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We assessed effects of feed restriction and inclusion of 200 g kg−1 extracted soybean meal in the diet on gene expression in Atlantic salmon using a cDNA microarray (SFA2.0) and real-time qPCR. The trial lasted for 54 days. Restricted feeding and soybean inclusion reduced the thermal growth coefficient by respectively 51% and 22% compared with fish fed with the fishmeal-based control diet to satiation. Soybean decreased distal intestinal expression of lysosomal (cathepsins C, D, L, Y and Z) and extracellular proteases while genes involved in responses to cellular stress were up-regulated. Expression changes of immune genes suggested both pro- and anti-inflammatory regulation. The hepatic responses to soybean and restricted feeding were highly similar, which could be because of negative effects of soybean meal on digestion and nutrient absorption. We observed up-regulation of ribosomal proteins and down-regulation of genes involved in lipid and steroid metabolism. Of note, growth reduction in both study groups was associated with coordinated down-regulation of genes involved in oxidative and cellular stress responses, metabolism of xenobiotics and protein degradation. High expression of stress-related genes in salmon fed with the control diet suggests that maximum growth rates can be associated with health problems. [ABSTRACT FROM AUTHOR]

Abstract Background Use of plant ingredients in aquaculture feeds is impeded by high contents of antinutritional factors such as saponins, which may cause various pharmacological and biological effects. In this study, transcriptome changes were analyzed using a 21 k oligonucleotide microarray and qPCR in the distal intestine of Atlantic salmon fed diets based on five plant protein sources combined with soybean saponins. Results Diets with corn gluten, sunflower, rapeseed or horsebean produced minor effects while the combination of saponins with pea protein concentrate caused enteritis and major transcriptome changes. Acute inflammation was characterised by up-regulation of cytokines, NFkB and TNFalpha related genes and regulators of T-cell function, while the IFN-axis was suppressed. Induction of lectins, complement, metalloproteinases and the respiratory burst complex parallelled a down-regulation of genes for free radical scavengers and iron binding proteins. Marked down-regulation of xenobiotic metabolism was also observed, possibly increasing vulnerability of the intestinal tissue. A hallmark of metabolic changes was dramatic down-regulation of lipid, bile and steroid metabolism. Impairment of digestion was further suggested by expression changes of nutrient transporters and regulators of water balance (e.g. aquaporin, guanylin). On the other hand, microarray profiling revealed activation of multiple mucosal defence processes. Annexin-1, with important anti-inflammatory and gastroprotective properties, was markedly up-regulated. Furthermore, augmented synthesis of polyamines needed for cellular proliferation (up-regulation of arginase and ornithine decarboxylase) and increased mucus production (down-regulation of glycan turnover and goblet cell hyperplasia) could participate in mucosal healing and restoration of normal tissue function. Conclusion The current study promoted understanding of salmon intestinal pathology and establishment of a model for feed induced enteritis. Multiple gene expression profiling further characterised the inflammation and described the intestinal pathology at the molecular level. Ethical approval The present experiment was approved by the Norwegian Animal Research Authority and conducted according to prevailing animal welfare regulations: FOR-1996-01-15-23 (Norway), European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Strasbourg, 18.III.1986) and COUNCIL DIRECTIVE of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes (86/609/EEC).

Abstract Background The salmon louse is an ectoparasitic copepod that causes major economic losses in the aquaculture industry of Atlantic salmon. This host displays a high level of susceptibility to lice which can be accounted for by several factors including stress. In addition, the parasite itself acts as a potent stressor of the host, and outcomes of infection can depend on biotic and abiotic factors that stimulate production of cortisol. Consequently, examination of responses to infection with this parasite, in addition to stress hormone regulation in Atlantic salmon, is vital for better understanding of the host pathogen interaction. Results Atlantic salmon post smolts were organised into four experimental groups: lice + cortisol, lice + placebo, no lice + cortisol, no lice + placebo. Infection levels were equal in both treatments upon termination of the experiment. Gene expression changes in skin were assessed with 21 k oligonucleotide microarray and qPCR at the chalimus stage 18 days post infection at 9°C. The transcriptomic effects of hormone treatment were significantly greater than lice-infection induced changes. Cortisol stimulated expression of genes involved in metabolism of steroids and amino acids, chaperones, responses to oxidative stress and eicosanoid metabolism and suppressed genes related to antigen presentation, B and T cells, antiviral and inflammatory responses. Cortisol and lice equally down-regulated a large panel of motor proteins that can be important for wound contraction. Cortisol also suppressed multiple genes involved in wound healing, parts of which were activated by the parasite. Down-regulation of collagens and other structural proteins was in parallel with the induction of proteinases that degrade extracellular matrix (MMP9 and MMP13). Cortisol reduced expression of genes encoding proteins involved in formation of various tissue structures, regulators of cell differentiation and growth factors. Conclusions These results suggest that cortisol-induced stress does not affect the level of infection of Atlantic salmon with the parasite, however, it may retard repair of skin. The cortisol induced changes are in close concordance with the existing concept of wound healing cascade.

Abstract Background The salmon louse (Lepeophtheirus salmonis Krøyer), an ectoparasitic copepod with a complex life cycle causes significant losses in salmon aquaculture. Pesticide treatments against the parasite raise environmental concerns and their efficacy is gradually decreasing. Improvement of fish resistance to lice, through biological control methods, needs better understanding of the protective mechanisms. We used a 21 k oligonucleotide microarray and RT-qPCR to examine the time-course of immune gene expression changes in salmon skin, spleen, and head kidney during the first 15 days after challenge, which encompassed the copepod and chalimus stages of lice development. Results Large scale and highly complex transcriptome responses were found already one day after infection (dpi). Many genes showed bi-phasic expression profiles with abrupt changes between 5 and 10 dpi (the copepod-chalimus transitions); the greatest fluctuations (up- and down-regulation) were seen in a large group of secretory splenic proteases with unknown roles. Rapid sensing was witnessed with induction of genes involved in innate immunity including lectins and enzymes of eicosanoid metabolism in skin and acute phase proteins in spleen. Transient (1-5 dpi) increase of T-cell receptor alpha, CD4-1, and possible regulators of lymphocyte differentiation suggested recruitment of T-cells of unidentified lineage to the skin. After 5 dpi the magnitude of transcriptomic responses decreased markedly in skin. Up-regulation of matrix metalloproteinases in all studied organs suggested establishment of a chronic inflammatory status. Up-regulation of putative lymphocyte G0/G1 switch proteins in spleen at 5 dpi, immunoglobulins at 15 dpi; and increase of IgM and IgT transcripts in skin indicated an onset of adaptive humoral immune responses, whereas MHCI appeared to be down-regulated. Conclusions Atlantic salmon develops rapid local and systemic reactions to L. salmonis, which, however, do not result in substantial level of protection. The dramatic changes observed after 5 dpi can be associated with metamorphosis of copepod, immune modulation by the parasite, or transition from innate to adaptive immune responses.

