaluated regarding its protective effect in gills parasitized by Sparicotyle chrysophrii in Sparus aurata. A nutritional trial (65 days) followed by a cohabitation challenge with parasitized fish (39 days) were performed. Transcriptomic analysis by microarrays of gills of fish fed the EOs diet showed an up-regulation of genes related to biogenesis, vesicular transport and exocytosis, leukocyte-mediated immunity, oxidation-reduction and overall metabolism processes. The functional network obtained indicates a tissue-specific pro-inflammatory immune response arbitrated by degranulating acidophilic granulocytes, sustained by antioxidant and anti-inflammatory responses. The histochemical study of gills also showed an increase of carboxylate glycoproteins containing sialic acid in mucous and epithelial cells of fish fed the EOs diet, suggesting a mucosal defence mechanism through the modulation of mucin secretions. The outcomes of the in vivo challenge supported the transcriptomic results obtained from the nutritional trial, where a significant reduction of 78% in the abundance of S. chrysophrii total parasitation and a decrease in the prevalence of most parasitic developmental stages evaluated were observed in fish fed the EOs diet. These results suggest that the microencapsulation of garlic, carvacrol and thymol EOs could be considered an effective natural dietary strategy with antiparasitic properties against the ectoparasite S. chrysophrii.

Skin Multi-Omics-Based Interactome Analysis: Integrating the Tissue and Mucus Exuded Layer for a Comprehensive Understanding of the Teleost Mucosa Functionality as Model of Study.

From a general structural perspective, a mucosal tissue is constituted by two main matrices: the tissue and the secreted mucus. Jointly, they fulfill a wide range of functions including the protection of the epithelial layer. In this study, we simultaneously analyzed the epithelial tissue and the secreted mucus response using a holistic interactome-based multi-omics approach. The effect of the gilthead sea bream (Sparus aurata) skin mucosa to a dietary inclusion of spray-dried porcine plasma (SDPP) was evaluated. The epithelial skin microarrays-based transcriptome data showed 194 differentially expressed genes, meanwhile the exuded mucus proteome analysis 35 differentially synthesized proteins. Separately, the skin transcripteractome revealed an expression profile that favored biological mechanisms associated to gene expression, biogenesis, vesicle function, protein transport and localization to the membrane. Mucus proteome showed an enhanced protective role with putatively higher antioxidant and antimicrobial properties. The integrated skin mucosa multi-interactome analysis evidenced the interrelationship and synergy between the metabolism and the exuded mucus functions improving specifically the tissue development, innate defenses, and environment recognition. Histologically, the skin increased in thickness and in number of mucous cells. A positive impact on animal performance, growth and feed efficiency was also registered. Collectively, the results suggest an intimate crosstalk between skin tissue and its exuded mucus in response to the nutritional stimulus (SDPP supplementation) that favors the stimulation of cell protein turnover and the activation of the exudation machinery in the skin mucosa. Thus, the multi-omics-based interactome analysis provides a comprehensive understanding of the biological context of response that takes place in a mucosal tissue. In perspective, this strategy is applicable for evaluating the effect of any experimental variable on any mucosal tissue functionality, including the benefits this assessment may provide on the study of the mammalian mucosa.

Netting the Stress Responses in Fish.

