Characterization of the impact of dietary immunostimulant CpG on the expression of mRNA biomarkers involved in the immune responses in Atlantic salmon (Salmo salar).

Infectious diseases have significantly impacted Atlantic salmon aquaculture worldwide. Modulating fish immunity with immunostimulant-containing functional feeds could be an effective strategy in mitigating disease problems. Previously, we characterized the impact of polyriboinosinic polyribocytidylic acid (pIC) and formalin-killed typical Aeromonas salmonicida bacterin on miRNA expression in Atlantic salmon fed a commercial diet with and without immunostimulant CpG. A set of miRNA biomarkers of Atlantic salmon head kidney responding to pIC and/or bacterin immune stimulations was identified (Xue et al., 2019) [1]. Herein, we report a complementary qPCR study that investigated the impact of the pIC, bacterin and dietary CpG on the expression of immune-relevant mRNAs (n = 31) using the same samples as in the previous study (Xue et al., 2019) [1]. Twenty-six of these genes were predicted target transcripts of the pIC- and/or bacterin-responsive miRNAs identified in the earlier study. The current data showed that pIC and/or bacterin stimulations significantly modulated the majority of the qPCR-analyzed genes involved in various immune pathways. Some genes responded to both stimulations (e.g. tnfa, il10rb, ifng, irf9, cxcr3, campb) while others appeared to be stimulation specific [e.g. irf3, irf7a, il1r1, mxa, mapk3 (pIC only); clra (bacterin only)]. A. salmonicida bacterin stimulation produced a strong inflammatory response (e.g. higher expression of il1b, il8a and tnfa), while salmon stimulated with pIC showed robust interferon responses (both type I and II). Furthermore, the current data indicated significant down-regulation of immune-relevant transcripts (e.g. tlr9, irf5, il1r1, hsp90ab1, itgb2) by dietary immunostimulant CpG, especially among pre-injection and PBS-injected fish. Together with our prior miRNA study, the present research provided complementary information on Atlantic salmon anti-viral and anti-bacterial immune responses and on how dietary CpG may modulate these responses.

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.

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.

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.

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.

Variations of physiological and innate immunological responses in goldfish (Carassius auratus) subjected to recurrent acute stress.

This study was undertaken to investigate the influence of repeated acute stress on the physiological status and non-specific immune response of goldfish, Carassius auratus. The acute stress was a succession of a 3 min-chasing period followed by a 2 min-air exposure. The goldfish in triplicate tanks were subjected 3 times daily to this stress for one (S3) or three (S9) days. A separate group of unstressed fish was used as control for each sampling time. Blood samples were collected 12, 48 and 120 h after the last stress procedure. Variations of globulin levels, plasma anti-protease and bactericidal activities were not significant in the present study. The haematological parameters and plasma total protein and albumin strongly declined in S9 fish 12 h post-stress compared to control fish. However, plasma cortisol, glucose and lactate levels in both S3 and S9 transiently increased compared to the control fish. Similarly, plasma peroxidase activity transiently increased in both stressed groups 12 h after stress. An increase in plasma lysozyme and complement activities suggested a hormesis-like effect with one-day acute stress improving the immunological response of goldfish while an extension of the stress period to three days impaired physiology and immunity for up to 5 days. This study revealed that recurrent acute stress could immunosuppress goldfish as usually expected of chronic stress.

Characterization and transcript expression analyses of four Atlantic salmon (Salmo salar) serpinh1 paralogues provide evidence of evolutionary divergence.

