Impact of stress phenotype, elevated temperature, and bacterin exposure on male Atlantic salmon (Salmo salar) growth, stress, and immune biomarker gene expression.

In this study, postsmolt male Atlantic salmon, previously identified as low responders (LRs) or high responders (HRs) based on poststress cortisol levels, had their head kidney and liver sampled at 12°C and 20°C before injection (time 0) and after injection (i.e., at 12- and 24-h postinjection, respectively) with either Forte Micro (a multivalent vaccine containing bacterin, to capture peak antibacterial responses) or an equal volume of PBS. Quantitative real-time PCR (qPCR) was then used to measure the expression of 15 biomarker genes in the head kidney and 12 genes in the liver at each temperature/sampling point. Target transcripts were chosen that were related to growth, stress, and innate antibacterial immune responses. Many temperature, phenotype, and injection effects were found for individual genes within these three broad categories, and multivariate statistical analyses (i.e., principal component analysis and permutational multivariate analysis of variance) were used to look for overall patterns in transcript expression. These analyses revealed that HR salmon at 20°C mounted a more robust response (P < 0.05) for the 10 head kidney immune-related transcripts when injected with Forte Micro than LR salmon. In contrast, the seven liver stress-related transcripts displayed a greater response (P = 0.057) in LR versus HR fish with Forte Micro at 12°C. Overall, although this research did find some differences between LR and HR fish, it does not provide strong (conclusive) evidence that the selection of a particular phenotype would have major implications for the health of salmon over the temperature range examined.NEW & NOTEWORTHY This is the first paper to describe the impact of both temperature and bacterial stimulation on head kidney and liver transcript expression in Atlantic salmon characterized as LRs versus HRs. Notably, we found that HR salmon at 20°C mounted a more robust innate antibacterial immune response than LR salmon. In addition, LR fish at 12°C may (P = 0.057) exhibit higher expression of stress-related transcripts in response to vaccine injection relative to HR fish.

The late-evolving salmon and trout join the GnRH1 club.

Although it is known that the whitefish, an ancient salmonid, expresses three distinct gonadotropin-releasing hormone (GnRH) forms in the brain, it has been thought that the later-evolving salmonids (salmon and trout) had only two types of GnRH: GnRH2 and GnRH3. We now provide evidence for the expression of GnRH1 in the gonads of Atlantic salmon by rapid amplification of cDNA ends, real-time quantitative PCR and immunohistochemistry. We examined six different salmonid genomes and found that each assembly has one gene that likely encodes a viable GnRH1 prepropeptide. In contrast to both functional GnRH2 and GnRH3 paralogs, the GnRH1 homeolog can no longer express the hormone. Furthermore, the viable salmonid GnRH1 mRNA is composed of only three exons, rather than the four exons that build the GnRH2 and GnRH3 mRNAs. Transcribed gnrh1 is broadly expressed (in 17/18 tissues examined), with relative abundance highest in the ovaries. Expression of the gnrh2 and gnrh3 mRNAs is more restricted, primarily to the brain, and not in the gonads. The GnRH1 proximal promoter presents composite binding elements that predict interactions with complexes that contain diverse cell fate and differentiation transcription factors. We provide immunological evidence for GnRH1 peptide in the nucleus of 1-year-old type A spermatogonia and cortical alveoli oocytes. GnRH1 peptide was not detected during other germ cell or reproductive stages. GnRH1 activity in the salmonid gonad may occur only during early stages of development and play a key role in a regulatory network that controls mitotic and/or meiotic processes within the germ cell.

Ancestral reconstruction reveals catalytic inactivation of activation-induced cytidine deaminase concomitant with cold water adaption in the Gadiformes bony fish.

BACKGROUND: Antibody affinity maturation in vertebrates requires the enzyme activation-induced cytidine deaminase (AID) which initiates secondary antibody diversification by mutating the immunoglobulin loci. AID-driven antibody diversification is conserved across jawed vertebrates since bony and cartilaginous fish. Two exceptions have recently been reported, the Pipefish and Anglerfish, in which the AID-encoding aicda gene has been lost. Both cases are associated with unusual reproductive behavior, including male pregnancy and sexual parasitism. Several cold water fish in the Atlantic cod (Gadinae) family carry an aicda gene that encodes for a full-length enzyme but lack affinity-matured antibodies and rely on antibodies of broad antigenic specificity. Hence, we examined the functionality of their AID. RESULTS: By combining genomics, transcriptomics, immune responsiveness, and functional enzymology of AID from 36 extant species, we demonstrate that AID of that Atlantic cod and related fish have extremely lethargic or no catalytic activity. Through ancestral reconstruction and functional enzymology of 71 AID enzymes, we show that this enzymatic inactivation likely took place relatively recently at the emergence of the true cod family (Gadidae) from their ancestral Gadiformes order. We show that this AID inactivation is not only concordant with the previously shown loss of key adaptive immune genes and expansion of innate and cell-based immune genes in the Gadiformes but is further reflected in the genomes of these fish in the form of loss of AID-favored sequence motifs in their immunoglobulin variable region genes. CONCLUSIONS: Recent demonstrations of the loss of the aicda gene in two fish species challenge the paradigm that AID-driven secondary antibody diversification is absolutely conserved in jawed vertebrates. These species have unusual reproductive behaviors forming an evolutionary pressure for a certain loss of immunity to avoid tissue rejection. We report here an instance of catalytic inactivation and functional loss of AID rather than gene loss in a conventionally reproducing vertebrate. Our data suggest that an expanded innate immunity, in addition to lower pathogenic pressures in a cold environment relieved the pressure to maintain robust secondary antibody diversification. We suggest that in this unique scenario, the AID-mediated collateral genome-wide damage would form an evolutionary pressure to lose AID function.

