Importance of environmental signals for cardiac morphological development in Atlantic salmon.

The hearts of salmonids display remarkable plasticity, adapting to various environmental factors that influence cardiac function and demand. For instance, in response to cold temperature, the salmonid heart undergoes growth and remodeling to counterbalance the reduced contractile function associated with dropping temperatures. Alongside heart size, the distinct pyramidal shape of the wild salmonid heart is essential for optimal cardiac performance, yet the environmental drivers behind this optimal cardiac morphology remain to be fully understood. Intriguingly, farmed salmonids often have rounded, asymmetrical ventricles and misaligned bulbi from an early age. These deformities are noteworthy given that farmed salmon are often not exposed to natural cues, such as a gradual temperature increase and changing day lengths, during critical developmental stages. In this study, we investigated whether natural environmental conditions during early life stages are pivotal for proper cardiac morphology. Atlantic salmon were raised under simulated natural conditions (low temperature with a natural photoperiod; SIMNAT) and compared with those reared under simulated farming conditions (SIMFARM). Our findings reveal that the ventricle shape and bulbus alignment in SIMNAT fish closely resemble those of wild salmon, while functional analyses indicate significant differences between SIMNAT and SIMFARM hearts, suggesting diastolic dysfunction and higher cardiac workload in SIMFARM hearts. These findings highlight the profound influence of environmental factors such as water temperature and photoperiod on the structural development of the salmonid heart, underscoring the importance of early environmental conditions for cardiac health.

Acute freshwater CO2 exposure does not impair seawater transfer in three different sizes of Atlantic salmon (Salmo salar) subjected to different photoperiod manipulations.

There is a growing interest in Atlantic salmon (Salmo salar) aquaculture to extend the time fish are reared in freshwater (FW) recirculating aquaculture systems (RAS), producing larger FW salmon that can then be induced to undergo smoltification before transfer into marine net pens for grow-out and harvest. Smolts can be produced by photoperiod (PT) manipulation in RASs, but little is known about how delaying smoltification to larger body sizes affects susceptibility to elevated CO2 levels (hypercapnia), which can occur at high stocking densities in FW RAS or during transport from FW RAS rearing facilities to marine net pens. To address this, Atlantic salmon were reared from hatch to one of three different sizes (~230, ~580, or ~1300 g) in FW (3 ppt) under continuous light (24:0, light:dark). Once fish reached the desired sizes, a group of salmon were maintained on continuous light 24L:0D to serve as a control salmon. A second group of salmon were exposed to 8 weeks of 12L:12D and then to 4 weeks of 24L:0D to serve as PT treatment salmon, which is the PT manipulation commonly used in Atlantic salmon aquaculture to induce smoltification. At the end of PT manipulation, both control and PT treatment salmon were exposed to 0% or 1.5% CO2 (30 mg/L) for 96 h in FW and then transferred to air-equilibrated seawater (SW, 35 ppt, normocapnia). Salmon were sampled at the end of the 96-h FW CO2 exposure and at 24 h and 7 days in SW for measurements of blood ion/acid-base status, muscle water content (MWC), and gill and kidney Na+/K+ ATPase (NKA) activity. Exposure to 96 h of CO2 in FW resulted in acid-base disturbances in fish from all three size classes, with decreases in blood pH and increases in blood PCO2 and plasma [HCO3 -] but no mortality. Despite these large acid-base disturbances in FW, after transfer to normocapnic SW, there were no significant effects of CO2 exposure on extracellular blood pH, intracellular red blood cell pH, or plasma osmoregulatory status for all three sizes of post-smolt salmon. In general, SW transfer was associated with significant increases in plasma ions and osmolality, as well as gill and kidney NKA activity after 24 h and 1 week in SW with no significant impacts between different sizes of salmon. Thus, exposure to 30 mg CO2/L that mimics levels experienced during transport from FW RAS to an SW transfer site may have minimal effects on Atlantic salmon smolts up to 1300 g.

Change in Activities of Enzymes of Energy and Carbohydrate Metabolism in Pink Salmon Oncorhynchus gorbuscha (Walb.) Smolts with Change in Environmental Salinity.