Atlantic salmon (Salmo salar) are not only the world's most economically important farmed fish in terms of total value, but also a salmonid, which means that they are invaluable for studies of the evolutionary fate of genes following multiple whole-genome duplication (WGD) events. In this study, four paralogues of the molecular chaperone serpinh1 were characterized in Atlantic salmon, as while this gene is considered to be a sensitive biomarker of heat stress in salmonids, mammalian studies have also identified it as being essential for collagen structural assembly and integrity. The four salmon paralogues were cloned and sequenced so that in silico analyses at the nucleotide and deduced amino acid levels could be performed. In addition, qPCR was used to measure: paralogue- and sex-specific constitutive serpinh1 expression across 17 adult tissues; and their expression in the liver and head kidney of male Atlantic salmon as affected by stress phenotype (high vs. low responder), increased temperature, and injection with a multi-valent vaccine. Compared to the other three paralogues, serpinh1a-2 had a unique constitutive expression profile across the 17 tissues. Although stress phenotype had minimal impact on the transcript expression of the four paralogues, injection with a commercial vaccine containing several formalin inactivated bacterins increased the expression of most paralogues (by 1.1 to 4.5-fold) across both tissues. At 20 °C, the expression levels of serpinh1a-1 and serpinh1a-2 were generally lower (by -1.1- to -1.6-fold), and serpinh1b-1 and serpinh1b-2 were 10.2- to 19.0-fold greater, in comparison to salmon held at 12 °C. With recent studies suggesting a putative link between serpinh1 and upper thermal tolerance in salmonids, the current research is a valuable first step in elucidating the potential mechanisms involved. This research: supports the use of serpinh1b-1 and serpinh1b-2 as a biomarkers of heat stress in salmon; and provides evidence of neo- and/or subfunctionalization between the paralogues, and important insights into how multiple genome duplication events can potentially lead to evolutionary divergence.

Vegetable omega-3 and omega-6 fatty acids differentially modulate the antiviral and antibacterial immune responses of Atlantic salmon.

The immunomodulatory effects of omega-3 and omega-6 fatty acids are a crucial subject of investigation for sustainable fish aquaculture, as fish oil is increasingly replaced by terrestrial vegetable oils in aquafeeds. Unlike previous research focusing on fish oil replacement with vegetable alternatives, our study explored how the omega-6 to omega-3 polyunsaturated fatty acid (PUFA) ratio in low-fish oil aquafeeds influences Atlantic salmon's antiviral and antibacterial immune responses. Atlantic salmon were fed aquafeeds rich in soy oil (high in omega-6) or linseed oil (high in omega-3) for 12 weeks and then challenged with bacterial (formalin-killed Aeromonas salmonicida) or viral-like (polyriboinosinic polyribocytidylic acid) antigens. The head kidneys of salmon fed high dietary omega-3 levels exhibited a more anti-inflammatory fatty acid profile and a restrained induction of pro-inflammatory and neutrophil-related genes during the immune challenges. The high-omega-3 diet also promoted a higher expression of genes associated with the interferon-mediated signaling pathway, potentially enhancing antiviral immunity. This research highlights the capacity of vegetable oils with different omega-6 to omega-3 PUFA ratios to modulate specific components of fish immune responses, offering insights for future research on the intricate lipid nutrition-immunity interplay and the development of novel sustainable low-fish oil clinical aquaculture feeds.

Transcriptomic response of lumpfish (Cyclopterus lumpus) head kidney to viral mimic, with a focus on the interferon regulatory factor family.

The economic importance of lumpfish (Cyclopterus lumpus) is increasing, but several aspects of its immune responses are not well understood. To discover genes and mechanisms involved in the lumpfish antiviral response, fish were intraperitoneally injected with either the viral mimic polyinosinic:polycytidylic acid [poly(I:C)] or phosphate-buffered saline (PBS; vehicle control), and head kidneys were sampled 24 hours post-injection (hpi) for transcriptomic analyses. RNA sequencing (RNA-Seq) (adjusted p-value <0.05) identified 4,499 upregulated and 3,952 downregulated transcripts in the poly(I:C)-injected fish compared to the PBS-injected fish. Eighteen genes identified as differentially expressed by RNA-Seq were included in a qPCR study that confirmed the upregulation of genes encoding proteins with antiviral immune response functions (e.g., rsad2) and the downregulation of genes (e.g., jarid2b) with potential cellular process functions. In addition, transcript expression levels of 12 members of the interferon regulatory factor (IRF) family [seven of which were identified as poly(I:C)-responsive in this RNA-Seq study] were analyzed using qPCR. Levels of irf1a, irf1b, irf2, irf3, irf4b, irf7, irf8, irf9, and irf10 were significantly higher and levels of irf4a and irf5 were significantly lower in the poly(I:C)-injected fish compared to the PBS-injected fish. This research and associated new genomic resources enhance our understanding of the genes and molecular mechanisms underlying the lumpfish response to viral mimic stimulation and help identify possible therapeutic targets and biomarkers for viral infections in this species.