Distinct early life stage gene expression effects of hybridization among European and North American farmed and wild Atlantic salmon populations.

Due to multigeneration domestication selection, farmed and wild Atlantic salmon diverge genetically, which raises concerns about potential genetic interactions among escaped farmed and wild populations and disruption of local adaptation through introgression. When farmed strains of distant geographic origin are used, it is unknown whether the genetic consequences posed by escaped farmed fish will be greater than if more locally derived strains are used. Quantifying gene transcript expression differences among divergent farmed, wild and F1  hybrids under controlled conditions is one of the ways to explore the consequences of hybridization. We compared the transcriptomes of fry at the end of yolk sac absorption of a European (EO) farmed ("StofnFiskur", Norwegian strain), a North American (NA) farmed (Saint John River, NB strain), a Newfoundland (NF) wild population with EO ancestry, and related F1  hybrids using 44 K microarrays. Our findings indicate that the wild population showed greater transcriptome differences from the EO farmed strain than that of the NA farmed strain. We also found the largest differences in global gene expression between the two farmed strains. We detected the fewest differentially expressed transcripts between F1  hybrids and domesticated/wild maternal strains. We also found that the differentially expressed genes between cross types over-represented GO terms associated with metabolism, development, growth, immune response, and redox homeostasis processes. These findings suggest that the interbreeding of escaped EO/NA farmed and NF wild population would alter gene transcription, and the consequences of hybridization would be greater from escaped EO farmed than NA farmed salmon, resulting in potential effects on the wild populations.

Atlantic Salmon (Salmo salar) bacterial and viral innate immune responses are not impaired by florfenicol or tetracycline administration.

Antibiotics are used to treat bacterial infections in fish aquaculture, and these drugs can interact with immune cells/the immune system and potentially leave fish vulnerable to viral, fungal, parasitic, or other bacterial infections. However, the effects of antibiotics on fish immunity have largely been overlooked by the aquaculture industry. We tested, at 12 and 20 °C, whether tetracycline and florfenicol (the most commonly used antibiotics in commercial aquaculture), affected the Atlantic salmon's capacity to respond to bacterial or viral stimulation. Atlantic salmon were acclimated to 12 or 20 °C and fed with tetracycline or florfenicol (100 and 10 mg kg of body weight-1 day-1, respectively) medicated feed for 15 or 10 days, respectively. Thereafter, we evaluated their immune function prior to, and after, an intraperitoneal injection of Forte Micro (containing inactivated cultures of Aeromonas salmonicida, Vibrio anguillarum, Vibrio ordalii and Vibrio salmonicida) or the viral mimic polyriboinosinic polyribocytidylic acid (pIC). We measured the transcript expression levels of 8 anti-bacterial and 8 anti-viral putative biomarker genes, and the innate (leukocyte respiratory burst, plasma lysozyme activity and hemolytic activity of the alternative complement pathway) and cellular (relative number of erythrocytes, lymphocytes and thrombocytes, and granulocytes such as monocytes and neutrophils) responses to these challenges. Overall, we only found a few minor effects of either tetracycline or florfenicol on immune gene expression or function at either temperature. Although several studies have reported that antibiotics may negatively affect fish immune responses, our results show that industry-relevant dietary tetracycline and florfenicol treatments do not substantially impact the salmon's innate immune responses. Currently, this is the most comprehensive study on the effects of antibiotics administrated according to industry protocols on immune function in Atlantic salmon.

Influence of Supplemental Dietary Cholesterol on Growth Performance, Indices of Stress, Fillet Pigmentation, and Upper Thermal Tolerance of Female Triploid Atlantic Salmon (Salmo salar).