Activities of key enzymes of energy and carbohydrate metabolism (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), aldolase, glucose 6-phosphate dehydrogenase (G6PDH), and 1-glycerophosphate dehydrogenase (1-GPDH)) were studied in pink salmon Oncorhynchus gorbuscha (Walb.) smolts from the White Sea in a cage experiment simulating the transition from a freshwater to a marine environment. A decrease in COX, G6PDH, and 1-GPDH activities and an increase in LDH and aldolase activities were observed in juveniles with an increase in water salinity. Based on the findings, a redistribution of energy substrates between the reactions of aerobic and anaerobic metabolism towards higher anaerobic ATP synthesis was assumed for pink salmon. This may indicate that adaptive mechanisms rearrange metabolism to provide energy for osmoregulation in pink salmon juveniles when the salinity changes in their habitat.

Dietary plant oil supplemented with arachidonic acid and eicosapentaenoic acid affects the fatty acid composition and eicosanoid metabolism of Atlantic salmon (Salmo salar L.) during smoltification.

This study sought to investigate whether a "natural diet" (mimicking the fatty acid composition of freshwater aquatic insects eaten by salmon parr) during the freshwater (FW) life stage of pre-smolt Atlantic salmon (Salmo salar L.) affected red blood cells and gill fatty acid composition as well as eicosanoid metabolism in gill during smolting at different temperatures. Before being transferred to seawater (SW), salmon parr were fed with a modified (MO) diet containing vegetable oils (rapeseed, palm, and linseed oils) supplemented with eicosapentaenoic acid (EPA) and arachidonic acid (ARA) to completely replace the fish oil (FO). Fatty acid composition in red blood cells and gill tissues was determined before SW transfer and six weeks after. Additionally, the expression of genes associated with eicosanoid metabolism and Na+/K+-ATPase (NKA) activity in salmon gill was examined at different temperatures before SW transfer and 24 h after. The results showed the changes in fatty acid composition, including sum monounsaturated fatty acids (MUFAs), docosahexaenoic acid (DHA), ARA, EPA, and sum n-6 polyunsaturated fatty acids (n-6 PUFA) in both red blood cells and gill tissues at the FW stage were consistent with the fatty acid profiles of the supplied MO and FO fish diets; however sum EPA and DHA composition exhibited opposite trends to those of the FO diet. The proportion of ARA, EPA, and n-6 PUFA increased, whereas sum MUFAs and DHA decreased in the red blood cells and gill tissues of MO-fed fish compared to those fed with the FO diet at FW stage. Additionally, 5-lipoxygenase-activating protein (Flap) expression was downregulated in MO-fed fish prior to SW transfer. During the process of SW transfer at different temperatures, the MO diet remarkably suppressed NKAα1a expression in MO-fed fish both at 12 and 16 °C. The MO diet also upregulated phospholipase A2 group IV (PLA2g4) expression in gills at 8, 12, and 16 °C, but suppressed phospholipase A2 group VI (PLA2g6) expression in gills at 12 °C compared to FO-fed fish at 12 °C and MO-fed fish at 8 °C. The MO diet also upregulated Cyclooxygenase 2 (Cox-2) expression at 8 °C compared to FO-fed fish and increased Arachidonate 5-lipoxygenase (5-Lox) expression in MO-fed fish at 16 °C compared to both FO-fed fish at 16 °C and MO-fed fish at 8 °C. Our study also determined that both SW transfer water temperatures and diets during the FW period jointly influenced the mRNA expression of PLA2g4, PLA2g6, and Lpl, whereas 5-Lox was more sensitive to dietary changes. In conclusion, the MO diet affected the fatty acid composition in gill and in red blood cells. When transferred to SW, dietary ARA supplementation could promote the bioavailability for eicosanoid synthesis in gill mainly via PLA2g4 activation, and potentially inhibit the stress and inflammatory response caused by different water temperatures through dietary EPA supplementation.

Changes in circulating insulin-like growth factor-1 and its binding proteins in yearling rainbow trout during spring under natural and manipulated photoperiods and their relationships with gill Na+, K+-ATPase and body size.

