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.

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.

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.

RNA-Seq Analysis of the Growth Hormone Transgenic Female Triploid Atlantic Salmon (Salmo salar) Hepatic Transcriptome Reveals Broad Temperature-Mediated Effects on Metabolism and Other Biological Processes.

This study examined the impact of rearing temperature (10.5, 13.5 or 16.5°C) on the hepatic transcriptome of AquAdvantage Salmon (growth hormone transgenic female triploid Atlantic salmon) at an average weight of 800 g. Six stranded PE libraries were Illumina-sequenced from each temperature group, resulting in an average of over 100 M raw reads per individual fish. RNA-sequencing (RNA-seq) results showed the greatest difference in the number of differentially expressed transcripts (1750 DETs), as revealed by both DESeq2 and edgeR (q < 0.05; fold-change > |1.5|), was between the 10.5 and 16.5°C temperature groups. In contrast, 172 and 52 DETs were found in the 10.5 vs. 13.5°C and the 13.5 vs. 16.5°C comparisons, respectively. Considering the DETs between the 10.5 and 16.5°C groups, 282 enriched gene ontology (GO) terms were identified (q < 0.05), including "response to stress", "immune system process", "lipid metabolic process", "oxidation-reduction process", and "cholesterol metabolic process", suggesting elevated temperature elicited broad effects on multiple biological systems. Pathway analysis using ClueGO showed additional impacts on amino acid and lipid metabolism. There was a significant positive correlation between RNA-seq and real-time quantitative polymerase chain reaction (RT-qPCR) results for 8 of 9 metabolic-related transcripts tested. RT-qPCR results also correlated to changes in fillet tissue composition previously reported in these salmon (e.g., methionine and lysine concentrations positively correlated with hsp90ab1 transcript expression), suggesting that rearing temperature played a significant role in mediating metabolic/biosynthetic pathways of AquAdvantage Salmon. Many transcripts related to lipid/fatty acid metabolism (e.g., elovl2, fabpi, hacd2, mgll, s27a2, thrsp) were downregulated at 16.5°C compared to both other temperature groups. Additionally, enrichment of stress-, apoptosis- and catabolism-relevant GO terms at 16.5°C suggests that this temperature may not be ideal for commercial production when using freshwater recirculating aquaculture systems (RAS). This study relates phenotypic responses to transcript-specific findings and therefore aids in the determination of an optimal rearing temperature for AquAdvantage Salmon. With approval to grow and sell AquAdvantage Salmon in the United States and Canada, the novel insights provided by this research can help industry expansion by promoting optimal physiological performance and health.

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.

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.

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.