The salmon aquaculture industry must be proactive at developing mitigation tools/strategies to offset the potential negative impacts of climate change. Therefore, this study examined if additional dietary cholesterol could enhance salmon production at elevated temperatures. We hypothesized that supplemental cholesterol could aid in maintaining cell rigidity, reducing stress and the need to mobilize astaxanthin muscle stores, and improving salmon growth and survival at high rearing temperatures. Accordingly, postsmolt female triploid salmon were exposed to an incremental temperature challenge (+0.2°C day-1) to mimic conditions that they experience in sea cages in the summer, with temperature held at both 16 and 18°C for several weeks [i.e., 3 weeks at 16°C, followed by an increase at 0.2°C day-1 to 18°C (10 days), then 5 weeks at 18°C] to prolong their exposure to elevated temperatures. From 16°C onwards, the fish were fed either a control diet, or one of two nutritionally equivalent experimental diets containing supplemental cholesterol [+1.30%, experimental diet #1 (ED1); or +1.76%, experimental diet #2 (ED2)]. Adding cholesterol to the diet did not affect the salmon's incremental thermal maximum (ITMax), growth, plasma cortisol, or liver stress-related transcript expression. However, ED2 appeared to have a small negative impact on survival, and both ED1 and ED2 reduced fillet "bleaching" above 18°C as measured using SalmoFan™ scores. Although the current results suggest that supplementing salmon diets with cholesterol would have few/minimal benefits for the industry, ≤ 5% of the female triploid Atlantic salmon used in this study irrespective of diet died before temperature reached 22°C. These latter data suggest that it is possible to produce all female populations of reproductively sterile salmon that can withstand summer temperatures in Atlantic Canada.

The transcriptomic responses of Atlantic salmon (Salmo salar) to high temperature stress alone, and in combination with moderate hypoxia.

BACKGROUND: Increases in ocean temperatures and in the frequency and severity of hypoxic events are expected with climate change, and may become a challenge for cultured Atlantic salmon and negatively affect their growth, immunology and welfare. Thus, we examined how an incremental temperature increase alone (Warm & Normoxic-WN: 12 → 20 °C; 1 °C week- 1), and in combination with moderate hypoxia (Warm & Hypoxic-WH: ~ 70% air saturation), impacted the salmon's hepatic transcriptome expr\ession compared to control fish (CT: 12 °C, normoxic) using 44 K microarrays and qPCR. RESULTS: Overall, we identified 2894 differentially expressed probes (DEPs, FDR < 5%), that included 1111 shared DEPs, while 789 and 994 DEPs were specific to WN and WH fish, respectively. Pathway analysis indicated that the cellular mechanisms affected by the two experimental conditions were quite similar, with up-regulated genes functionally associated with the heat shock response, ER-stress, apoptosis and immune defence, while genes connected with general metabolic processes, proteolysis and oxidation-reduction were largely suppressed. The qPCR assessment of 41 microarray-identified genes validated that the heat shock response (hsp90aa1, serpinh1), apoptosis (casp8, jund, jak2) and immune responses (apod, c1ql2, epx) were up-regulated in WN and WH fish, while oxidative stress and hypoxia sensitive genes were down-regulated (cirbp, cyp1a1, egln2, gstt1, hif1α, prdx6, rraga, ucp2). However, the additional challenge of hypoxia resulted in more pronounced effects on heat shock and immune-related processes, including a stronger influence on the expression of 14 immune-related genes. Finally, robust correlations between the transcription of 19 genes and several phenotypic traits in WH fish suggest that changes in gene expression were related to impaired physiological and growth performance. CONCLUSION: Increasing temperature to 20 °C alone, and in combination with hypoxia, resulted in the differential expression of genes involved in similar pathways in Atlantic salmon. However, the expression responses of heat shock and immune-relevant genes in fish exposed to 20 °C and hypoxia were more affected, and strongly related to phenotypic characteristics (e.g., growth). This study provides valuable information on how these two environmental challenges affect the expression of stress-, metabolic- and immune-related genes and pathways, and identifies potential biomarker genes for improving our understanding of fish health and welfare.

Phenotypic stress response does not influence the upper thermal tolerance of male Atlantic salmon (Salmo salar).