Smoltification in salmonids occurs during spring in response to increasing photoperiod to prepare for marine life. Smoltification is associated with increased hypo-osmoregulatory ability and enhanced growth potential, mediated by growth hormone and insulin-like growth factor (IGF)-1. Rainbow trout is uniquely insensitive to the induction of smoltification-associated changes by photoperiod, such as the activation of gill Na+,K+-ATPase (NKA). We measured the circulating IGF-1 and IGF-binding protein (IGFBP)-2b levels in yearling rainbow trout exposed to natural and manipulated photoperiods during spring and correlated these with gill NKA activity and body size. Although the effect of photoperiod manipulation on body size and circulating IGF-1 and IGFBP-2b was negligible, they were positively correlated with gill NKA activity in fish under simulated natural photoperiod. We next pit-tagged yearling rainbow trout and fed them a restricted ration or to satiation under a natural photoperiod. In April, gill NKA activity was higher in the satiation group than in the restricted group and positively correlated with body size and growth rate. In addition, circulating IGFBP-2b was positively correlated with gill NKA, size and growth, whereas circulating IGF-1 was correlated only with size and growth. The relationship between circulating IGF-1 and growth intensified from May to June, suggesting that the IGF-1-growth relationship was disrupted in April when gill NKA was activated. Two additional IGFBPs were related to growth parameters but not to gill NKA activity. The present study suggests that circulating IGFBP-2b and IGF-1 mediate the size-dependent activation of gill NKA in yearling rainbow trout during spring.

Differential Expression of miRNAs and Their Predicted Target Genes Indicates That Gene Expression in Atlantic Salmon Gill Is Post-Transcriptionally Regulated by miRNAs in the Parr-Smolt Transformation and Adaptation to Sea Water.

Smoltification (parr-smolt transformation) is a complex developmental process consisting of developmental changes that lead to remodeling of the Atlantic salmon gill. Here, the expression changes of miRNAs and mRNAs were studied by small-RNA sequencing and microarray analysis, respectively, to identify miRNAs and their predicted targets associated with smoltification and subsequent sea water adaptation (SWA). In total, 18 guide miRNAs were identified as differentially expressed (gDE miRNAs). Hierarchical clustering analysis of expression changes divided these into one cluster of 13 gDE miRNAs with decreasing expression during smoltification and SWA that included the miRNA-146, miRNA-30 and miRNA-7132 families. Another smaller cluster that showed increasing expression consisted of miR-101a-3p, miR-193b-5p, miR-499a-5p, miR-727a-3p and miR-8159-5p. The gDE miRNAs were predicted to target 747 of the genes (DE mRNAs), showing expression changes in the microarray analysis. The predicted targets included genes encoding NKA-subunits, aquaporin-subunits, cystic fibrosis transmembrane conductance regulator and the solute carrier family. Furthermore, the predicted target genes were enriched in biological processes associated with smoltification and SWA (e.g., immune system, reactive oxygen species, stress response and extracellular matrix organization). Collectively, the results indicate that remodeling of the gill involves the post-transcriptional regulation of gene expression by the characterized gDE miRNAs.

Endoplasmic reticulum stress as a key mechanism in stunted growth of seawater rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Rainbow trout (Oncorhynchus mykiss) is a salmonid species with a complex life-history. Wild populations are naturally divided into freshwater residents and sea-run migrants. Migrants undergo an energy-demanding adaptation for life in seawater, known as smoltification, while freshwater residents display these changes in an attenuated magnitude and rate. Despite this, in seawater rainbow trout farming all fish are transferred to seawater. Under these circumstances, weeks after seawater transfer, a significant portion of the fish die (around 10%) or experience growth stunting (GS; around 10%), which represents an important profitability and welfare issue. The underlying causes leading to GS in seawater-transferred rainbow trout remain unknown. In this study, we aimed at characterising the GS phenotype in seawater-transferred rainbow trout using untargeted and targeted approaches. To this end, the liver proteome (LC-MS/MS) and lipidome (LC-MS) of GS and fast-growing phenotypes were profiled to identify molecules and processes that are characteristic of the GS phenotype. Moreover, the transcription, abundance or activity of key proteins and hormones related to osmoregulation (Gill Na+, K + -ATPase activity), growth (plasma IGF-I, and liver igf1, igfbp1b, ghr1 and ctsl) and stress (plasma cortisol) were measured using targeted approaches. RESULTS: No differences in Gill Na+, K + -ATPase activity and plasma cortisol were detected between the two groups. However, a significant downregulation in plasma IGF-I and liver igf1 transcription pointed at this growth factor as an important pathomechanism for GS. Changes in the liver proteome revealed reactive-oxygen-species-mediated endoplasmic reticulum stress as a key mechanism underlying the GS phenotype. From the lipidomic analysis, key observations include a reduction in triacylglycerols and elevated amounts of cardiolipins, a characteristic lipid class associated with oxidative stress, in GS phenotype. CONCLUSION: While the triggers to the activation of endoplasmic reticulum stress are still unknown, data from this study point towards a nutritional deficiency as an underlying driver of this phenotype.

