In recent years there have been a number of instances where declines in water temperature are suspected to have contributed to winter mortalities at salmon cage-sites in Atlantic Canada. In addition, it is expected that temperature variability due to more intense and frequent winter storms will increase with climate change, and this may further threaten Atlantic salmon health and welfare in sea-cages. To better understand cage-site conditions and aspects of salmon behaviour and physiology during the winter, we fitted Atlantic salmon in commercial size sea-cages in Newfoundland (Canada) for two consecutive years with internal and external data storage tags (DSTs) that recorded depth, temperature, heart rate (fH) and 3D-acceleration (swimming activity). Tags were surgically attached / implanted in late October / early November when water temperatures were 8-10 degrees C, and the DSTs were collected at the time of harvest. Although tag retrieval was limited due to some early mortality, challenges due to Covid-19 and with fish identification during harvest, the data collected for both years were comparable. Water temperature decreased at a rate of 0.35-0.57 degrees C week(-1) from October / November to March, and the coolest average weekly temperature experienced by the fish was 1.10 degrees C in early March of both years. Average f H decreased with temperature starting in the fall, and the fish primarily occupied the upper 5 m of the cage for the duration of this study, despite the fact that temperatures were generally homogeneous with depth. Nonetheless, vertical migrations to deeper depths were observed during some periods (e.g., at night during the coldest months), and thus, the average depth of the fish was greater during these times. These data reveal that temperatures in sea-cages in Newfoundland remain below 5 degrees C for 5 months and are below 2 degrees C for 1.5 months. Thus, understanding how cold temperatures affect various aspects of salmon biology, and monitoring these fish during this period, is critical to the sustainability and future of this industry.

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 degrees 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 degrees 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.

ABSTRACTIn recent lab‐based experiments, some post‐smolt Atlantic salmon (Salmo salar) held at 3°C for 5 weeks exhibited a range of clinical signs. They became lethargic and swam at the water's surface, developed ulcers to the head and jaw (clinical signs similar to tenacibaculosis in Norwegian salmon aquaculture) and had fin erosion, and this was associated with significant mortalities. In addition, when fish with ‘early’ and ‘advanced’ stages of these different clinical signs were further examined, their livers were found to be large, pale and friable. Fish with this aetiology also had elevated aspartate aminotransferase levels (indicative of liver damage), elevated plasma [Na+], [Cl−] and osmolality (indicating osmoregulatory impairment), low glucose levels (likely limiting metabolic responses to maintain homeostasis) and high circulating cortisol levels (∼100 ng/mL). This suite of physiological disturbances is very similar to that observed in a condition referred to as ‘Winter Syndrome’ or ‘Winter Disease’ (WS/WD) in cultured gilthead sea bream (Sparus aurata) and other fish species. Thus, it appears that WS/WD described here for the first time in Atlantic salmon, alone or in combination with opportunistic infections, results in lipid deposition in the liver, compromising liver function and osmoregulatory capacity, and metabolic collapse that ultimately results in significant losses.

Escape of genetically distinct farmed Atlantic salmon (Salmo salar) raises concerns about their potential interactions with wild populations and the disruption of local adaptation through genetic admixture. It is often unknown whether genetic origin or common domestication effects will have a greater influence on consequences posed by escaped farmed fish. Previous work showed that domestication could have prevalent effects on the behaviour and growth of farmed salmon, independent of their genetic origin. Yet, less is known whether this extends more broadly to gene expression, particularly at critical early life stages. Thus, we compared the expression of 24 transcripts related to the immune response, structural maintenance, stress response and iron metabolism among distinct farmed (North American [NA] and European [EO]), wild (Newfoundland) and F1 hybrid salmon at hatching under controlled conditions using qPCR analyses. A slightly higher number of transcripts were differentially expressed between the wild population relative to EO (i.e. atf3a, atf3b, bnip3, trim37a, ftm, hp and gapdh) than NA-farmed salmon (i.e. epdl2, hba1a, hba1b, hbb4 and ftm). The most differences existed between the two farmed strains themselves (11 of 24 transcripts), with the fewest differentially expressed transcripts found between the F1 hybrids and the domesticated/wild maternal strains (4 of 24 transcripts). Interestingly, despite similarities in the overall extent of gene expression differences among cross types, the expression patterns differed relative to a past study that compared fry from the same cross types at the end of yolk sac absorption. Overall, our findings suggest that interbreeding of escaped farmed salmon with wild Newfoundland populations would alter transcript expression levels and that developmental stage influences these changes.

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.