Abstract Background Excessive fat deposition is one of the largest problems faced by salmon aquaculture industries, leading to production losses due to high volume of adipose tissue offal. In addition, increased lipid accumulation may impose considerable stress on adipocytes leading to adipocyte activation and production and secretion of inflammatory mediators, as observed in mammals. Results Microarray and qPCR analyses were performed to follow transcriptome changes during adipogenesis in the primary culture of adipose stromo-vascular fraction (aSVF) of Atlantic salmon. Cellular heterogeneity decreased by confluence as evidenced by the down-regulation of markers of osteo/chondrogenic, myogenic, immune and vasculature lineages. Transgelin (TAGLN), a marker of the multipotent pericyte, was prominently expressed around confluence while adipogenic PPARγ was up-regulated already in subconfluent cells. Proliferative activity and subsequent cell cycle arrest were reflected in the fluctuations of pro- and anti-mitotic regulators. Marked regulation of genes involved in lipid and glucose metabolism and pathways producing NADPH and glycerol-3-phosphate (G3P) was seen during the terminal differentiation, also characterised by diverse stress responses. Activation of the glutathione and thioredoxin antioxidant systems and changes in the iron metabolism suggested the need for protection against oxidative stress. Signs of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) occured in parallel with the increased lipid droplet (LD) formation and production of secretory proteins (adipsin, visfatin). The UPR markers XBP1 and ATF6 were induced together with genes involved in ubiquitin-proteasome and lysosomal proteolysis. Concurrently, translation was suppressed as evidenced by the down-regulation of genes encoding elongation factors and components of the ribosomal machinery. Notably, expression changes of a panel of genes that belong to different immune pathways were seen throughout adipogenesis. The induction of AP1 (Jun, Fos), which is a master regulator of stress responses, culminated by the end of adipogenesis, concurrent with the maximal observed lipid deposition. Conclusions Our data point to an intimate relationship between metabolic regulation and immune responses in white adipocytes of a cold-blooded vertebrate. Stress imposed on adipocytes by LD formation and expansion is prominently reflected in the ER compartment and the activated UPR response could have an important role at visceral obesity in fish.

Abstract Background Furunculosis, a disease caused with gram negative bacteria Aeromonas salmonicida produces heavy losses in aquaculture. Vaccination against furunculosis reduces mortality of Atlantic salmon but fails to eradicate infection. Factors that determine high individual variation of vaccination efficiency remain unknown. We used gene expression analyses to search for the correlates of vaccine protection against furunculosis in Atlantic salmon. Results Naïve and vaccinated fish were challenged by co-habitance. Fish with symptoms of furunculosis at the onset of mass mortality (LR - low resistance) and survivors (HR - high resistance) were sampled. Hepatic gene expression was analyzed with microarray (SFA2.0 - immunochip) and real-time qPCR. Comparison of LR and HR indicated changes associated with the protection and results obtained with naïve fish were used to find and filter the vaccine-independent responses. Genes involved in recruitment and migration of immune cells changed expression in both directions with greater magnitude in LR. Induction of the regulators of immune responses was either equal (NFkB) or greater (Jun) in LR. Expression levels of proteasome components and extracellular proteases were higher in LR while protease inhibitors were up-regulated in HR. Differences in chaperones and protein adaptors, scavengers of reactive oxygen species and genes for proteins of iron metabolism suggested cellular and oxidative stress in LR. Reduced levels of free iron and heme can be predicted in LR by gene expression profiles with no protection against pathogen. The level of complement regulation was greater in HR, which showed up-regulation of the components of membrane attack complex and the complement proteins that protect the host against the auto-immune damages. HR fish was also characterized with up-regulation of genes for proteins involved in the protection of extracellular matrix, lipid metabolism and clearance of endogenous and exogenous toxic compounds. A number of genes with marked expression difference between HR and LR can be considered as positive and negative correlates of vaccine protection against furunculosis. Conclusion Efficiency of vaccination against furunculosis depends largely on the ability of host to neutralize the negative impacts of immune responses combined with efficient clearance and prevention of tissue damages.

Abstract Background The salmon louse (SL) is an ectoparasitic caligid crustacean infecting salmonid fishes in the marine environment. SL represents one of the major challenges for farming of salmonids, and veterinary intervention is necessary to combat infection. This study addressed gene expression responses of Atlantic salmon infected with SL, which may account for its high susceptibility. Results The effects of SL infection on gene expression in Atlantic salmon were studied throughout the infection period from copepodids at 3 days post infection (dpi) to adult lice (33 dpi). Gene expression was analyzed at three developmental stages in damaged and intact skin, spleen, head kidney and liver, using real-time qPCR and a salmonid cDNA microarray (SFA2). Rapid detection of parasites was indicated by the up-regulation of immunoglobulins in the spleen and head kidney and IL-1 receptor type 1, CD4, beta-2-microglobulin, IL-12β, CD8α and arginase 1 in the intact skin of infected fish. Most immune responses decreased at 22 dpi, however, a second activation was observed at 33 dpi. The observed pattern of gene expression in damaged skin suggested the development of inflammation with signs of Th2-like responses. Involvement of T cells in responses to SL was witnessed with up-regulation of CD4, CD8α and programmed death ligand 1. Signs of hyporesponsive immune cells were seen. Cellular stress was prevalent in damaged skin as seen by highly significant up-regulation of heat shock proteins, other chaperones and mitochondrial proteins. Induction of the major components of extracellular matrix, TGF-β and IL-10 was observed only at the adult stage of SL. Taken together with up-regulation of matrix metalloproteinases (MMP), this classifies the wounds afflicted by SL as chronic. Overall, the gene expression changes suggest a combination of chronic stress, impaired healing and immunomodulation. Steady increase of MMP expression in all tissues except liver was a remarkable feature of SL infected fish. Conclusion SL infection in Atlantic salmon is associated with a rapid induction of mixed inflammatory responses, followed by a period of hyporesponsiveness and delayed healing of injuries. Persistent infection may lead to compromised host immunity and tissue self-destruction.