Effect of two constant light regimens on antibody profiles and immune gene expression in Atlantic salmon following vaccination and experimental challenge with salmonid alphavirus.

Before seawater transfer, farmed Atlantic salmon are subjected to treatments that may affect the immune system and susceptibility to pathogens. E.g., exposure to constant light (CL) stimulates smoltification, which prepares salmon to life in sea water, but endocrine changes in this period are associated with suppression of immune genes. Salmon are vaccinated towards end of the freshwater period to safeguard that adequate vaccine efficacy is achieved by the time the fish is transferred to sea. In the present study, we investigated how the responses to vaccination and viral infection varied depending on the time of CL onset relative to vaccination. The salmon were either exposed to CL two weeks prior to vaccination (2-PRI) or exposed to CL at the time of vaccination (0-PRI). A cohabitant challenge with salmonid alphavirus, the causative agent of pancreatic disease, was performed 9 weeks post vaccination. The immunological effects of the different light manipulation were examined at 0- and 6-weeks post vaccination, and 6 weeks post challenge. Antibody levels in serum were measured using a serological bead-based multiplex panel as well as ELISA, and 92 immune genes in heart and spleen were measured using an integrated fluidic circuit-based qPCR array for multiple gene expression. The 2-PRI group showed a moderate transcript down-regulation of genes in the heart at the time of vaccination, which were restored 6 weeks after vaccination (WPV). Conversely, at 6WPV a down-regulation was seen for the 0-PRI fish. Moreover, the 2-PRI group had significantly higher levels of antibodies binding to three of the vaccine components at 6WPV, compared to 0-PRI. In response to SAV challenge, transcription of immune genes between 2-PRI and 0-PRI was markedly dissimilar in the heart and spleen of control fish, but no difference was found between vaccinated salmon from the two CL regimens. Thus, by using labor-saving high throughput detection methods, we demonstrated that light regimens affected antibody production and transcription of immune genes in non-vaccinated and virus challenged salmon, but the differences between the light treatment groups appeared eliminated by vaccination.

Testing of NKA expression by mobile real time PCR is an efficient indicator of smoltification status of farmed Atlantic salmon.

Assessment of seawater readiness of freshwater salmon smolts is a crucial husbandry step with economic implications in salmon aquaculture but current methods rely on delayed centralised enzymic activity measurement. The efficiency of a qRT-PCR assay for sodium potassium ATPase (NKA) α1a mRNA was tested in a 3-year study on 19 hatcheries across Scotland incorporating environmental factors such as temperature and metal contamination. The NKA qRT-PCR assay was transferred to a mobile laboratory and on-site testing was carried out at 3 hatchery sites. For the first two years standard enzymatic and gene expression assays had similar success rates in detecting smoltification (NKA activity 60%, qRT-PCR 57%). In the third year, all but one site were determined as sea water ready by qRT-PCR but only at 4 by enzymatic testing. On site testing with mobile qRT-PCR was successfully performed on four farm sites. Altogether, high sensitivity was shown for the in lab (98.9%, SE 0.24) and mobile (93.43%, SE 0.119) assays when tested using a quantitative RNA standard. Some indication for obscured smoltification assay results due to environmental increased heavy metal contamination was observed. Our results prove it is possible to test a smoltification marker on site and provide results on the day of testing during the smolt period allowing for informed decisions on seawater transfer.

