All fish handling procedures employed in the study were in accordance with national (Approval ID 11814) and EU legislation (2010/63/EU) on animal experimentation. Directly after fertilization, Atlantic salmon (AquaGen strain) eggs were kept at 60% (LO) and 80–100% NO DO levels for approximately 10 months (Figure 1). Oxygen levels above 80% are relevant to aquaculture operation and do not affect salmon growth. Eggs from each of the two groups were kept in triplicate tanks (for more details, see Kelly et al., 2020). After 10 months, the fish were PIT-tagged for individual identification and kept at 80–100% DO. At 14 months, smoltification was induced by the following light regimen: 6 weeks of 18 h darkness and 6 h light (winter), followed by 6 weeks of 24 h light (summer). Temperature during the first month after hatching was 7°C, after that the fish were kept at 10°C for the rest of the experimental period. The smoltification process was followed by seawater challenge tests (n = 10 per group) at 0, 3, and 6 weeks after the start of summer period. After 24 h, they were sacrificed using an overdose of Benzoak (0.3 mL/L) and blood was sampled for serum cortisol concentration and gill Na ATPase activity (Eurofins) analyses, to assess the development of hypo-osmoregulatory capacity. Both groups were adapted to seawater after the summer period, and no difference in plasma cortisol levels and ATPase activity was observed between the groups during smoltification. In the part of the trial described here, a total of 276 fish were used: 60 for the seawater challenge tests and 216 fish for the following bacterial challenge test. No mortalities were registered before the bacterial challenge test was started. No significant weight differences between the two groups were registered during the experimental period.

A bacterial challenge test with M. viscosa was performed after smoltification, at 17 months and at mean weight 81.2 ± 10.2 g. A total of 108 fish from each of the two groups were transferred to seawater in two 900-l circular tanks, 54 fish from each group per tank, at 10°C, and acclimated for 5 days before bath challenge (stagnant water, density 52 kg/m³, oxygenated, duration 1 h) with M. viscosa (LFI5006/2), originally isolated from the head kidney of farmed Atlantic salmon in northern Norway suffering from winter ulcer. Bacteria from frozen stock culture were grown at 12°C on blood agar (Oxoid) with 2% NaCl for 48 h and single colonies transferred to 20 ml Marine Broth (2216 Difco) and grown for 48 h at 12°C with shaking before inoculation of 600 ml MB and further growth for 48 h at 12°C. Before challenge, the OD_{600 nm} was measured and challenge dose used was 10⁷ cfu/ml. Challenge dose was confirmed by titration of the bacterial culture used for challenge on blood agar plates with 2% NaCl and counting of colonies. After the end of bath challenge, the fish were kept in running water at 10°C and density ca 30 kg/m³. Any background mortality during the challenge experiment was monitored in uninfected fish from the same groups and kept under the same conditions as the infected fish. Samples of gill, head kidney, and spleen were collected, at day 0 (start of continuous light of the smoltification process), day 42 of light stimulation (smolts), and 5 days before pathogen challenge, and at the end of the challenge trial. Mortality was registered for 36 days. Verification of M. viscosa as the cause of death was done by gross pathology (specifically wound development) and isolation of the pathogen from the head kidney samples of moribund fish on blood agar with 2% NaCl. At termination, all fish from the two tanks were scored for wounds, and 30 fish from each tank (five per group) were sampled for blood (plasma, erythrocytes), gill, skin, head kidney, and spleen in RNAlater.

Total RNA and DNA were isolated and purified using AllPrep DNA/RNA/miRNA Universal Kit (Qiagen) according to the manufacturer’s protocol. RNA quantity and quality were determined with Nanodrop (Thermo Scientific) and Bioanalyzer (Agilent).