Disease and parasitism cause major welfare, environmental and economic concerns for global aquaculture. In this review, we examine the status and potential of technologies that exploit genetic variation in host resistance to tackle this problem. We argue that there is an urgent need to improve understanding of the genetic mechanisms involved, leading to the development of tools that can be applied to boost host resistance and reduce the disease burden. We draw on two pressing global disease problems as case studies-sea lice infestations in salmonids and white spot syndrome in shrimp. We review how the latest genetic technologies can be capitalised upon to determine the mechanisms underlying inter- and intra-species variation in pathogen/parasite resistance, and how the derived knowledge could be applied to boost disease resistance using selective breeding, gene editing and/or with targeted feed treatments and vaccines. Gene editing brings novel opportunities, but also implementation and dissemination challenges, and necessitates new protocols to integrate the technology into aquaculture breeding programmes. There is also an ongoing need to minimise risks of disease agents evolving to overcome genetic improvements to host resistance, and insights from epidemiological and evolutionary models of pathogen infestation in wild and cultured host populations are explored. Ethical issues around the different approaches for achieving genetic resistance are discussed. Application of genetic technologies and approaches has potential to improve fundamental knowledge of mechanisms affecting genetic resistance and provide effective pathways for implementation that could lead to more resistant aquaculture stocks, transforming global aquaculture., (© 2022 The Authors. Reviews in Aquaculture published by John Wiley & Sons Australia, Ltd.)

Lepeophtheirus salmonis (sea lice) and bacterial co-infection threatens wild and farmed Atlantic salmon performance and welfare. In the present study, pre-adult L. salmonis -infected and non-infected salmon were intraperitoneally injected with either formalin-killed Aeromonas salmonicida bacterin (ASAL) or phosphate-buffered saline (PBS). Dorsal skin samples from each injection/infection group (PBS/no lice, PBS/lice, ASAL/no lice, and ASAL/lice) were collected at 24 h post-injection and used for transcriptome profiling using a 44K salmonid microarray platform. Microarray results showed no clear inflammation gene expression signatures and revealed extensive gene repression effects by pre-adult lice (2,189 down and 345 up-regulated probes) in the PBS-injected salmon (PBS/lice vs . PBS/no lice), which involved basic cellular (e.g., RNA and protein metabolism) processes. Lice repressive effects were not observed within the group of ASAL-injected salmon (ASAL/lice vs . ASAL/no lice); on the contrary, the observed skin transcriptome changes -albeit of lesser magnitude (82 up and 1 down-regulated probes)- suggested the activation in key immune and wound healing processes (e.g., neutrophil degranulation, keratinocyte differentiation). The molecular skin response to ASAL was more intense in the lice-infected (ASAL/lice vs . PBS/lice; 272 up and 11 down-regulated probes) than in the non-infected fish (ASAL/no lice vs . PBS/no lice; 27 up-regulated probes). Regardless of lice infection, the skin's response to ASAL was characterized by the putative activation of both antibacterial and wound healing pathways. The transcriptomic changes prompted by ASAL+lice co-stimulation (ASAL/lice vs . PBS/no lice; 1878 up and 3120 down-regulated probes) confirmed partial mitigation of lice repressive effects on fundamental cellular processes and the activation of pathways involved in innate (e.g., neutrophil degranulation) and adaptive immunity (e.g., antibody formation), as well as endothelial cell migration. The qPCR analyses evidenced immune-relevant genes co-stimulated by ASAL and lice in an additive (e.g., mbl2b , bcl6 ) and synergistic (e.g., hampa , il4r ) manner. These results provided insight on the physiological response of the skin of L. salmonis -infected salmon 24 h after ASAL stimulation, which revealed immunostimulatory properties by the bacterin with potential applications in anti-lice treatments for aquaculture. As a simulated co-infection model, the present study also serves as a source of candidate gene biomarkers for sea lice and bacterial co-infection., Competing Interests: RT and SS are former and current employees of Cargill Inc., respectively, but did not participate in the microarray/qPCR study design, the result interpretation, and the decision to submit the manuscript for publication. NU participated in this study as a postdoctoral fellow at Memorial University but was recently employed by Aquabounty Canada Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Caballero-Solares, Umasuthan, Xue, Katan, Kumar, Westcott, Chen, Fast, Skugor, Taylor and Rise.)