The effects of temperature and salinity on the endocrinology in two life stages of juvenile rainbow/steelhead trout (Oncorhynchus mykiss).

The San Francisco Bay Delta is experiencing seasonally warmer waters and saltwater intrusion into historically freshwater ecosystems due to climate change. Steelhead/rainbow trout (Oncorhynchus mykiss) are resident in the Bay-Delta from juvenile development through the smoltification process. Due to increases in sea level, premature seawater (SW) acclimation may co-occur with increased temperatures on pre-smolt juveniles. To evaluate the interactive effects of salinity and temperature on juvenile life stages of salmonids, rainbow trout alevin (3 days post-hatching) were exposed to 13, 16.4 and 19°C for 10 days and then challenged for 24 h to 18 parts per thousand SW. Similarly, fry (4 weeks post-hatching) were exposed to 13, 16.4 and 19°C for 2 weeks (14 days) and then challenged to SW. Estradiol-17ß (E2 ), cortisol, triiodothyronine (T3 ) and thyroxine (T4 ) were measured in whole animal homogenates and muscle tissue using enzyme-linked immunosorbent assays. Transcripts of gill Na+ /K+ ATPase ß (NKAα1b), brain growth hormone I (gh1) and brain gonadotropin-releasing hormone receptor 2 (gnrh2) were also measured. Alevin exhibited a significant temperature-dependent decrease in survival, and fry showed a temperature-dependent decrease in condition factor. The gene expression of NKAα1b, gh1 and gnrh2 significantly decreased in all SW-challenged alevin, and a significant decrease in gnrh2 expression was observed in fry with temperature. Alevin T3 and T4 concentrations significantly increased with increasing temperature. There was a temperature-dependent increase in E2 of fry but not of alevin. The results of this study demonstrate that increasing temperature and SW exposure may adversely affect the survival and SW acclimation of alevin and fry stages of salmonids and that the tolerances of younger juvenile stages should be considered when assessing the response of salmonid populations to climate change stressors.

Na+/K+-ATPase Activity in Smolts of Pink Salmon Oncorhynchus gorbuscha (Walbaum, 1792) from the White Sea Exposed to Fresh, Estuarine, and Sea Water.

The Na+/K+-ATPase (NKA) activity in smolts of pink salmon Oncorhynchus gorbuscha (a salmon species introduced in 1959 into the rivers of the Kola Peninsula) was studied in a ten-day cage experiment with fresh, estuarine, and sea water; the fish were caught during seaward migration in the Indera River of the White Sea basin. The development of tolerance to increased salinity in pink salmon smolts was accompanied by NKA activation. In estuarine water characterized by salinity fluctuations (from fresh to sea water) and in the marine environment (28‰), the NKA activity in pink salmon smolts was significantly higher than in the individuals kept in the cages installed in fresh water. The hypoosmoregulatory ability of pink salmon fry was registered, these data indicated that smoltification in this fish species took place in early ontogenesis. The changes in NKA activity evidenced the readiness of migrating pink salmon fry for the marine phase of their life cycle.

Impact of temperature shift on gill physiology during smoltification of Atlantic salmon smolts (Salmo salar L.).

Exposure to a temperature increase may disrupt smoltification and delay or stop the downstream migration of smolts. Thermal regimes are often different between a river and its tributaries, but the effects of a relative temperature shift are not well described. We used expression of smoltification genes coupled with gill Na+/K+-ATPase activity (NKA) and plasma cortisol and growth hormone (GH) levels to investigate the impact of a 5 °C difference between tributary and river on salmon juveniles. Responses to a temperature challenge were examined at four time points during the smoltification period, with juveniles reared under three regimes including control, early and late temperature increase. The temperature shifts reduced gill NKA, plasma GH and cortisol levels which indicate hypo-osmoregulation impairment and may reduce the survival of smolts. Out of the 22 genes examined, the expression of six genes was influenced by the temperature treatments, while changes in further eleven genes were influenced by the date of sampling. Genes usually known to be upregulated during smoltification were downregulated after the temperature increase, notably nkaα1b, nkcc1a and igf1r. Upregulation of some genes involved in the hormonal regulation and acid-base equilibrium in early June may indicate a switch towards desmoltification. This study gives further insights about the impact of temperature increase on the molecular processes underlying smoltification and possible responses to human-related water temperature increase. The data also suggest dual roles in the smoltification and desmoltification for GH and IGF1 and points to the implication of genes in the smoltification process, that have previously been unstudied (nbc) or with little data available (igf2).