Basal and immune-responsive transcript expression of two Atlantic salmon interferon regulatory factor 2 (irf2) paralogues.

Atlantic salmon (Salmo salar) is one of the most economically important aquaculture species globally. However, disease has become a prevalent threat to this industry. A thorough understanding of the genes and molecular pathways involved in the immune responses of Atlantic salmon is imperative for selective breeding of disease-resistant broodstock, as well as developing new diets and vaccines to mitigate the impact of disease. Members of the interferon regulatory factor (IRF) family of transcription factors play roles in the induction of interferons and other cytokines involved in host immune responses to intracellular and parasitic pathogens. IRF family members also play diverse roles in other biological processes, such as stress response, reproduction and development. The current study focused on one member of the IRF family: interferon regulatory factor 2 (irf2). As previously shown, due to the genome duplication that occurred ∼80 million years ago in the salmonid lineage, there are two irf2 paralogues in the Atlantic salmon genome. In silico analyses at the cDNA and deduced amino acid levels were conducted followed by phylogenetic tree construction with IRF2 amino acid sequences from various ray-finned fishes, cartilaginous fish and tetrapods. qPCR was then used to analyze paralogue-specific irf2 constitutive expression across 17 adult tissues, as well as responses to the viral mimic pIC (i.e., synthetic double-stranded RNA analog) in cultured macrophage-like cells (in vitro) and to infection with the Gram-negative bacterium Moritella viscosa in skin samples (in vivo). The qPCR studies showed sex- and paralogue-specific differences in expression across tissues. For example, expression of both paralogues was higher in ovary than in testes; expression (considering both sexes together) was highest for irf2-1 in gonad and for irf2-2 in hindgut. Both irf2 paralogues were responsive to pIC stimulation, but varied in their induction level, with irf2-1 having an overall stronger response than irf2-2. Only one paralogue, irf2-2, was significantly responsive to M. viscosa infection. Differences in irf2-1 and irf2-2 transcript expression levels constitutively across tissues, and in response to pIC and M. viscosa, may suggest neo- or subfunctionalization of the duplicated genes. This novel information expands current knowledge and provides insight into how genome duplication events may impact host regulation of important immune markers.

Global gene expression responses of Atlantic salmon skin to Moritella viscosa.

Moritella viscosa is a Gram-negative pathogen that causes large, chronic ulcers, known as winter-ulcer disease, in the skin of several fish species including Atlantic salmon. We used a bath challenge approach to profile the transcriptome responses of M. viscosa-infected Atlantic salmon skin at the lesion (Mv-At) and away from the lesion (Mv-Aw) sites. M. viscosa infection was confirmed through RNA-based qPCR assays. RNA-Seq identified 5212 and 2911 transcripts differentially expressed in the Mv-At compared to no-infection control and Mv-Aw groups, respectively. Also, there were 563 differentially expressed transcripts when comparing the Mv-Aw to control samples. Our results suggest that M. viscosa caused massive and strong, but largely infection site-focused, transcriptome dysregulations in Atlantic salmon skin, and its effects beyond the skin lesion site were comparably subtle. The M. viscosa-induced transcripts of Atlantic salmon were mainly involved in innate and adaptive immune response-related pathways, whereas the suppressed transcripts by this pathogen were largely connected to developmental and cellular processes. As validated by qPCR, M. viscosa dysregulated transcripts encoding receptors, signal transducers, transcription factors and immune effectors playing roles in TLR- and IFN-dependent pathways as well as immunoregulation, antigen presentation and T-cell development. This study broadened the current understanding of molecular pathways underlying M. viscosa-triggered responses of Atlantic salmon, and identified biomarkers that may assist to diagnose and combat this pathogen.