Salmonid rickettsial septicemia (SRS), caused by Piscirickettsia salmonis , is one of the most devastating diseases of salmonids. However, the transcriptomic responses of Atlantic salmon ( Salmon salar ) in freshwater to an EM-90-like isolate have not been explored. Here, we infected Atlantic salmon parr with an EM-90-like isolate and conducted time-course qPCR analyses of pathogen load and four biomarkers ( campb , hampa , il8a , tlr5a ) of innate immunity on the head kidney samples. Transcript expression of three of these genes (except hampa ), as well as pathogen level, peaked at 21 days post-injection (DPI). Multivariate analyses of infected individuals at 21 DPI revealed two infection phenotypes [lower (L-SRS) and higher (H-SRS) infection level]. Five fish from each group (Control, L-SRS, and H-SRS) were selected for transcriptome profiling using a 44K salmonid microarray platform. We identified 1,636 and 3,076 differentially expressed probes (DEPs) in the L-SRS and H-SRS groups compared with the control group, respectively (FDR = 1%). Gene ontology term enrichment analyses of SRS-responsive genes revealed the activation of a large number of innate (e.g. "phagocytosis", "defense response to bacterium", "inflammatory response") and adaptive (e.g. "regulation of T cell activation", "antigen processing and presentation of exogenous antigen") immune processes, while a small number of general physiological processes (e.g. "apoptotic process", development and metabolism relevant) was enriched. Transcriptome results were confirmed by qPCR analyses of 42 microarray-identified transcripts. Furthermore, the comparison of individuals with differing levels of infection (H-SRS vs. L-SRS) generated insights into the biological processes possibly involved in disease resistance or susceptibility. This study demonstrated a low mortality (~30%) EM-90-like infection model and broadened the current understanding of molecular pathways underlying P. salmonis -triggered responses of Atlantic salmon, identifying biomarkers that may assist to diagnose and combat this pathogen., Competing Interests: Authors EJ, SS, CH and RGT were employed by company Cargill Incorporated. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from EWOS Innovation (now part of Cargill, Incorporated). Authors EJ, SS, CH, and RGT, in the representation of Cargill, Incorporated, participated in the design of infection trial, sample collection, data interpretation, and manuscript preparation. However, they had no role in the design, data collection and analysis of gene expression experiments, and the decision to submit the manuscript for publication., (Copyright © 2021 Xue, Caballero-Solares, Hall, Umasuthan, Kumar, Jakob, Skugor, Hawes, Santander, Taylor and Rise.)

Background: Vertebrate hosts limit the availability of iron to microbial pathogens in order to nutritionally starve the invaders. The impact of iron deficiency induced by the iron chelator deferoxamine mesylate (DFO) was investigated in Atlantic salmon SHK-1 cells infected with the facultative intracellular bacterium Piscirickettsia salmonis., Results: Effects of the DFO treatment and P. salmonis on SHK-1 cells were gaged by assessing cytopathic effects, bacterial load and activity, and gene expression profiles of eight immune biomarkers at 4- and 7-days post infection (dpi) in the control group, groups receiving single treatments (DFO or P. salmonis) and their combination. The chelator appears to be well-tolerated by host cells, while it had a negative impact on the number of bacterial cells and associated cytotoxicity. DFO alone had minor effects on gene expression of SHK-1 cells, including an early activation of IL-1β at 4 dpi. In contrast to few moderate changes induced by single treatments (either infection or chelator), most genes had highest upregulation in the infected groups receiving DFO. The mildest induction of hepcidin-1 (antimicrobial peptide precursor and regulator of iron homeostasis) was observed in cells exposed to DFO alone, followed by P. salmonis infected cells while the addition of DFO to infected cells further increased the mRNA abundance of this gene. Transcripts encoding TNF-α (immune signaling) and iNOS (immune effector) showed sustained increase at both time points in this group while cathelicidin-1 (immune effector) and IL-8 (immune signaling) were upregulated at 7 dpi. The stimulation of protective gene responses seen in infected cultures supplemented with DFO coincided with the reduction of bacterial load and activity (judged by the expression of P. salmonis 16S rRNA), and damage to cultured host cells., Conclusion: The absence of immune gene activation under normal iron conditions suggests modulation of host responses by P. salmonis. The negative effect of iron deficiency on bacteria likely allowed host cells to respond in a more protective manner to the infection, further decreasing its progression. Presented findings encourage in vivo exploration of iron chelators as a promising strategy against piscirickettsiosis.

Parasitic sea lice (e.g., Lepeophtheirus salmonis < 0.0001). Most immune/defense-relevant transcripts were downregulated in both ATT and ADJ sites compared to PRE, suggesting that chalimus exerts immunosuppressive effects in the salmon's fins. The comparison between ATT and ADJ sites demonstrated the upregulation of a suite of immune-relevant transcripts, evidencing the salmon's attempt to mount an anti-lice response. We hypothesize that an imbalance between immunomodulation caused by chalimus during the early phase of infection and weak defense response manifested by Atlantic salmon makes it a susceptible host for p < 0.0001). Most immune/defense-relevant transcripts were downregulated in both ATT and ADJ sites compared to PRE, suggesting that chalimus exerts immunosuppressive effects in the salmon's fins. The comparison between ATT and ADJ sites demonstrated the upregulation of a suite of immune-relevant transcripts, evidencing the salmon's attempt to mount an anti-lice response. We hypothesize that an imbalance between immunomodulation caused by chalimus during the early phase of infection and weak defense response manifested by Atlantic salmon makes it a susceptible host for L. salmonis ., Competing Interests: Authors S.S. and R.G.T. in the representation of Cargill Innovation, participated in the data interpretation and discussion, but had no role in the design of the microarray experiment and transcript expression experiment, the data collection and analysis, the preparation of this manuscript, and the decision to submit the manuscript for publication. All authors declare no conflict of interest.