Fish can be identified as either low responders (LR) or high responders (HR) based on post-stress cortisol levels and whether they exhibit a proactive or reactive stress coping style, respectively. In this study, male Atlantic salmon (Salmo salar) from 17 families reared at 9 °C were repeatedly exposed to an acute handling stress over a period of four months, with plasma cortisol levels measured at 1 h post-stress. Fish were identified as either LR or HR if the total Z-score calculated from their cortisol responses fell into the lower or upper quartile ranges, respectively; with intermediate responders (IR) classified as the remainder. Salmon characterized as LR, IR or HR were then subjected to an incremental thermal challenge, where temperature was raised at 0.2 °C day-1 from their acclimation temperature (12 °C) to mimic natural sea-cage farming conditions during the summer in Newfoundland. Interestingly, feed intake remained high up to 22 °C, while previous studies have shown a decrease in salmon appetite after ∼16-18 °C. After the first three mortalities were recorded at elevated temperature, a subset of LR and HR salmon were exposed to another acute handling stress event at 23.6 °C. Basal and post-stress measurements of plasma cortisol, glucose and lactate did not differ between stress response phenotypes at this temperature. In the end, the average incremental thermal maximum (ITMax) of LR and HR fish was not different (25.1 °C). In comparison, the critical thermal maximum (CTMax; temperature increased at 2 °C h-1) of the remaining IR fish that had been held at 12 °C was 28.5 °C. Collectively, these results: 1) show that this population of Atlantic salmon is very thermally tolerant, and further question the relevance of CTMax in assessing responses to real-world temperature changes; and 2) indicate that characterization of stress phenotype at 9 °C is not predictive of their stress response or survival at high temperatures. Therefore, selection of fish based on phenotypic stress response at low temperatures may not be beneficial to incorporate into Atlantic salmon breeding programs, especially if the goal is to improve growth performance and survival at high temperatures in sea-cages.

Profiling the transcriptome response of Atlantic salmon head kidney to formalin-killed Renibacterium salmoninarum.

Renibacterium salmoninarum is a Gram-positive, intracellular bacterial pathogen that causes Bacterial Kidney Disease (BKD) in Atlantic salmon (Salmo salar). The host transcriptomic response to this immune-suppressive pathogen remains poorly understood. To identify R. salmoninarum-responsive genes, Atlantic salmon were intraperitoneally injected with a low (5 × 105 cells/kg, Low-Rs) or high (5 × 107 cells/kg; High-Rs) dose of formalin-killed R. salmoninarum bacterin or phosphate-buffered saline (PBS control); head kidney samples were collected before and 24 h after injection. Using 44K microarray analysis, we identified 107 and 345 differentially expressed probes in response to R. salmoninarum bacterin (i.e. High-Rs vs. PBS control) by Significance Analysis of Microarrays (SAM) and Rank Products (RP), respectively. Twenty-two microarray-identified genes were subjected to qPCR assays, and 17 genes were confirmed as being significantly responsive to the bacterin. There was an up-regulation in expression of genes playing putative roles as immune receptors and antimicrobial effectors. Genes with putative roles as pathogen recognition (e.g. clec12b and tlr5) or immunoregulatory (e.g. tnfrsf6b and tnfrsf11b) receptors were up-regulated in response to R.salmoninarum bacterin. Also, chemokines and a chemokine receptor showed opposite regulation [up-regulation of effectors (i.e. ccl13 and ccl) and down-regulation of cxcr1] in response to the bacterin. The present study identified and validated novel biomarker genes (e.g. ctsl1, lipe, cldn4, ccny) that can be used to assess Atlantic salmon response to R. salmoninarum, and will be valuable in the development of tools to combat BKD.

Impact of rearing temperature on the innate antiviral immune response of growth hormone transgenic female triploid Atlantic salmon (Salmo salar).

AquAdvantage Salmon (growth hormone transgenic female triploid Atlantic salmon) are a faster-growing alternative to conventional farmed diploid Atlantic salmon. To investigate optimal rearing conditions for their commercial production, a laboratory study was conducted in a freshwater recirculating aquaculture system (RAS) to examine the effect of rearing temperature (10.5 °C, 13.5 °C, 16.5 °C) on their antiviral immune and stress responses. When each temperature treatment group reached an average weight of 800 g, a subset of fish were intraperitoneally injected with either polyriboinosinic polyribocytidylic acid (pIC, a viral mimic) or an equal volume of sterile phosphate-buffered saline (PBS). Blood and head kidney samples were collected before injection and 6, 24 and 48 h post-injection (hpi). Transcript abundance of 7 antiviral biomarker genes (tlr3, lgp2, stat1b, isg15a, rsad2, mxb, ifng) was measured by real-time quantitative polymerase chain reaction (qPCR) on head kidney RNA samples. Plasma cortisol levels from blood samples collected pre-injection and from pIC and PBS groups at 24 hpi were quantified by ELISA. While rearing temperature and treatment did not significantly affect circulating cortisol, all genes tested were significantly upregulated by pIC at all three temperatures (except for tlr3, which was only upregulated in the 10.5 °C treatment). Target gene activation was generally observed at 24 hpi, with most transcript levels decreasing by 48 hpi in pIC-injected fish. Although a high amount of biological variability in response to pIC was evident across all treatments, rearing temperature significantly influenced transcript abundance and/or fold-changes comparing time- and temperature-matched pIC- and PBS-injected fish for several genes (tlr3, lgp2, stat1b, isg15a, rsad2 and ifng) at 24 hpi. As an example, significantly higher fold-changes of rsad2, isg15a and ifng were found in fish reared at 10.5 °C when compared to 16.5 °C. Multivariate analysis confirmed that rearing temperature modulated antiviral immune response. The present experiment provides novel insight into the relationship between rearing temperature and innate antiviral immune response in AquAdvantage Salmon.