Differences in smolt status affect the resistance of Atlantic salmon (Salmo salar L.) against infectious pancreatic necrosis, while vaccine-mediated protection is unaffected.

In today's aquaculture of Atlantic salmon (Salmo salar L.), a majority of viral disease outbreaks occur after seawater transfer. A relevant question is how the parr-smolt transformation influences the efficacy of viral vaccines and the innate resistance against viral diseases. In this study, vaccinated and unvaccinated A. salmon parr were exposed to different photoperiodic regimens (1-, 3- or 6-week continuous light-WCL). Fish groups at different stages in the smoltification process were induced, as demonstrated by differences in morphological and physiological smolt parameters. At the time of seawater transfer, the 6-WCL group had reached a more pronounced stage in the smoltification process than the 1-WCL group. In unvaccinated fish, the subsequent cohabitation challenge with infectious pancreatic necrosis virus (IPNV) gave a significantly higher accumulated mortality in the 6-WCL group (87%) compared to the 1-WCL group (39%). In the vaccinated groups, this effect was not apparent and there were no differences in accumulated mortality between the 1 WCL, 3 WCL and 6-WCL groups. These data suggest that the resistance to IPN in A. salmon was negatively influenced by smoltification, while vaccine-mediated protection to IPN was maintained equally well irrespective of smolt status.

A temperature shift on the migratory route similarly impairs hypo-osmoregulatory capacities in two strains of Atlantic salmon (Salmo salar L.) smolts.

Anthropogenic use of water systems may cause temperature fluctuations between tributaries and large rivers for which physiological population related-effects on osmoregulatory capacity of Atlantic salmon are not well described. We simulated the downstream route in the case of the River Meuse basin to investigate the impact of a 5 °C temperature shift during smoltification on hypo-osmoregulatory capacities of smolts. Three temperature regimes were tested: control temperature-treatment (T1) without temperature shift, early (T2) or late (T3) temperature shift-treatment. Moreover, fish were subjected to seawater challenge during and after the downstream migration peak time. Two allochtonous strains were used: Loire-Allier (LA) and Cong (CG). Without temperature shift (T1), significant differences between the strains were noticed in the peak date and maximum activity of gill Na+/K+ATPase as well as in plasma sodium and potassium concentrations. For early (T2) and late (T3) temperature shift-treatments, gill Na+/K+ATPase activity, plasma osmolality and ion concentrations were negatively influenced in both strains. After salinity challenge, the highest osmolality was measured in smolts subjected to the temperature shift. Predictably circulating levels of GH and IGF-1 changed over the smolting period but they did not explain the observed modifications in hypo-osmoregulatory abilities whatever the population. The results show a negative impact of a temperature shift on hypo-osmoregulatory capacities of smolts regardless of population differences in smoltification timing under conditions without temperature shift. The resilience of such physiological impact was sustained at least for 1 week, comforting the role of high temperature in influencing the rate of changes occurring during smoltification. Therefore, favouring the downstream migration to help smolts reach the sea faster may mitigate the impact of a rapid temperature increase.

Atlantic salmon adapted to seawater for 9 weeks develop a robust immune response to salmonid alphavirus upon bath challenge.