Background: Reduction of Lepeophtheirus salmonis infection in Atlantic salmon achieved by glucosinolates (GLs) from Brassica plants was recently reported. However, wider application of functional feeds based on GLs requires better knowledge of their positive and adverse effects., Methods: Liver, distal kidney and muscle transcriptomes of salmon exposed to the extreme dose of GLs were profiled by microarray, while qPCR analysis followed up selected hepatic and renal responses under the extreme and moderate GLs dose during the L. salmonis challenge. Transcriptional analysis were complemented with measurements of organ indices, liver steatosis and plasma profiling, including indicators of cytolysis and bilirubin. Finally, the third trial was performed to quantify the effect of lower GLs doses on growth., Results: The extreme GLs dose caused a decrease in hepatic fat deposition and growth, in line with microarray findings, which suggested tissue remodeling and reduction of cellular proliferation in the skeletal muscle and liver. Lower GLs inclusion levels in a follow-up trial did not show negative effects on growth. Microarray analysis of the distal kidney pointed to activation of anti-fibrotic responses under the overexposure. However, analyses of ALT, CK and AST enzymes in plasma provided no evidence of increased cytolysis and organ damage. Prevalent activation of phase-2 detoxification genes that occurred in all three tissues could be considered part of beneficial effects caused by the extreme dose of GLs. In addition, transcriptomic evidence suggested GLs-mediated iron and heme withdrawal response, including increased heme degradation in muscle (upregulation of heme oxygenase-1), decrease of its synthesis in liver (downregulation of porphobilinogen deaminase) and increased iron sequestration from blood (hepatic induction of hepcidin-1 and renal induction of intracellular storage protein ferritin). This response could be advantageous for salmon upon encountering lice, which depend on the host for the provision of iron carrying heme. Most of the hepatic genes studied by qPCR showed similar expression levels in fish exposed to GLs, lice and their combination, while renal induction of leptin suggested heightened stress by the combination of extreme dose of GLs and lice. High expression of interferon γ (cytokine considered organ-protective in mammalian kidney) was detected at the moderate GLs level. This fish also showed highest plasma bilirubin levels (degradation product of heme), and had lowest number of attached lice, further supporting hypothesis that making heme unavailable to lice could be part of an effective anti-parasitic strategy., Conclusions: Modulation of detoxification and iron metabolism in Atlantic salmon tissues could be beneficial prior and during lice infestations. Investigation of anti-lice functional feeds based on low and moderate GLs inclusion levels thus deserves further attention.

Background: The use of phytochemicals is a promising solution in biological control against salmon louse (Lepeophtheirus salmonis). Glucosinolates belong to a diverse group of compounds used as protection against herbivores by plants in the family Brassicaceae, while in vertebrates, ingested glucosinolates exert health-promoting effects due to their antioxidant and detoxifying properties as well as effects on cell proliferation and growth. The aim of this study was to investigate if Atlantic salmon fed two different doses of glucosinolate-enriched feeds would be protected against lice infection. The effects of feeding high dose of glucosinolates before the infection, and of high and low doses five weeks into the infection were studied., Methods: Skin was screened by 15 k oligonucleotide microarray and qPCR., Results: A 25 % reduction (P < 0.05) in lice counts was obtained in the low dose group and a 17 % reduction in the high dose group compared to fish fed control feed. Microarray analysis revealed induction of over 50 interferon (IFN)-related genes prior to lice infection. Genes upregulated five weeks into the infection in glucosinolate-enriched dietary groups included Type 1 pro-inflammatory factors, antimicrobial and acute phase proteins, extracellular matrix remodeling proteases and iron homeostasis regulators. In contrast, genes involved in muscle contraction, lipid and glucose metabolism were found more highly expressed in the skin of infected control fish., Conclusions: Atlantic salmon fed glucosinolates had a significantly lower number of sea lice at the end of the experimental challenge. Feeding glucosinolates coincided with increased expression of IFN-related genes, and higher expression profiles of Type 1 immune genes late into the infection. In addition, regulation of genes involved in the metabolism of iron, lipid and sugar suggested an interplay between metabolism of nutrients and mechanisms of resistance.