Characterization of miRNAs in Cultured Atlantic Salmon Head Kidney Monocyte-Like and Macrophage-Like Cells.

Macrophages are among the first cells to respond to infection and disease. While microRNAs (miRNAs) are involved in the process of monocyte-to-macrophage differentiation in mammals, less is known in teleost fish. Here, Atlantic salmon head kidney leukocytes (HKLs) were used to study the expression of miRNAs in response to in vitro culture. The morphological analysis of cultures showed predominantly monocyte-like cells on Day 1 and macrophage-like cells on Day 5, suggesting that the HKLs had differentiated from monocytes to macrophages. Day 5 HKLs also contained a higher percentage of phagocytic cells. Small RNA sequencing and qPCR analysis were applied to examine the miRNA diversity and expression. There were 370 known mature Atlantic salmon miRNAs in HKLs. Twenty-two miRNAs (15 families) were downregulated while 44 miRNAs (25 families) were upregulated on Day 5 vs. Day 1. Mammalian orthologs of many of the differentially expressed (DE) miRNAs are known to regulate macrophage activation and differentiation, while the teleost-specific miR-2188, miR-462 and miR-731 were also DE and are associated with immune responses in fish. In silico predictions identified several putative target genes of qPCR-validated miRNAs associated with vertebrate macrophage differentiation. This study identified Atlantic salmon miRNAs likely to influence macrophage differentiation, providing important knowledge for future functional studies.

Transcriptomic Profiling in Fins of Atlantic Salmon Parasitized with Sea Lice: Evidence for an Early Imbalance Between Chalimus-Induced Immunomodulation and the Host's Defense Response.

Parasitic sea lice (e.g., Lepeophtheirus salmonis) cause costly outbreaks in salmon farming. Molecular insights into parasite-induced host responses will provide the basis for improved management strategies. We investigated the early transcriptomic responses in pelvic fins of Atlantic salmon parasitized with chalimus I stage sea lice. Fin samples collected from non-infected (i.e. pre-infected) control (PRE) and at chalimus-attachment sites (ATT) and adjacent to chalimus-attachment sites (ADJ) from infected fish were used in profiling global gene expression using 44 K microarrays. We identified 6568 differentially expressed probes (DEPs, FDR < 5%) that included 1928 shared DEPs between ATT and ADJ compared to PRE. The ATT versus ADJ comparison revealed 90 DEPs, all of which were upregulated in ATT samples. Gene ontology/pathway term network analyses revealed profound changes in physiological processes, including extracellular matrix (ECM) degradation, tissue repair/remodeling and wound healing, immunity and defense, chemotaxis and signaling, antiviral response, and redox homeostasis in infected fins. The QPCR analysis of 37 microarray-identified transcripts representing these functional themes served to confirm the microarray results with a significant positive correlation (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.

Minimizing marine ingredients in diets of farmed Atlantic salmon (Salmo salar): effects on liver and head kidney lipid class and fatty acid composition.

Limited fish meal and fish oil supplies have necessitated research on alternatives for aquafeeds. Seven dietary treatments with different protein and lipid sources were formulated for farmed Atlantic salmon, and their effects on liver and head kidney lipid class, fatty acid, and elemental composition were studied. Fish meal, fish oil, and EPA + DHA content ranged from 5-35%, 0-12%, and 0.1-3%, respectively. Elemental analysis showed that the C to N ratio was higher in the head kidney than in the liver, which is consistent with higher content of total lipids in the head kidney compared with the liver. There was a greater susceptibility to dietary lipid alterations in the liver compared with the head kidney despite liver having a greater proportion of phospholipid and a much lower proportion of triacylglycerol. So long as fish oil levels were 5% or more of the diet, arachidonic acid (ARA) and docosahexaenoic acid (DHA) proportions were the same for each tissue as with feeding the marine diet with 12% fish oil; however, livers and head kidneys from fish fed the lowest amount of fish meal and fish oil had the lowest levels of eicosapentaenoic (EPA) and DHA and the highest ARA levels. Removal of fish oil and reduction of fish meal to 5% in diets of farmed Atlantic salmon affected elemental and lipid compositions of the liver and head kidney tissues potentially increasing susceptibility to inflammation. However, with 10% of the diet comprising fish meal and fish oil, lipid contents were comparable with fish fed marine-based diets.