Pancreas disease (PD) caused by salmonid alphavirus (SAV) is the most serious viral disease in Norwegian aquaculture. Study of the immune response to SAV will aid preventative measures including vaccine development. The innate immune response was studied in Atlantic salmon infected by either bath immersion (BI) or by intra-muscular (i.m.) injection (IM) with SAV subtype 3, two and nine weeks after seawater transfer (Phases A and B respectively). Phase A results have been previously published (Moore et al., 2017) and Phase B results are presented here together with a comparison of results achieved in Phase A. There was a rapid accumulation of infected fish in the IM-B (IM Phase B) group and all fish sampled were SAV RNA positive by 7 dpi (days post infection). In contrast, only a few SAV RNA positive (infected) fish were identified at 14, 21 and 28 dpi in the BI-B (BI Phase B) group. Differences in the transcription of several immune genes were apparent when compared between the infected fish in the IM-B and BI-B groups. Transcription of the analysed genes peaked at 7 dpi in the IM-B group and at 14 dpi in the BI-B group. However, this latter finding was difficult to interpret due to the low prevalence of SAV positive fish in this group. Additionally, fish positive for SAV RNA in the BI-B group showed higher transcription of IL-1ß, IFNγ and CXCL11_L1, all genes associated with the inflammatory response, compared to the IM-B group. Histopathological changes in the heart were restricted to the IM-B group, while (immune) cell filtration into the pancreas was observed in both groups. Compared to the Phase A fish that were exposed to SAV3 two weeks after seawater transfer, the Phase B fish in the current paper, showed a higher and more sustained innate immune gene transcription in response to the SAV3 infection. In addition, the basal transcription of several innate immune genes in non-infected control fish in Phase B (CT-B) was also significantly different when compared to Phase A control fish (CT-A).

Atlantic salmon post-smolts adapted for a longer time to seawater develop an effective humoral and cellular immune response against Salmonid alphavirus.

Salmonid alphavirus (SAV) causes pancreas disease (PD) in Atlantic salmon (Salmo salar L.) and disease outbreaks are mainly detected after seawater transfer. The influence of the smoltification process on the immune responses, specifically the adaptive response of Atlantic salmon after SAV infection, is not fully understood. In this study, Atlantic salmon post-smolts were infected by either bath immersion (BI) or intramuscular injection (IM) with SAV subtype 3, 2 weeks (Phase A) or 9 weeks (Phase B) after seawater transfer. The transcript levels of genes related to cellular, humoral and inflammatory responses were evaluated on head kidney samples collected at 3, 7, 14, 21, and 28 days post-infection (dpi). Corresponding negative control groups (CT) were established accordingly. Significant differences were found between both phases and between the IM and BI groups. The anti-inflammatory cytokine IL-10 was up-regulated in Phase A at a higher level than in Phase B. High mRNA levels of the genes RIG-1, SOCS1 and STAT1 were observed in all groups except the BI-B group (BI-Phase B). Moreover, the IM-B group showed a higher regulation of genes related to cellular responses, such as CD40, MHCII, and IL-15, that indicated the activation of a strong cell-mediated immune response. CD40 mRNA levels were elevated one week earlier in the BI-B group than in the BI-A group (BI-Phase A). A significant up-regulation of IgM and IgT genes was seen in both IM groups, but the presence of neutralizing antibodies to SAV was detected only in Phase B fish at 21 and 28 dpi. In addition, we found differences in the basal levels of some of the analysed genes between non-infected control groups of both phases. Findings suggest that Atlantic salmon post-smolts adapted for a longer time to seawater before they come into contact with SAV, developed a stronger humoral and cell-mediated immune response during a SAV infection.

Influence of strain origin on osmoregulatory and endocrine parameters of two non-native strains of Atlantic salmon (Salmo salar L.).

Non-native strains of Atlantic salmon are used in reinstatement trials where populations are extinct. Environmental cues like photoperiod and temperature are known to influence the smolting process and there is evidence of strain-, stock- or population-specific differences associated with seaward migration or smoltification. The objective of this study was to compare morphological, osmoregulatory and endorcrine features between two strains, one originating from a cold and short river in Ireland (Cong) and another from a long and warm river in France (Loire-Allier), reared under Belgian conditions in order to highlight major differences in restocking adaptability. Comprehensive endocrine profiles, consistent with their interactive role of mediating changes associated with smolting, have been observed. Na+/K+ATPase activity (1.3-10.5 µmol ADP∗mg prot.-1∗h-1) and hormone plasma levels (e.g. 55-122 ng∗mL-1 of cortisol and 4.5-6.4 ng∗mL-1 of GH) were consistent with reported values. We observed strain-related differences of the influence of temperature and daylength on cortisol, GH and sodium plasma levels. These may be related to the respective environmental conditions prevailing in the river of origin, which have impacted the genetic background for smoltification. Using Na+/K+ATPase activity as an indicator, both strains smoltified successfully and simultaneously testifying a prevailing influence of environmental cues over genetic factors for smoltification.