Atlantic salmon is susceptible to the salmon louse (Lepeophtheirus salmonis) and the variation in susceptibility within the species can be exploited in selective breeding programs for louse resistant fish. In this study, lice counts were completed on 3000 siblings from 150 families of Atlantic salmon identified as high resistant (HR) and low resistant (LR) families in two independent challenge trials. Skin samples behind the dorsal fin (nearby lice attachment) were collected from ten extreme families (HR or LR) and analyzed by qPCR for the expression of 32 selected genes, including a number of genes involved in T helper cell (Th) mediated immune responses, which have been previously implied to play important roles during salmon louse infections. Most genes showed lower expression patterns in the LR than in HR fish, suggesting an immunosuppressed state in LR families. The average number of lice (chalimi) was 9 in HR and 15 in LR fish. Large variation in lice counts was seen both within resistant and susceptible families, which enabled us to subdivide the groups into HR < 10 and HR > 10, and LR < 10 and LR > 10 to better understand the effect of lice burden per se. As expected, expression patterns were influenced both by genetic background and the number of attached parasites. Higher number of lice (>10) negatively affected gene expression in both HR and LR families. In general, strongest down-regulation was seen in LR > 10 and lesser down-regulation in HR < 10. HR in general and especially HR < 10 fish were better at resisting suppression of expression of both Th1 and Th2 genes. However, the best inverse correlation with infection level was seen for the prototypical Th1 genes, including several members from the interferon pathways. In addition, skin histomorphometry suggests that infected LR salmon had thicker epidermis in the area behind the dorsal fin and larger mucous cell size compared to infected HR fish, however marginally significant (p = 0.08). This histomorphometric finding was in line with the immune response being skewed in LR towards the Th2 rather than a Th1 profile. Our findings suggest that the ability to resist lice infection depends on the ability to avoid immunosuppression and not as much on the physical tissue barrier functions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The crustacean ectoparasitic salmon louse (Lepeophtheirus salmonis) is a major problem of Atlantic salmon aquaculture in the Northern hemisphere. Host-pathogen interactions in this system are highly complex. Resistance to the parasite involves variations in genetic background, nutrition, properties of skin, and status of the endocrine and immune systems. This study addressed the relationship between sex hormones and lice infection. Field observation revealed a sharp reduction of lice prevalence during sexual maturation with no difference between male and female fish. To determine if higher resistance against lice was related to sex hormones, post-smolt salmon were administered control feed and feeds containing 17β-estradiol (20 mg/kg) and testosterone (25 mg/kg) during a 3-week pre-challenge period. After challenge with lice, counts were reduced 2-fold and 1.5-fold in fish that received 17β-estradiol and testosterone, respectively. Gene expression analyses were performed from skin of salmon collected in the field trial and from the controlled lab experiment at three time points (end of feeding-before challenge, 3 days post challenge (dpc) and 16 dpc) using oligonucleotide microarray and qPCR. Differential expression was observed in genes associated with diverse biological processes. Both studies revealed similar changes of several antibacterial acute phase proteins; of note was induction of cathelicidin and down-regulation of a defensin gene. Treatment with hormones revealed their ability to modulate T helper cell (Th)-mediated immunity in skin. Enhanced protection achieved by 17β-estradiol administration might in part be due to the skewing of Th responses away from the prototypic anti-parasitic Th2 immunity and towards the more effective Th1 responses. Multiple genes involved in wound healing, differentiation and remodelling of skin tissue were stimulated during maturation but suppressed with sex hormones. Such opposite regulation suggested that these processes were not associated with resistance to the parasite under the studied conditions. Both studies revealed regulation of a suite of genes encoding putative large mucosal proteins found exclusively in fish. Marked decrease of erythrocyte markers indicated reduced circulation while down-regulation of multiple zymogen granule membrane proteins and transporters of cholesterol and other compounds suggested limited availability of nutrients for the parasites., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Genome sequencing combined with transcriptome profiling promotes exploration of defence against pathogens and discovery of immune genes. Based on sequences from the recently released genome of Atlantic cod, a genome-wide oligonucleotide microarray (ACIQ-1) was designed and used for analyses of gene expression in the brain during infection with nervous necrosis virus (NNV). A challenge experiment with NNV was performed with Atlantic cod juveniles and brain samples from virus infected and uninfected fish were used for microarray analysis. Expression of virus induced genes increased at 5 days post challenge and persisted at stable level to the last sampling at 25 days post challenge. A large fraction of the up-regulated genes (546 features) were known or expected to have immune functions and most of these have not previously been characterized in Atlantic cod. Transcriptomic changes induced by the virus involved strong activation of genes associated with interferon and tumour necrosis factor related responses and acute inflammation. Up-regulation of genes involved in adaptive immunity suggested a rapid recruitment of B and T lymphocytes to the NNV infected brain. QPCR analyses of 15 candidate genes of innate immunity showed rapid induction by poly(I:C) in Atlantic cod larvae cells suggesting an antiviral role. Earliest and greatest expression changes after poly I:C stimulation was observed for interferon regulatory factors IRF4 and IRF7. Comparative studies between teleost species provided new knowledge about the evolution of innate antiviral immunity in fish. A number of genes is present or responds to viruses only in fish. Innate immunity of Atlantic cod is characterized by selective expansion of several medium-sized multigene families with ribose binding domains. An interesting finding was the high representation of three large gene families among the early antiviral genes, including tripartite motif proteins (TRIM) and proteins with PRY-SPRY and NACHT domains. The latter two with respectively 52 and 114 members in Atlantic cod have gone through expansions in different groups of fish. These proteins most likely have ligand binding properties and their propagation could be linked to the loss of MHC class II in the Atlantic cod genome., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In the aquatic environment, fish are exposed to various stimuli at once and have developed different response mechanisms to deal with these multiple stimuli. The current study assessed the combined impacts of estrogens and bacterial infection on the physiological status of fish. Juvenile rainbow trout were exposed to two different concentrations of 17β-estradiol (E2) (2 or 20 mg/kg feed) and then infected with three concentrations of Yersinia ruckeri, a bacterial pathogen causing massive losses in wild and farmed salmonid populations. Organism-level endpoints to assess the impact of the single and combined treatments included hepatic vitellogenin transcript expression to evaluate the E2 exposure efficiency and survival rate of pathogen-challenged fish. The two E2 doses increased vitellogenin levels within the physiological range. Infection with Y. ruckeri caused mortality of trout, and this effect was significantly enhanced by a simultaneous exposure to high E2 dose. The hormone reduced survival at intermediate and high (10(4) and 10(6) colony forming units, cfu) bacterial concentrations, but not for a low one (10(2) cfu). Analysis of hepatic gene expression profiles by a salmonid 2 k cDNA microarray chip revealed complex regulations of pathways involved in immune responses, stress responses, and detoxicification pathways. E2 markedly reduced the expression of several genes implicated in xenobiotic metabolism. The results suggest that the interaction between pathogen and E2 interfered with the fish's capability of clearing toxic compounds. The findings of the current study add to our understanding of multiple exposure responses in fish.

Background: The salmon louse (Lepeophtheirus salmonis Krøyer), an ectoparasitic copepod with a complex life cycle causes significant losses in salmon aquaculture. Pesticide treatments against the parasite raise environmental concerns and their efficacy is gradually decreasing. Improvement of fish resistance to lice, through biological control methods, needs better understanding of the protective mechanisms. We used a 21 k oligonucleotide microarray and RT-qPCR to examine the time-course of immune gene expression changes in salmon skin, spleen, and head kidney during the first 15 days after challenge, which encompassed the copepod and chalimus stages of lice development., Results: Large scale and highly complex transcriptome responses were found already one day after infection (dpi). Many genes showed bi-phasic expression profiles with abrupt changes between 5 and 10 dpi (the copepod-chalimus transitions); the greatest fluctuations (up- and down-regulation) were seen in a large group of secretory splenic proteases with unknown roles. Rapid sensing was witnessed with induction of genes involved in innate immunity including lectins and enzymes of eicosanoid metabolism in skin and acute phase proteins in spleen. Transient (1-5 dpi) increase of T-cell receptor alpha, CD4-1, and possible regulators of lymphocyte differentiation suggested recruitment of T-cells of unidentified lineage to the skin. After 5 dpi the magnitude of transcriptomic responses decreased markedly in skin. Up-regulation of matrix metalloproteinases in all studied organs suggested establishment of a chronic inflammatory status. Up-regulation of putative lymphocyte G0/G1 switch proteins in spleen at 5 dpi, immunoglobulins at 15 dpi; and increase of IgM and IgT transcripts in skin indicated an onset of adaptive humoral immune responses, whereas MHCI appeared to be down-regulated., Conclusions: Atlantic salmon develops rapid local and systemic reactions to L. salmonis, which, however, do not result in substantial level of protection. The dramatic changes observed after 5 dpi can be associated with metamorphosis of copepod, immune modulation by the parasite, or transition from innate to adaptive immune responses.