Sockeye salmon demonstrate robust yet distinct transcriptomic kidney responses to rhabdovirus (IHNV) exposure and infection.

Aquatic rhabdoviruses are globally significant pathogens associated with disease in both wild and cultured fish. Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that causes the internationally regulated disease infectious hematopoietic necrosis (IHN) in most species of salmon. Yet not all naïve salmon exposed to IHNV become diseased, and the mechanisms by which some individuals evade or rapidly clear infection following exposure are poorly understood. Here we used RNA-sequencing to evaluate transcriptomic changes in sockeye salmon, a keystone species in the North Pacific and natural host for IHNV, to evaluate the consequences of IHNV exposure and/or infection on host cell transcriptional pathways. Immersion challenge of sockeye salmon smolts with IHNV resulted in approximately 33% infection prevalence, where both prevalence and viral kidney load peaked at 7 days post challenge (dpc). De novo assembly of kidney transcriptomes at 7 dpc revealed that both infected and exposed but noninfected individuals experienced substantial transcriptomic modification; however, stark variation in gene expression patterns were observed between exposed but noninfected, infected, and unexposed populations. GO and KEGG pathway enrichment in concert with differential expression analysis identified that kidney responses in exposed but noninfected fish emphasised a global pattern of transcriptional down-regulation, particularly for pathways involved in DNA transcription, protein biosynthesis and macromolecule metabolism. In contrast, transcriptomes of infected fish demonstrated a global emphasis of transcriptional up-regulation highlighting pathways involved in antiviral response, inflammation, apoptosis, and RNA processing. Quantitative PCR was subsequently used to highlight differential and time-specific regulation of acute phase, antiviral, inflammatory, cell boundary, and metabolic responsive transcripts in both infected and exposed but noninfected groups. This data demonstrates that waterborne exposure with IHNV has a dramatic effect on the sockeye salmon kidney transcriptome that is discrete between resistant and acutely susceptible individuals. We identify that metabolic, acute phase and cell boundary pathways are transcriptionally affected by IHNV and kidney responses to local infection are highly divergent from those generated as part of a disseminated response. These data suggest that primary resistance of naïve fish to IHNV may involve global responses that encourage reduced cellular signaling rather than promoting classical innate antiviral responses.

Changes in the liver transcriptome of farmed Atlantic salmon (Salmo salar) fed experimental diets based on terrestrial alternatives to fish meal and fish oil.

BACKGROUND: Dependence on marine natural resources threatens the sustainability of Atlantic salmon aquaculture. In the present study, Atlantic salmon fed for 14 weeks with an experimental diet based on animal by-products and vegetable oil (ABP) exhibited reduced growth performance compared with others fed a fish meal/fish oil based experimental diet (MAR) and a plant protein/vegetable oil-based experimental diet (VEG). To characterize the molecular changes underlying the differences in growth performance, we conducted a 44 K microarray study of the liver transcriptome of the three dietary groups. RESULTS: The microarray experiment identified 122 differentially expressed features (Rank Products, PFP < 10%). Based on their associated Gene Ontology terms, 46 probes were classified as metabolic and growth-relevant genes, 25 as immune-related, and 12 as related to oxidation-reduction processes. The microarray results were validated by qPCR analysis of 29 microarray-identified transcripts. Diets significantly modulated the transcription of genes involved in carbohydrate metabolism (gck and pfkfb4), cell growth and proliferation (sgk2 and htra1), apoptosis (gadd45b), lipid metabolism (fabp3, idi1, sqs), and immunity (igd, mx, ifit5, and mhcI). Hierarchical clustering and linear correlation analyses were performed to find gene expression patterns among the qPCR-analyzed transcripts, and connections between them and muscle and liver lipid composition. Overall, our results indicate that changes in the liver transcriptome and tissue lipid composition were driven by cholesterol synthesis up-regulation by ABP and VEG diets, and the lower carbohydrate intake in the ABP group. Two of the microarray-identified genes (sgk2 and htra1) might be key to explaining glucose metabolism regulation and the dietary-modulation of the immune system in fish. To evaluate the potential of these genes as predictive biomarkers, we subjected the qPCR data to a stepwise discriminant analysis. Three sets of no more than four genes were found to be able to predict, with high accuracy (67-94%), salmon growth and fatty acid composition. CONCLUSIONS: This study provides new findings on the impact of terrestrial animal and plant products on the nutrition and health of farmed Atlantic salmon, and a new method based on gene biomarkers for potentially predicting desired phenotypes, which could help formulate superior feeds for the Atlantic salmon aquaculture industry.