Nutritional immunomodulation of Atlantic salmon response to Renibacterium salmoninarum bacterin.

We investigated the immunomodulatory effect of varying levels of dietary ω6/ω3 fatty acids (FA) on Atlantic salmon (Salmo salar) antibacterial response. Two groups were fed either high-18:3ω3 or high-18:2ω6 FA diets for 8 weeks, and a third group was fed for 4 weeks on the high-18:2ω6 diet followed by 4 weeks on the high-18:3ω3 diet and termed "switched-diet". Following the second 4 weeks of feeding (i.e., at 8 weeks), head kidney tissues from all groups were sampled for FA analysis. Fish were then intraperitoneally injected with either a formalin-killed Renibacterium salmoninarum bacterin (5 × 107 cells mL-1) or phosphate-buffered saline (PBS control), and head kidney tissues for gene expression analysis were sampled at 24 h post-injection. FA analysis showed that the head kidney profile reflected the dietary FA, especially for C18 FAs. The qPCR analyses of twenty-three genes showed that both the high-ω6 and high-ω3 groups had significant bacterin-dependent induction of some transcripts involved in lipid metabolism (ch25ha and lipe), pathogen recognition (clec12b and tlr5), and immune effectors (znrf1 and cish). In contrast, these transcripts did not significantly respond to the bacterin in the "switched-diet" group. Concurrently, biomarkers encoding proteins with putative roles in biotic inflammatory response (tnfrsf6b) and dendritic cell maturation (ccl13) were upregulated, and a chemokine receptor (cxcr1) was downregulated with the bacterin injection regardless of the experimental diets. On the other hand, an inflammatory regulator biomarker, bcl3, was only significantly upregulated in the high-ω3 fed group, and a C-type lectin family member (clec3a) was only significantly downregulated in the switched-diet group with the bacterin injection (compared with diet-matched PBS-injected controls). Transcript fold-change (FC: bacterin/PBS) showed that tlr5 was significantly over 2-fold higher in the high-18:2ω6 diet group compared with other diet groups. FC and FA associations highlighted the role of DGLA (20:3ω6; anti-inflammatory) and/or EPA (20:5ω3; anti-inflammatory) vs. ARA (20:4ω6; pro-inflammatory) as representative of the anti-inflammatory/pro-inflammatory balance between eicosanoid precursors. Also, the correlations revealed associations of FA proportions (% total FA) and FA ratios with several eicosanoid and immune receptor biomarkers (e.g., DGLA/ARA significant positive correlation with pgds, 5loxa, 5loxb, tlr5, and cxcr1). In summary, dietary FA profiles and/or regimens modulated the expression of some immune-relevant genes in Atlantic salmon injected with R. salmoninarum bacterin. The modulation of Atlantic salmon responses to bacterial pathogens and their associated antigens using high-ω6/high-ω3 diets warrants further investigation.

Liver Transcriptome Profiling Reveals That Dietary DHA and EPA Levels Influence Suites of Genes Involved in Metabolism, Redox Homeostasis, and Immune Function in Atlantic Salmon (Salmo salar).