To assess the immune competence (ImCom) of Atlantic salmon smolts and growers, a multigene expression assay representing the key functional groups of the immune system was designed on Biomark HD platform (Fluidigm; Krasnov et al., 2020). The first version of ImCom was used that includes 92 immune and stress genes, which were selected by the expression profiles in many challenge trials with pathogens and under inflammatory conditions, and reference genes. Analyses were performed in the gill, spleen, and head kidney of fish collected at the start and end of constant light stimulation (days 0 and 42, n = 6, totally 72 samples). The extracted RNA was adjusted at 10 ng/5 μl and reverse-transcribed using the Reverse transcription master mix (Fluidigm). Subsequently, the individual cDNA samples were added to the aforementioned 96 primer pairs (100 μM) and the PreAmp master mix (Fluidigm) and subjected to 12 pre-amplification cycles in a standard thermocycler (TAdvanced, Biometra). The pre-amplified products were treated with exonuclease I (New England BioLabs) and diluted in a SoFast EvaGreen supermix with Low ROX (Bio-Rad) and 20× DNA-binding dye sample loading reagent. The sample and primer mixes were transferred to the respective inlets of two 48.48 dynamic array IFC chips. These chips were individually primed in the BioMark IFC controller MX (Fluidigm) according to the Load mix 48.48 GE script. The loaded array chips were then placed in the BioMark HD system (Fluidigm) to proceed with the qPCR according to the GE 48 × 48 Fast PCR + Melt v2.pcl cycling program. Fluidigm RealTime PCR analysis software v. 3.0.2 was used to retrieve raw qPCR results, which were transferred in a relational database. The geometric means of two reference genes: elongation factor 1-alpha 1 and 40s ribosomal protein s20 (eef1a1b and rps20), which showed stability across samples, were used for calculation of ΔCt values. Further, the average for each gene was calculated for the entire data set and subtracted from each data point. Differential expression between the treatment groups and time-points was assessed by criteria: difference of ΔΔCt > | 0.8| and p < 0.05 (t-test).

Transcriptome analyses were carried out on gill and spleen of uninfected and challenged salmon with Nofima’s Atlantic salmon genome-wide 42.5 k DNA oligonucleotide microarray Salgeno-2 (GPL28080), totally 41 microarrays were used (n = 5). Microarrays were manufactured by Agilent Technologies, and the reagents and equipment were purchased from the same provider. Genes are annotated in Nofima’s bioinformatic pipeline STARS using GO, KEGG, and custom vocabulary (Krasnov et al., 2011a). RNA amplification and labeling were performed with a One-Color Quick Amp Labeling Kit and a Gene Expression Hybridization kit was used for fragmentation of labeled RNA. Total RNA input for each reaction was 500 ng. After overnight hybridization in an oven (17 h, 65°C, rotation speed 0.01 g), arrays were washed with Gene Expression Wash Buffers 1 and 2 and scanned with Agilent scanner. Subsequent data analyses were carried out with STARS. Global normalization was performed by equalizing the mean intensities of all microarrays. The individual values for each feature were divided by the mean value of all samples thus producing expression ratios (ER). The log₂-ER were calculated and normalized with the locally weighted non-linear regression (Lowess). The differentially expressed genes (DEG) were selected by criteria: log₂-ER > 0.8 (1.75-fold) and p < 0.05 (t-test). Enrichment of functional categories of gene Ontology (GO) was evaluated by comparing the numbers of genes per term in the lists of DEG, and on the microarray platform, significance was assessed with Yates’ corrected chi-square test. Data were submitted to NCBI GEO Omnibus (GSE171693).

The expression profiles of immune and stress genes in the lymphatic organs and gill were overall similar although the numbers and composition of DEG varied (Figures 2, 3). At day 0, the numbers of immune genes with lower expression in NO compared to LO ranged from 11 genes in the spleen to 19 genes in the head kidney and gill, while only one gene was upregulated in the spleen. During smoltification, many immune genes were downregulated, which is in concordance with our previous observations (Johansson et al., 2016; Robinson et al., 2017; Karlsen et al., 2018). The number of genes with decreased expression at day 42 was larger in tissues of salmon that had not been exposed to hypoxia: 42 versus 14 genes in the spleen and 28 versus 7 genes in the gill. Similar numbers of genes were downregulated in the head kidneys of LO and NO and only a few genes showed significant difference between the treatment groups at day 42, which was explained with high variance in LO. Although the decrease of expression in the end of smoltification clearly prevailed, a suite of genes showed upregulation at day 42, especially in the gill (Figure 3). A group of 15 genes included several markers of acute inflammation, such as chemokine lect2 (Mutoloki et al., 2010), a component of oxidative burst complex neutrophil cytosolic factor 1 and collagen degrading matrix metalloproteinases mmp9 and mmp13. Macrophage receptor marco (Poynter et al., 2017) and immunoglobulin receptor are involved in phagocytosis. Upregulation of these genes has been observed in Atlantic salmon under various diseases and inflammatory conditions (submitted manuscript). At day 0, the mean expression was 1.9-fold higher in LO. This group also showed a smaller reduction associated with smoltification (1.9-fold in NO and 1.5-fold in LO), the difference between the groups increased to 2.4-fold at day 42.