Rising sea surface temperatures and heat waves are a challenge to salmon aquaculture, and the industry must endeavour to mitigate their impacts. To investigate genetic-based differences in upper thermal tolerance, 20 salmon families were exposed to an incremental temperature increase (+0.2 degrees C per day from 12 degrees C) to mimic the rise in temperatures experienced at sea-cages in Atlantic Canada during the summer, or held at 10 degrees C. Post-smolt Atlantic salmon exposed to the incremental temperature increase up to 20 degrees C gained more weight than fish reared at 10 degrees C over the same period, and there were family-specific differences (p < 0.05) in growth and body morphometrics. Significant differences were also detected between the families' incremental thermal maximum (ITMax), with average family ITMax values ranging from 23.3 to 25.0 degrees C. Fish weight and growth rate were not significantly related to ITMax, but a negative correlation (p < 0.01) was found between hepatosomatic index (HSI) and ITMax. In contrast, the critical thermal maximum (CTMax, measured using a temperature increase of 2 degrees C h(-1) from 10 degrees C) of the bottom and top four temperature tolerant families (as determined by ITMax) did not differ (similar to 28.0 degrees C). A negative correlation (p < 0.01) between HSI and CTMax was also found. However, no relationship was evident between relative ventricular mass (RVM) and ITMax or CTMax. Our data show that ITMax is a more sensitive metric of a salmon's upper temperature tolerance than even an 'environmentally relevant/realistic' CTMax test, and suggest that it could be used in breeding programs that aim to enhance the salmon's tolerance to withstand rising ocean temperatures.

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.

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.

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.

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.

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 degrees 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 degrees C day(-1) from their acclimation temperature (12 degrees C) to mimic natural sea-cage farming conditions during the summer in Newfoundland. Interestingly, feed intake remained high up to 22 degrees C, while previous studies have shown a decrease in salmon appetite after similar to 16-18 degrees 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 degrees 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 degrees C). In comparison, the critical thermal maximum (CTMax; temperature increased at 2 degrees C h(-1)) of the remaining IR fish that had been held at 12 degrees C was 28.5 degrees 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 degrees 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.

This study examined the effect of rearing temperature on growth performance and nutrient utilization of AquAdvantage Salmon (growth hormone transgenic female triploid Atlantic salmon) reared in a land-based freshwater recirculating aquaculture system (RAS) at three temperatures (10.5 degrees C, 13.5 degrees C, 16.5 degrees C) from first-feeding to 1500 g. All fish were fed the same commercial diets, which were analyzed for proximate, amino acid, fatty acid and mineral composition, and daily feed consumption was recorded for the calculation of feed conversion ratios (FCRs). Whole-body samples were collected at 300, 500, 800 and 1500 g for proximate composition, and fillet samples were collected at 500, 800 and 1500 g for amino acid, fatty acid, and mineral composition. Fillet yield, astaxanthin concentration and DSM SalmoFan (TM) colour were determined at 1500 g. Results indicated that fish reared at 16.5 degrees C required more feed per unit of biomass gain, and were less efficient at depositing important nutrients such as omega-3 fatty acids, compared to salmon reared at 10.5 degrees C and 13.5 degrees C. With higher viscerosomatic index (VSI) and condition factor (K) scores at 16.5 degrees C, these fish also diverted more dietary energy to lipid deposition in the viscera instead of muscle tissue compared to the lower temperature treatments. Fish reared at 16.5 degrees C had lighter fillet colouration compared to salmon reared at 13.5 degrees C, and exhibited lower fillet yields than the colder temperature groups. Fish reared at 16.5 degrees C reached the end target weight (1500 g) in 36 fewer days compared to 13.5 degrees C and 93 fewer days in contrast to 10.5 degrees C. However, thermal-unit growth coefficient (TGC) results indicated that growth rate was higher in fish reared at 10.5 degrees C compared to 16.5 degrees C for the majority of the trial. Collectively, these results suggest that it is preferable to rear AquAdvantage Salmon at either 10.5 degrees C or 13.5 degrees C in freshwater RAS. The information generated from this study is the first to provide insight into the relationships between rearing temperature and nutrient utilization in AquAdvantage Salmon for commercial application.

This study describes the process of developing an enzymatic pretreatment to improve the nutritional value of feather meal (FeM). In a first experiment, a full factorial design was used to examine the effects of various incubation conditions on the solubilization of nitrogen in FeM. We incubated FeM for 3 h with various levels of a commercial alkaline serine protease (Savinase® 16L), sodium sulphite (Na2SO3), and digestion buffer. A Savinase® 16L level of 3% (%FeM v/w), Na2SO3 level of 3% (%FeM w/w), and digestion buffer level of 500% (%FeM w/w) were identified as the optimal conditions. Under these optimal conditions, 45% of the nitrogen in FeM was solubilized. In a second experiment, we evaluated the effect of more economically sustainable incubation conditions on the in vitro digestibility of protein (pepsin-HCl digestibility and multistep protein evaluation) in FeM. Two FeMs were incubated with 0.5% Savinase® 16L (%FeM v/w), 2% Na2SO3 (%FeM w/w), and 200% buffer (%FeM w/w) for 24 h. The pretreatment improved pepsin-HCl digestibility by 7–16% and the total tract degradable protein content by 14–50%. Accordingly, this novel pretreatment could be applied in the animal feed industry to improve the nutritional value of FeM.

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.