The optimal dietary requirement of omega-3 long-chain polyunsaturated fatty acids (ω3 LC-PUFA), namely docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), for Atlantic salmon that promotes growth and health warrants careful investigation. We used 44K microarrays to study the influence of increasing levels of dietary DHA + EPA (0, 1.0, and 1.4% of the diet, as formulated) in the presence of high linoleic acid (LA) on Atlantic salmon growth and liver transcriptome. After a 14-week feeding trial, Atlantic salmon fed diet ω3LC0 (i.e. 0% of DHA + EPA) showed significantly lower final weight and weight gain, and higher feed conversion ratio compared with ω3LC1.0 and ω3LC1.4 diet groups. The microarray experiment identified 55 and 77 differentially expressed probes (Rank Products analyses; PFP < 10%) in salmon fed diets ω3LC1.4 and ω3LC1.0 compared with those fed diet ω3LC0, respectively. The comparison between ω3LC1.4 and ω3LC1.0 revealed 134 differentially expressed probes. The microarray results were confirmed by qPCR analyses of 22 microarray-identified transcripts. Several key genes involved in fatty acid metabolism including LC-PUFA synthesis were upregulated in fish fed ω3LC0 compared with both other groups. Hierarchical clustering and linear regression analyses of liver qPCR and fatty acid composition data demonstrated significant correlations. In the current study, 1.0% ω3 LC-PUFA seemed to be the minimum requirement for Atlantic salmon based on growth performance; however, multivariate statistical analyses (PERMANOVA and SIMPER) showed that fish fed ω3LC1.0 and ω3LC1.4 diets had similar hepatic fatty acid profiles but marked differences in the transcript expression of biomarker genes involved in redox homeostasis (mgst1), immune responses (mxb, igmb, irf3, lect2a, srk2, and lyz2), and LC-PUFA synthesis (srebp1, fadsd5, and elovl2). This research has provided new insights into dietary requirement of DHA and EPA and their impact on physiologically important pathways in addition to lipid metabolism in Atlantic salmon.

Transcriptomic Profiling of the Adaptive and Innate Immune Responses of Atlantic Salmon to Renibacterium salmoninarum Infection.

Bacterial Kidney Disease (BKD), which is caused by a Gram-positive, intracellular bacterial pathogen (Renibacterium salmoninarum), affects salmonids including Atlantic salmon (Salmo salar). However, the transcriptome response of Atlantic salmon to BKD remained unknown before the current study. We used a 44K salmonid microarray platform to characterise the global gene expression response of Atlantic salmon to BKD. Fish (~54 g) were injected with a dose of R. salmoninarum (H-2 strain, 2 × 108 CFU per fish) or sterile medium (control), and then head kidney samples were collected at 13 days post-infection/injection (dpi). Firstly, infection levels of individuals were determined through quantifying the R. salmoninarum level by RNA-based TaqMan qPCR assays. Thereafter, based on the qPCR results for infection level, fish (n = 5) that showed no (control), higher (H-BKD), or lower (L-BKD) infection level at 13 dpi were subjected to microarray analyses. We identified 6,766 and 7,729 differentially expressed probes in the H-BKD and L-BKD groups, respectively. There were 357 probes responsive to the infection level (H-BKD vs. L-BKD). Several adaptive and innate immune processes were dysregulated in R. salmoninarum-infected Atlantic salmon. Adaptive immune pathways associated with lymphocyte differentiation and activation (e.g., lymphocyte chemotaxis, T-cell activation, and immunoglobulin secretion), as well as antigen-presenting cell functions, were shown to be differentially regulated in response to BKD. The infection level-responsive transcripts were related to several mechanisms such as the JAK-STAT signalling pathway, B-cell differentiation and interleukin-1 responses. Sixty-five microarray-identified transcripts were subjected to qPCR validation, and they showed the same fold-change direction as microarray results. The qPCR-validated transcripts studied herein play putative roles in various immune processes including pathogen recognition (e.g., tlr5), antibacterial activity (e.g., hamp and camp), regulation of immune responses (e.g., tnfrsf11b and socs1), T-/B-cell differentiation (e.g., ccl4, irf1 and ccr5), T-cell functions (e.g., rnf144a, il13ra1b and tnfrsf6b), and antigen-presenting cell functions (e.g., fcgr1). The present study revealed diverse immune mechanisms dysregulated by R. salmoninarum in Atlantic salmon, and enhanced the current understanding of Atlantic salmon response to BKD. The identified biomarker genes can be used for future studies on improving the resistance of Atlantic salmon to BKD.

Characterization and Transcript Expression Analyses of Atlantic Cod Viperin.