Four DEG with different immune roles showed similar expression profiles in the gill, head kidney, and spleen: an antibacterial peptide cathelicidin (Shinnar et al., 2003), diamine acetyltransferase 1, an enzyme of polyamine metabolism, apolipoprotein c-I, a cholesterol transporter involved in protection against pathogens (Fuior and Gafencu, 2019), and cd40 (tnfr5) a costimulatory protein of antigen presenting cells (Figure 3). Despite similar trends with respect to the treatment groups and time-points, the composition of DEG in the tissues was different. The number of DEG encoding cytokines, chemokines, and receptors was highest in the gill, similar to the genes involved in antigen presentation (respectively, six and four genes). Innate antiviral responses usually show the strongest suppression during smoltification. Four genes from this group [interferon a, and highly active virus responsive genes – VRG (Krasnov et al., 2011b) receptor transporting protein, viperin and isg15] were downregulated in the spleen. Several markers of T cells showed lower expression in the lymphatic organs of NO: cd4 and cd28 in the head kidney and cd28, cd152, and cd274 in the spleen. Difference between the treatment groups involved diverse functional groups and pathways of the immune system.

The first mortality in challenged salmon was recorded at 5 dpi in LO fish and at 8 dpi in NO fish (Figure 4). The difference peaked at 18% at 20–25 dpi (p < 0.05). At the end of trial at 36 dpi, mortality remained 10% lower in NO, although not significantly (Figure 3). Overall, LO showed a trend toward higher mortality throughout the trial. No significant differences in the mortality were registered in the replicate tanks during the challenge period, and data are presented as mean cumulative mortality of the two tanks.

The magnitude of transcriptome responses to the infection with M. viscosa reflected by the numbers of DEG was similar in the experimental groups and expression changes in the spleen were stronger in comparison with gill (Table 1). In spite of relatively small numbers of immune genes (from 7.7 to 13.1% of all DEG), the terms related to the immune system prevailed among the enriched functional categories of GO (Table 2). The enrichment analysis and inspection of DEG suggested strong and complex defensive responses to the bacterial pathogen. Putative homologs of pathogen recognition Toll-like receptors (tlr12 and tlr8) were upregulated in both tissues (Figure 5) and spleen (Figure 6B), respectively. Lipid mediators seemed to play a key part in cell signaling and communication. Six genes involved in eicosanoid metabolism were upregulated in both tissues (Figure 5). Two genes including phospholipase a2, which releases fatty acids precursors of eicosanoids from phospholipids, were activated in the gill and seven genes were upregulated in the spleen (Figure 6). Concurrent downregulation of four chemokines in the spleen and 11 chemokines in the gill suggested a trade-off between these two signaling systems. Reduced levels of chemokines transcripts in gill also indicated possible migration of immune cells to the infected sites or redistribution of cell populations in this organ. This could explain an apparent downregulation of several immune genes, such as free radicals producing inducible nitric oxide synthase (22- and 10-fold in, respectively, NO and LO fish), cytochrome b245, and neutrophil cytosolic factors – ncf2 (two other ncf2 were upregulated). Similar changes were observed in the complement factors c7 and c8 beta, rnase zf-3, an antibacterial effector with strong expression changes in gill of Atlantic salmon (Zanfardino et al., 2010; Król et al., 2020), and several other immune genes (Figure 5A). The pathogen stimulated both humoral and cellular immune responses. Diverse immune effectors were involved including antibacterial and acute phase proteins, components of the oxidative burst complex, serine and matrix metalloproteases (mmp 9 and mmp 13), and TNF-inducible metalloreductase steap 4; a hallmark of Atlantic salmon responses to M. viscosa in this trial was upregulation of a large set of lectins in both tissues and especially in the spleen. Five genes showed opposite changes in tissues including three chemokines, cathelicidin, and differentially regulated trout protein (Figure 5).

Among genes that responded to M. viscosa, only a few showed differences between the treatment groups (highlighted in Figures 5, 6). The bacterial infection caused minor changes in expression of genes involved in antiviral immunity. However, differences between LO and NO fish increased after challenge, especially in the gill. A panel of genes exceeded the threshold of differential expression; higher expression in LO was shown by three interferons and a number of VRG including highly specialized antiviral effectors, such as receptor transporting protein, gig1-1, ifit5, and very large inducible gtpase (Figure 7).