In addition to its central role of energy storage and release, white adipose tissue (WAT) performs complex endocrine and immune activities. WAT produces physiologically active secretory proteins, including cytokines and complement factors. Furthermore, treatment of mammalian adipocytes with cytokines and inflammatory stimulators induces immune genes, suppresses regulators of adipocyte differentiation and activates lipolysis. Previously we reported up-regulation of immune genes in the course of in vitro development of Atlantic salmon white adipocytes. If WAT is immunoactive tissue in fish, excessive deposition of fat resulting from lipid-rich diets may imply risk for health of farmed fish. In this paper we investigated how lipopolysaccharide (LPS) affects immune activity in the adipose tissue-derived stromo-vascular fraction (aSVF) of Atlantic salmon. Experiments were performed with confluent cultures of proliferating preadipocytes. Exposure to LPS induced expression of immune genes, including TNFalpha and TNF-dependent genes, chemokines and receptors, NFkappaB related genes, matrix metalloproteinases and genes involved in eicosanoid metabolism. LPS decreased expression of adipocyte markers and genes involved in lipid metabolism, however, in parallel, it accelerated a number of transcriptional events that take place during the adipogenic differentiation of aSVF., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Regulation of oxidative stress (OS) in adipocytes is an important mediator of their development and dysfunction. Highly unsaturated fatty acids (HUFAs) play essential roles in marine fish, where they have anti-lipogenic effects, but they are prone to peroxidation. The aim of this study was to investigate how the effects of HUFAs in fish adipocytes are modulated by changes in their intracellular redox status. Adipocytes from Atlantic salmon were cultivated on HUFA-rich media and treated with buthionine sulfoximine (BSO), which is known to deplete stores of the antioxidant glutathione (GSH) thus increasing OS, and alpha-tocopherol (alpha-TOC), which protects from OS. Gene expression was assessed by qPCR. In addition, phospholipid composition, total fatty acid (FA) composition, TBARS, the activities of pro-apoptotic caspase 3 (CASP3) and antioxidant superoxide dismutase (SOD) were determined. BSO treatment decreased the expression of genes encoding GSH-based antioxidant enzymes glutathione peroxidase (GPX) 2 and GPX3. Consequently, depletion of GSH resulted in the highest level of peroxidation products TBARS despite the increased activity of SOD in this group. Significant reduction of TBARS was achieved by alpha-TOC. Further, in comparison to two alpha-TOC supplemented groups, GSH-depleted cells accumulated less fat and their gene expression profile of adipogenic markers was lower. The formation of large intracellular vesicles was prominent in the control and BSO groups while reduction of OS by alpha-TOC coincided with the increased gene expression of the activating transcription factor 6 (ATF6), a transducer of the endoplasmic reticulum (ER)-stress response. CASP3 assay showed no difference between groups; however, depletion of GSH resulted in the increased gene expression of several apoptotic markers. Up-regulation of the apoptosis inducible factor (AIF) implied higher probability of CASP3-independent apoptosis in cells under increased OS. In conclusion, the study provides several lines of evidence in favour of anti-adipogenic effects of OS in a cold blooded vertebrate., (2010 Elsevier Inc. All rights reserved.)

Background: Excessive fat deposition is one of the largest problems faced by salmon aquaculture industries, leading to production losses due to high volume of adipose tissue offal. In addition, increased lipid accumulation may impose considerable stress on adipocytes leading to adipocyte activation and production and secretion of inflammatory mediators, as observed in mammals., Results: Microarray and qPCR analyses were performed to follow transcriptome changes during adipogenesis in the primary culture of adipose stromo-vascular fraction (aSVF) of Atlantic salmon. Cellular heterogeneity decreased by confluence as evidenced by the down-regulation of markers of osteo/chondrogenic, myogenic, immune and vasculature lineages. Transgelin (TAGLN), a marker of the multipotent pericyte, was prominently expressed around confluence while adipogenic PPARgamma was up-regulated already in subconfluent cells. Proliferative activity and subsequent cell cycle arrest were reflected in the fluctuations of pro- and anti-mitotic regulators. Marked regulation of genes involved in lipid and glucose metabolism and pathways producing NADPH and glycerol-3-phosphate (G3P) was seen during the terminal differentiation, also characterised by diverse stress responses. Activation of the glutathione and thioredoxin antioxidant systems and changes in the iron metabolism suggested the need for protection against oxidative stress. Signs of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) occured in parallel with the increased lipid droplet (LD) formation and production of secretory proteins (adipsin, visfatin). The UPR markers XBP1 and ATF6 were induced together with genes involved in ubiquitin-proteasome and lysosomal proteolysis. Concurrently, translation was suppressed as evidenced by the down-regulation of genes encoding elongation factors and components of the ribosomal machinery. Notably, expression changes of a panel of genes that belong to different immune pathways were seen throughout adipogenesis. The induction of AP1 (Jun, Fos), which is a master regulator of stress responses, culminated by the end of adipogenesis, concurrent with the maximal observed lipid deposition., Conclusions: Our data point to an intimate relationship between metabolic regulation and immune responses in white adipocytes of a cold-blooded vertebrate. Stress imposed on adipocytes by LD formation and expansion is prominently reflected in the ER compartment and the activated UPR response could have an important role at visceral obesity in fish.

Background: Furunculosis, a disease caused with gram negative bacteria Aeromonas salmonicida produces heavy losses in aquaculture. Vaccination against furunculosis reduces mortality of Atlantic salmon but fails to eradicate infection. Factors that determine high individual variation of vaccination efficiency remain unknown. We used gene expression analyses to search for the correlates of vaccine protection against furunculosis in Atlantic salmon., Results: Naïve and vaccinated fish were challenged by co-habitance. Fish with symptoms of furunculosis at the onset of mass mortality (LR - low resistance) and survivors (HR - high resistance) were sampled. Hepatic gene expression was analyzed with microarray (SFA2.0 - immunochip) and real-time qPCR. Comparison of LR and HR indicated changes associated with the protection and results obtained with naïve fish were used to find and filter the vaccine-independent responses. Genes involved in recruitment and migration of immune cells changed expression in both directions with greater magnitude in LR. Induction of the regulators of immune responses was either equal (NFkB) or greater (Jun) in LR. Expression levels of proteasome components and extracellular proteases were higher in LR while protease inhibitors were up-regulated in HR. Differences in chaperones and protein adaptors, scavengers of reactive oxygen species and genes for proteins of iron metabolism suggested cellular and oxidative stress in LR. Reduced levels of free iron and heme can be predicted in LR by gene expression profiles with no protection against pathogen. The level of complement regulation was greater in HR, which showed up-regulation of the components of membrane attack complex and the complement proteins that protect the host against the auto-immune damages. HR fish was also characterized with up-regulation of genes for proteins involved in the protection of extracellular matrix, lipid metabolism and clearance of endogenous and exogenous toxic compounds. A number of genes with marked expression difference between HR and LR can be considered as positive and negative correlates of vaccine protection against furunculosis., Conclusion: Efficiency of vaccination against furunculosis depends largely on the ability of host to neutralize the negative impacts of immune responses combined with efficient clearance and prevention of tissue damages.