Viperin is a key antiviral effector in immune responses of vertebrates including the Atlantic cod (Gadus morhua). Using cloning, sequencing and gene expression analyses, we characterized the Atlantic cod viperin at the nucleotide and hypothetical amino acid levels, and its regulating factors were investigated. Atlantic cod viperin cDNA is 1,342 bp long, and its predicted protein contains 347 amino acids. Using in silico analyses, we showed that Atlantic cod viperin is composed of 5 exons, as in other vertebrate orthologs. In addition, the radical SAM domain and C-terminal sequences of the predicted Viperin protein are highly conserved among various species. As expected, Atlantic cod Viperin was most closely related to other teleost orthologs. Using computational modeling, we show that the Atlantic cod Viperin forms similar overall protein architecture compared to mammalian Viperins. qPCR revealed that viperin is a weakly expressed transcript during embryonic development of Atlantic cod. In adults, the highest constitutive expression of viperin transcript was found in blood compared with 18 other tissues. Using isolated macrophages and synthetic dsRNA (pIC) stimulation, we tested various immune inhibitors to determine the possible regulating pathways of Atlantic cod viperin. Atlantic cod viperin showed a comparable pIC induction to other well-known antiviral genes (e.g., interferon gamma and interferon-stimulated gene 15-1) in response to various immune inhibitors. The pIC induction of Atlantic cod viperin was significantly inhibited with 2-Aminopurine, Chloroquine, SB202190, and Ruxolitinib. Therefore, endosomal-TLR-mediated pIC recognition and signal transducers (i.e., PKR and p38 MAPK) downstream of the TLR-dependent pathway may activate the gene expression response of Atlantic cod viperin. Also, these results suggest that antiviral responses of Atlantic cod viperin may be transcriptionally regulated through the interferon-activated pathway.

Full characterization and transcript expression profiling of the interferon regulatory factor (IRF) gene family in Atlantic cod (Gadus morhua).

Atlantic cod (Gadus morhua) represents a unique immune system among teleost fish, making it a species of interest for immunological studies, and especially for investigating the evolutionary history of immune gene families. The interferon regulatory factor (IRF) gene family encodes transcription factors which function in the interferon pathway, but also in areas including leukocyte differentiation, cell growth, autoimmunity, and development. We previously characterized several IRF family members in Atlantic cod (Irf4a, Irf4b, Irf7, Irf8, and two Irf10 splice variants) at the cDNA and putative amino acid levels, and in the current study we took advantage of the new and improved Atlantic cod genome assembly in combination with rapid amplification of cDNA ends (RACE) to characterize the remaining family members (i.e. Irf3, Irf5, Irf6, Irf9, and two Irf2 splice variants). Real-time quantitative PCR (QPCR) was used to investigate constitutive expression of all IRF transcripts during embryonic development, suggesting several putative maternal transcripts, and potential stage-specific roles. QPCR studies also showed 11 of 13 transcripts were responsive to stimulation with poly(I:C), while 6 of 13 transcripts were responsive to lipopolysaccharide (LPS) in Atlantic cod head kidney macrophages, indicating roles for cod IRF family members in both antiviral and antibacterial responses. This study is the first to investigate expression of the complete IRF family in Atlantic cod, and suggests potential novel roles for several of these transcription factors within immunity as well as in early development of this species.

Discovery of microRNAs associated with the antiviral immune response of Atlantic cod macrophages.