Transcriptome profiling of antiviral immune and dietary fatty acid dependent responses of Atlantic salmon macrophage-like cells.

BACKGROUND: Due to the limited availability and high cost of fish oil in the face of increasing aquaculture production, there is a need to reduce usage of fish oil in aquafeeds without compromising farm fish health. Therefore, the present study was conducted to determine if different levels of vegetable and fish oils can alter antiviral responses of salmon macrophage-like cells (MLCs). Atlantic salmon (Salmo salar) were fed diets containing 7.4% (FO7) or 5.1% (FO5) fish oil. These diets were designed to be relatively low in EPA + DHA (i.e. FO7: 1.41% and FO5: 1%), but near the requirement level, and resulting in comparable growth. Vegetable oil (i.e. rapeseed oil) was used to balance fish oil in experimental diets. After a 16-week feeding trial, MLCs isolated from fish in these dietary groups were stimulated by a viral mimic (dsRNA: pIC) for 6 h (qPCR assay) and 24 h (microarray and qPCR assays). RESULTS: The fatty acid composition of head kidney leukocytes varied between the two dietary groups (e.g. higher 20:5n-3 in the FO7 group). Following microarray assays using a 44K salmonid platform, Rank Products (RP) analysis showed 14 and 54 differentially expressed probes (DEP) (PFP < 0.05) between the two diets in control and pIC groups (FO5 vs. FO7), respectively. Nonetheless, Significance Analysis of Microarrays (SAM, FDR < 0.05) identified only one DEP between pIC groups of the two diets. Moreover, we identified a large number (i.e. 890 DEP in FO7 and 1128 DEP in FO5 overlapping between SAM and RP) of pIC-responsive transcripts, and several of them were involved in TLR-/RLR-dependent and cytokine-mediated pathways. The microarray results were validated as significantly differentially expressed by qPCR assays for 2 out of 9 diet-responsive transcripts and for all of the 35 selected pIC-responsive transcripts. CONCLUSION: Fatty acid-binding protein adipocyte (fabp4) and proteasome subunit beta type-8 (psmb8) were significantly up- and down-regulated, respectively, in the MLCs of fish fed the diet with a lower level of fish oil, suggesting that they are important diet-responsive, immune-related biomarkers for future studies. Although the different levels of dietary fish and vegetable oils involved in this study affected the expression of some transcripts, the immune-related pathways and functions activated by the antiviral response of salmon MLCs in both groups were comparable overall. Moreover, the qPCR revealed transcripts responding early to pIC (e.g. lgp2, map3k8, socs1, dusp5 and cflar) and time-responsive transcripts (e.g. scarb1-a, csf1r, traf5a, cd80 and ctsf) in salmon MLCs. The present study provides a comprehensive picture of the putative molecular pathways (e.g. RLR-, TLR-, MAPK- and IFN-associated pathways) activated by the antiviral response of salmon MLCs.

Functional Annotation of All Salmonid Genomes (FAASG): an international initiative supporting future salmonid research, conservation and aquaculture.

We describe an emerging initiative - the 'Functional Annotation of All Salmonid Genomes' (FAASG), which will leverage the extensive trait diversity that has evolved since a whole genome duplication event in the salmonid ancestor, to develop an integrative understanding of the functional genomic basis of phenotypic variation. The outcomes of FAASG will have diverse applications, ranging from improved understanding of genome evolution, to improving the efficiency and sustainability of aquaculture production, supporting the future of fundamental and applied research in an iconic fish lineage of major societal importance.

The dietary replacement of marine ingredients by terrestrial animal and plant alternatives modulates the antiviral immune response of Atlantic salmon (Salmo salar).

The effects of replacing marine ingredients by terrestrial ingredients on the health of Atlantic salmon (Salmo salar) are poorly understood. During a 14-week trial, Atlantic salmon fed a fish meal-fish oil based diet (MAR) showed similar growth performance to others fed a plant protein/vegetable oil based diet (VEG), whereas poorer performance was observed in those fed an animal by-product meal/vegetable oil based diet (ABP). At the end of the trial, salmon were injected with either phosphate-buffered saline (PBS) or the viral mimic polyriboinosinic polyribocytidylic acid (pIC) and sampled for head kidney RNA after 24 h. The levels of 27 immune-related transcripts, and of 5 others involved in eicosanoid synthesis (including paralogues in both cases) were measured in the head kidney of the salmon using qPCR. All of the assayed immune-related genes and cox2 were pIC-induced, while the other eicosanoid synthesis-related genes were pIC-repressed. Linear regression was used to establish correlations between different immune transcripts, elucidating the cascade of responses to pIC and specialization among paralogues. Regarding the effect of diet on the antiviral immune response, pIC-treated fish fed diets ABP and VEG showed higher transcript levels of tlr3, irf1b, stat1a, isg15b, and gig1 compared to those fed diet MAR. We infer that the observed dietary immunomodulation could be due to the lower proportion of arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) in diets ABP and VEG. Furthermore, our results suggest a major role of dietary ARA in Atlantic salmon immunity, as low ARA proportion in diet VEG coincided with the highest pIC-induction of some immune transcripts (tlr7, stat1c, mxb, and gig1) and the lowest levels of transcripts encoding eicosanoid-synthesizing enzymes (5loxa, 5loxb, and pgds). In contrast, the high ARA/EPA ratio of diet ABP appeared to favor increased expression of transcripts involved in the synthesis of pro-inflammatory eicosanoids (5loxa and 5loxb) and chemotaxis (ccl19b). In conclusion, our findings show that nutritionally balanced plant-based diets may enhance the immune response of Atlantic salmon. Future studies should explore the possible advantages of plant-based diets in Atlantic salmon exposed to a viral infection.