Immunostimulants (IS) are considered a promising approach for improving resistance to pathogens in fish aquaculture. At present, development of IS are complicated due to limited knowledge on the mechanisms of their action. To assess the use of global gene expression analysis for screening of candidate IS we applied lentinan, a beta-glucan from the mushroom Lentinula edodes, as a model. After feeding rainbow trout (Oncorhynchus mykiss) with lentinan-supplemented (L) and control (C) diets for 37 days, fish were injected with bacterial lipopolysaccharide (LPS), a classical inducer of inflammation. Gene expression was analyzed in LPS-challenged compared to saline-injected fish using a salmonid 1.8k cDNA microarray (SFA2.0 immunochip) and real-time qPCR. Spleen was selected for data analyses due to highest magnitude of responses and its key role in the fish immune system. A group of genes implicated in acute inflammatory responses was higher induced in C versus L, including IFN-related and TNF-dependent genes (galectins and receptors, signal transducers and transcription factors), genes involved in MHC class I antigen presentation and leukocyte recruitment. A similar trend was observed in metabolism of iron and xenobiotics, markers of oxidative and cellular stress. Interestingly, differences between C and L were similar to those observed between salmon with low and high resistance to infectious salmon anemia virus. Genes with equal responses to LPS in L and C were related to cell communication (cytokines, chemokines and receptors), signal transduction, activation of immune cells, apoptosis, cellular maintenance and energy metabolism. In conclusion, lentinan decreased the expression of genes involved in acute inflammatory reactions to the inflammatory agent while major parts of the immune response remained unchanged. Such effects are expected for IS, which should modify immunity by enhancing beneficial and reducing detrimental responses.

Background: The salmon louse (SL) is an ectoparasitic caligid crustacean infecting salmonid fishes in the marine environment. SL represents one of the major challenges for farming of salmonids, and veterinary intervention is necessary to combat infection. This study addressed gene expression responses of Atlantic salmon infected with SL, which may account for its high susceptibility., Results: The effects of SL infection on gene expression in Atlantic salmon were studied throughout the infection period from copepodids at 3 days post infection (dpi) to adult lice (33 dpi). Gene expression was analyzed at three developmental stages in damaged and intact skin, spleen, head kidney and liver, using real-time qPCR and a salmonid cDNA microarray (SFA2). Rapid detection of parasites was indicated by the up-regulation of immunoglobulins in the spleen and head kidney and IL-1 receptor type 1, CD4, beta-2-microglobulin, IL-12beta, CD8alpha and arginase 1 in the intact skin of infected fish. Most immune responses decreased at 22 dpi, however, a second activation was observed at 33 dpi. The observed pattern of gene expression in damaged skin suggested the development of inflammation with signs of Th2-like responses. Involvement of T cells in responses to SL was witnessed with up-regulation of CD4, CD8alpha and programmed death ligand 1. Signs of hyporesponsive immune cells were seen. Cellular stress was prevalent in damaged skin as seen by highly significant up-regulation of heat shock proteins, other chaperones and mitochondrial proteins. Induction of the major components of extracellular matrix, TGF-beta and IL-10 was observed only at the adult stage of SL. Taken together with up-regulation of matrix metalloproteinases (MMP), this classifies the wounds afflicted by SL as chronic. Overall, the gene expression changes suggest a combination of chronic stress, impaired healing and immunomodulation. Steady increase of MMP expression in all tissues except liver was a remarkable feature of SL infected fish., Conclusion: SL infection in Atlantic salmon is associated with a rapid induction of mixed inflammatory responses, followed by a period of hyporesponsiveness and delayed healing of injuries. Persistent infection may lead to compromised host immunity and tissue self-destruction.

The peroxisomal enzyme urate oxidase plays a pivotal role in the degradation of purines in both prokaryotes and eukaryotes. However, knowledge about the purine-induced expression of the encoding gene is lacking in vertebrates. These are the first published sequences of fish urate oxidase, which were predicted from PCR amplified liver cDNAs of Atlantic salmon (Salmo salar), Atlantic cod (Gadus morhua), Atlantic halibut (Hippoglossus hippoglossus) and African lungfish (Protopterus annectens). Sequence alignment of different vertebrate urate oxidases revealed amino acid substitutions of putative functional importance in the enzyme of chicken and lungfish. In the adult salmon, expression of urate oxidase mRNA predominated in liver, but was also identified in several nonhepatic organs including brain, but not in skeletal muscle and kidney. Juvenile salmon fed diets containing bacterial protein meal (BPM) rich in nucleic acids showed a significant increase in liver urate oxidase enzyme activity, and urea concentrations in plasma, muscle and liver were elevated. Whereas salmon fed the 18% BPM diet showed a nonsignificant increase in liver mRNA levels of urate oxidase compared with the 0% BPM-fed fish, no further increase in mRNA levels was found in fish receiving 36% BPM. The discrepancy between urate oxidase mRNA and enzyme activity was explained by rapid mRNA degradation or alternatively, post-translational control of the activity. Although variable plasma and liver levels of urate were detected, the substrate increased only slightly in 36% BPM-fed fish, indicating that the uricolytic pathway of Atlantic salmon is intimately regulated to handle high dietary purine levels.