MicroRNAs (miRNAs) are known to play important immunoregulatory roles in teleosts, although miRNAs involved in the antiviral immune response of Atlantic cod (Gadus morhua) were previously uncharacterised. Using deep sequencing and qPCR, the present study was conducted to identify miRNAs responsive to the viral mimic, polyriboinosinic polyribocytidylic acid (pIC) in Atlantic cod macrophages. Macrophage samples isolated from Atlantic cod (n=3) and treated with pIC or phosphate buffered saline (PBS control) for 24 and 72h were used for miRNA profiling. Following deep sequencing, DESeq2 analyses identified four (miR-731-3p, miR-125b-3-3p, miR-150-3p and miR-462-3p) and two (miR-2188-3p and miR-462-3p) significantly differentially expressed miRNAs at 24 and 72h post-stimulation (HPS), respectively. Sequencing-identified miRNAs were subjected to qPCR validation using a larger number of biological replicates (n=6) exposed to pIC or PBS over time (i.e. 12, 24, 48 and 72 HPS). As in sequencing, miR-731-3p, miR-462-3p and miR-2188-3p showed significant up-regulation by pIC. The sequencing results were not qPCR-validated for miR-125b-3-3p and miR-150-3p as up- and down-regulated miRNAs at 24 HPS, respectively; however, qPCR results showed significant up-regulation in response to pIC stimulation at later time points (i.e. 48 and/or 72 HPS). We also used qPCR to assess the expression of other miRNAs that were previously shown as immune responsive in other vertebrates. qPCR results at 48 and/or 72 HPS revealed that miR-128-3-5p, miR-214-1-5p and miR-451-3p were induced by pIC, whereas miR-30b-3p and miR-199-1-3p expression were repressed in response to pIC. The present study identified ten pIC-stimulated miRNAs, suggesting them as important in antiviral immune responses of Atlantic cod macrophages. Some pIC-responsive miRNAs identified in this study were predicted to target putative immune-related genes of Atlantic cod (e.g. miR-30b-3p targeting herc4), although the regulatory functions of these miRNAs need to be validated by future studies.

Transcriptome profiling of the antiviral immune response in Atlantic cod macrophages.

A study was conducted to determine the transcriptome response of Atlantic cod (Gadus morhua) macrophages to the viral mimic, polyriboinosinic polyribocytidylic acid (pIC), using a 20K Atlantic cod microarray platform and qPCR. We identified 285 significantly up-regulated and 161 significantly down-regulated probes in cod macrophages 24 h after pIC stimulation. A subset of 26 microarray-identified transcripts was subjected to qPCR validation using samples treated with pIC or phosphate-buffered saline (control) over time (3, 6, 12, 24, 48 h), and 77% of them showed a significant response to pIC. The microarray and qPCR analyses in this study showed that pIC induced the expression of cod macrophage transcripts involved in RLR- and TLR-dependent pathogen recognition (e.g. tlr3, tlr7, mda5 and lgp2), as well as signal transducers (e.g. stat1 and nfkbia) and transcription activators (e.g. irf7 and irf10) in the MyD88-independent and dependent signalling pathways. Several immune effectors (e.g. isg15s, viperin, herc4, mip2 and ccl13) were significantly up-regulated in pIC-stimulated cod macrophages. The expression of some transcripts (e.g. irf7, irf10, viperin) was significantly up-regulated by pIC as early as 12 h. All pIC-induced transcripts had peak expression at either 24 h (e.g. tlr7, irf7, mip2) or 48 h (e.g. tlr3, lgp2, stat1). This study suggests possible roles of both vertebrate-conserved (e.g. tlr3 as an up-regulated gene) and fish-specific (tlr22g as a down-regulated gene) receptors in dsRNA recognition, and the importance of conserved and potentially fish-specific interferon stimulated genes in cod macrophages.

Variations of some physiological and immunological parameters in Siberian sturgeon (Acipenser baerii, Brandt, 1869) subjected to an acute stressor.

This study was carried out to investigate the effect of an acute stressor on the variation of some physiological and immunological parameters of Siberian sturgeon (Acipenser baerii) juveniles. Fish, reared in 3 tanks for 10 weeks, were used for this study. The acute stress of fish consisted of 2 min of air exposure stress. Plasma levels of cortisol, glucose, and lactate as well as lysozyme activity in plasma were measured before stress and 1 hr, 3 hr, 6 hr, 9 hr, 12 hr, and 24 hr after stress. The plasma cortisol significantly increased in the highest level 1 hr after stress, yet it gradually declined after 3 hr. The glucose significantly increased only 1 hr after stress. There was no significant difference between plasma lactate prestress and poststress. Moreover, lysozyme activity was enhanced by stress, thus reaching the highest level 9 hr after stress. The results of this study indicate that Siberian sturgeon not only have a rapid response to acute stress, but also a great capacity for recovery from stress, thus returning physiological parameters to prestress levels after 6 hr.