In the last decade, the concept of animal stress has been stressed thin to accommodate the effects of short-term changes in cell and tissue physiology, major behavioral syndromes in individuals and ecological disturbances in populations. Seyle's definition of stress as "the nonspecific (common) result of any demand upon the body" now encompasses homeostasis in a broader sense, including all the hierarchical levels in a networked biological system. The heterogeneity of stress responses thus varies within individuals, and stressors become multimodal in terms of typology, source and effects, as well as the responses that each individual elicits to cope with the disturbance. In fish, the time course of changes after stress strongly depends on several factors, including the stressful experiences in early life, the vertical transmission of stressful-prone phenotypes, the degree of individual phenotypic plasticity, the robustness and variety of the epigenetic network related to environmentally induced changes, and the intrinsic behavioral responses (individuality/personality) of each individual. The hierarchical heterogeneity of stress responses demands a code that may decrypt and simplify the analysis of both proximate and evolutionary causes of a particular stress phenotype. We propose an analytical framework, the stressotope, defined as an adaptive scenario dominated by common environmental selective pressures that elicit common multilevel acute stress-induced responses and produce a measurable allostatic load in the organism. The stressotope may constitute a blueprint of embedded interactions between stress-related variations in cell states, molecular mediators and systemic networks, a map of circuits that reflect the inherited and acquired stress responses in an ever-changing, microorganismal-loaded medium. Several features of the proposed model are discussed as a starting point to pin down the maximum common stress responses across immune-neuroendocrine relevant physiological levels and scenarios, including the characterization of behavioral responses, in fish.

Functional evidence for the inflammatory reflex in teleosts: A novel α7 nicotinic acetylcholine receptor modulates the macrophage response to dsRNA.

The inflammatory reflex modulates the innate immune system, keeping in check the detrimental consequences of overstimulation. A key player controlling the inflammatory reflex is the alpha 7 acetylcholine receptor (α7nAChR). This receptor is one of the signalling molecules regulating cytokine expression in macrophages. In this study, we characterize a novel teleost α7nAChR. Protein sequence analysis shows a high degree of conservation with mammalian orthologs and trout α7nAChR has all the features and essential amino acids to form a fully functional receptor. We demonstrate that trout macrophages can bind α-bungarotoxin (α-BTX), a competitive antagonist for α7nAChRs. Moreover, nicotine stimulation produces a decrease in pro-inflammatory cytokine expression after stimulation with poly(I:C). These results suggest the presence of a functional α7nAChR in the macrophage plasma membrane. Further, in vivo injection of poly(I:C) induced an increase in serum ACh levels in rainbow trout. Our results manifest for the first time the functional conservation of the inflammatory reflex in teleosts.

Comparative Immune- and Stress-Related Transcript Response Induced by Air Exposure and Vibrio anguillarum Bacterin in Rainbow Trout (Oncorhynchus mykiss) and Gilthead Seabream (Sparus aurata) Mucosal Surfaces.

Fish have to face various environmental challenges that may compromise the efficacy of the immune response in mucosal surfaces. Since the effect of acute stress on mucosal barriers in fish has still not been fully elucidated, we aimed to compare the short-term mucosal stress and immune transcriptomic responses in a freshwater (rainbow trout, Oncorhynchus mykiss) and a marine fish (gilthead seabream, Sparus aurata) to bacterial immersion (Vibrio anguillarum bacterin vaccine) and air exposure stress in skin, gills, and intestine. Air exposure and combined (vaccine + air) stressors exposure were found to be inducers of the cortisol secretion in plasma and skin mucus on both species in a time-dependent manner, while V. anguillarum bacterin exposure induced cortisol release in trout skin mucus only. This was coincident with a marked differential increase in transcriptomic patterns of stress- and immune-related gene expression profiles. Particularly in seabream skin, the expression of cytokines was markedly enhanced, whereas in gills the response was mainly suppressed. In rainbow trout gut, both air exposure and vaccine stimulated the transcriptomic response, whereas in seabream, stress and immune responses were mainly induced by air exposure. Therefore, our comparative survey on the transcriptomic mucosal responses demonstrates that skin and gut were generally more reactive in both species. However, the upregulation of immune transcripts was more pronounced in gills and gut of vaccinated trout, whereas seabream appeared to be more stress-prone and less responsive to V. anguillarum bacterin in gills and gut. When fish were subjected to both treatments no definite pattern was observed. Overall, the results indicate that (1) the immune response was not homogeneous among mucosae (2), it was greatly influenced by the specific traits of each stressor in each surface and (3) was highly species-specific, probably as a result of the adaptive life story of each species to the microbial load and environmental characteristics of their respective natural habitats.