Structure-function relationships in histidine-rich antimicrobial peptides from Atlantic cod.

Gad-1 and Gad-2 are antimicrobial peptide (AMP) sequences encoded by paralogous genes. They are rich in histidine, which suggests that their activity might be pH-dependent. We examined their structure-function relationships with a view to learning how to improve AMP therapeutic ratios. Activity assays with Gram-negative bacteria and cancer cell lines demonstrate that Gad-2 is substantially more active at slightly acidic pH than it is at neutral pH. By contrast, the activity of Gad-1 at lower pH is similar to its activity at pH7. Circular dichroism spectra indicate that the greater functional plasticity of Gad-2 correlates with a greater structural plasticity; Gad-2's percent helicity varies dramatically with altered pH and lipid environment. Interestingly, Gad-2's highest levels of helicity do not correspond to the conditions where it is most active. High resolution solution NMR structures were determined in SDS micelles at pH5, conditions that induce an intermediate level of helicity in the peptides. Gad-1 is more helical than Gad-2, with both peptides exhibiting the greatest helical tendencies in their central region and lowest helicity in their N-termini. The high resolution structures suggest that maximum activity relies on the appropriate balance between an N-terminal region with mixed hydrophobic/hydrophilic structure features and an amphipathic central and C-terminal region. Taken together with previous studies, our results suggest that to improve the therapeutic ratio of AMPs, consideration should be given to including sequential histidine-pairs, keeping the overall charge of the peptide modest, and retaining a degree of structural plasticity and imperfect amphipathicity.

De novo assembly of Sockeye salmon kidney transcriptomes reveal a limited early response to piscine reovirus with or without infectious hematopoietic necrosis virus superinfection.

BACKGROUND: Piscine reovirus (PRV) has been associated with the serious disease known as Heart and Skeletal Muscle Inflammation (HSMI) in cultured Atlantic salmon Salmo salar in Norway. PRV is also prevalent in wild and farmed salmon without overt disease manifestations, suggesting multifactorial triggers or PRV variant-specific factors are required to initiate disease. In this study, we explore the head kidney transcriptome of Sockeye salmon Oncorhynchus nerka during early PRV infection to identify host responses in the absence of disease in hopes of elucidating mechanisms by which PRV may directly alter host functions and contribute to the development of a disease state. We further investigate the role of PRV as a coinfecting agent following superinfection with infectious hematopoietic necrosis virus (IHNV) - a highly pathogenic rhabdovirus endemic to the west coast of North America. RESULTS: Challenge of Sockeye salmon with PRV resulted in high quantities of viral transcripts to become present in the blood and kidney of infected fish without manifestations of disease. De novo transcriptome assembly of over 2.3 billion paired RNA-seq reads from the head kidneys of 36 fish identified more than 320,000 putative unigenes, of which less than 20 were suggested to be differentially expressed in response to PRV at either 2 or 3 weeks post challenge by DESeq2 and edgeR analysis. Of these, only one, Ependymin, was confirmed to be differentially expressed by qPCR in an expanded sample set. In contrast, IHNV induced substantial transcriptional changes (differential expression of > 20,000 unigenes) which included transcripts involved in antiviral and inflammatory response pathways. Prior infection with PRV had no significant effect on host responses to superinfecting IHNV, nor did host responses initiated by IHNV exposure influence increasing PRV loads. CONCLUSIONS: PRV does not substantially alter the head kidney transcriptome of Sockeye salmon during early (2 to 3 week) infection and dissemination in a period of significant increasing viral load, nor does the presence of PRV change the host transcriptional response to an IHNV superinfection. Further, concurrent infections of PRV and IHNV do not appear to significantly influence the infectivity or severity of IHNV associated disease, or conversely, PRV load.