RNA-Seq reveals an integrated immune response in nucleated erythrocytes.

BACKGROUND: Throughout the primary literature and within textbooks, the erythrocyte has been tacitly accepted to have maintained a unique physiological role; namely gas transport and exchange. In non-mammalian vertebrates, nucleated erythrocytes are present in circulation throughout the life cycle and a fragmented series of observations in mammals support a potential role in non-respiratory biological processes. We hypothesised that nucleated erythrocytes could actively participate via ligand-induced transcriptional re-programming in the immune response. METHODOLOGY/PRINCIPAL FINDINGS: Nucleated erythrocytes from both fish and birds express and regulate specific pattern recognition receptor (PRR) mRNAs and, thus, are capable of specific pathogen associated molecular pattern (PAMP) detection that is central to the innate immune response. In vitro challenge with diverse PAMPs led to de novo specific mRNA synthesis of both receptors and response factors including interferon-alpha (IFNα) that exhibit a stimulus-specific polysomal shift supporting active translation. RNA-Seq analysis of the PAMP (Poly (I:C), polyinosinic:polycytidylic acid)-erythrocyte response uncovered diverse cohorts of differentially expressed mRNA transcripts related to multiple physiological systems including the endocrine, reproductive and immune. Moreover, erythrocyte-derived conditioned mediums induced a type-1 interferon response in macrophages thus supporting an integrative role for the erythrocytes in the immune response. CONCLUSIONS/SIGNIFICANCE: We demonstrate that nucleated erythrocytes in non-mammalian vertebrates spanning significant phylogenetic distance participate in the immune response. RNA-Seq studies highlight a mRNA repertoire that suggests a previously unrecognized integrative role for the erythrocytes in other physiological systems.

The effects of immunostimulation through dietary manipulation in the rainbow trout; evaluation of mucosal immunity.

Immunostimulant-containing diets are commonly used in aquaculture to enhance the resistance of cultured fish to disease and stress. Although widespread in use, there have been conflicting results published, and surprisingly little is known about the regulation of immune response-related genes in tissues key to mucosal immunity induced by immunostimulant dietary feeding. Using a salmonid-specific microarray platform enriched with immune-related genes and in situ hybridization, we investigated dietary acclimation in two organs relevant to mucosal immunity, the gills and the intestine, in the rainbow trout (Oncorhynchus mykiss). Immunostimulant diets significantly changed gene expression profiles and gene distribution in a tissue-specific manner: genes and functional Gene Ontology categories involved in immunity were differently expressed at portals of entry where significant changes in genes and functional groups related to remodeling processes and antigen presentation were observed. Furthermore, genes involved in chemotaxis, cell differentiation, antigen-presenting capacity and tissue remodeling were localized in both organs.

Characterization and expression of the transcription factor PU.1 during LPS-induced inflammation in the rainbow trout (Oncorhynchus mykiss).

The transcription factor PU.1 plays a key role in hematopoietic lineage development and therefore in determining immune cell fate. A full length cDNA transcript of 1237 nucleotides encoding a highly conserved putative protein of 293 amino acids was identified by EST analysis in lipopolysaccharide (LPS) activated trout macrophages. Phylogenetic analyses highlight the significant level of structural conservation of the PU.1 transcription factor reinforcing the importance of this molecule in animal immunity. In trout, the PU.1 mRNA shows a tissue-specific expression pattern and is induced in vivo by LPS in muscle, liver, intestine and brain. Furthermore PU.1 is highly expressed in trout macrophages in primary culture. In situ expression analysis in the head kidney describes a large number of PU.1+ve cells distributed through the tissue in both LPS-treated and control animals. Cellular proliferation examined by BrdU immunohistochemistry (IHC) shows that LPS regulates hematopoietic processes in adult fish by stimulating cellular proliferation 3 days after treatment. These studies provide initial insights into hematopoietic/cellular processes in the head kidney of rainbow trout after in vivo LPS challenge.