Assessment of the timing and degree of smolt development in southern populations of masu salmon Oncorhynchus masou.

The present study assessed whether non-anadromous masu salmon Oncorhynchus masou in Miyazaki, southern Japan, smoltify, and if so, at what time of the year. Yearling O. masou of Miyazaki and an anadromous population from Hokkaido, northern Japan, were reared in hatcheries in their respective regions and sampled monthly from February to June to examine the spring smoltification period. The Hokkaido population showed a peak of gill Na+ -K+ -ATPase (NKA) activity in May, which was accompanied with an increase in mRNA levels of the seawater (SW)-type NKA alpha subunit, nkaα1b. Increases in gill NKA activity and nkaa1b levels were not seen in Miyazaki populations. Transferring yearling Miyazaki population to 70% SW (salinity of 23) in mid-April resulted in an increased serum osmolality over 4 days. These results suggest that they do not smoltify in their second spring. Next, profiles of gill NKA activity and its subunit mRNA levels in under-yearling Miyazaki population in the autumn were examined. Two phenotypes differing in body color during this period were categorized as parr and smolt-like fish. Smolt-like fish had higher gill NKA activity than parr in December while there was no significant difference in gill nkaα1b levels. Smolt-like fish acclimated to 70% SW better than parr as judged by lower serum osmolality. However, serum osmolality in smolt-like fish did not return to the basal level 7 days after transfer to 70% SW, suggesting that their hypo-osmoregulatory ability was not fully developed to a level comparable to anadromous populations of this species. The present study suggests that, if O. masou in Miyazaki go though a smoltification process, it occurs in its first autumn instead of the second spring and is less pronounced compared with anadromous populations.

The physiology of saltwater acclimation in large juvenile Atlantic salmon Salmo salar.

The present study investigated the effects of transferring freshwater (FW) acclimated S. salar (678 g) that had been maintained under a constant photoperiod and thermal regime, into FW (salinity 0) and salt water (SW; salinity 35) on growth and physiological responses over a 28 day period. There were no mortalities observed throughout the study and no significant differences in mass or fork length between FW and SW groups after 28 days. Compared with fish transferred to FW, plasma osmolality and plasma chloride levels increased significantly in fish in SW by day 1. In the SW group, plasma chloride and osmolality had decreased significantly at day 14 when compared with day 1. Na+ -K+ -ATPase activity was significantly higher in SW compared with the FW group from day 7 and thereafter, but continued to increase until day 22. No differences in plasma cortisol and thyroxine were observed between FW and SW groups throughout the study. Plasma glucose significantly increased from day 1 to day 2 in SW but not in the FW group and levels were significantly reduced in SW compared with the FW group at day 28. Plasma cholesterol and triglyceride levels were significantly higher in FW at day 22 and day 14 to day 22, respectively, when compared with the SW group. In the SW group, plasma cholesterol and triglyceride levels did not change significantly throughout the study. The findings of this study suggest that large S. salar retained in FW maintain a high level of SW tolerance in the absence of photoperiod and thermal regimes necessary for smoltification, as demonstrated by 100% survival, unaffected growth performance, increased Na+ -K+ -ATPase activity and a capacity to regulate plasma chloride and osmolality for 28 days in the SW group.

Variation in branchial expression among insulin-like growth-factor binding proteins (igfbps) during Atlantic salmon smoltification and seawater exposure.

BACKGROUND: In preparation for migration from freshwater to marine habitats, Atlantic salmon (Salmo salar L.) undergo smoltification, a transformation that includes the acquisition of hyposmoregulatory capacity. The growth hormone (Gh)/insulin-like growth-factor (Igf) axis promotes the development of branchial ionoregulatory functions that underlie ion secretion. Igfs interact with a suite of Igf binding proteins (Igfbps) that modulate hormone activity. In Atlantic salmon smolts, igfbp4,-5a,-5b1,-5b2,-6b1 and-6b2 transcripts are highly expressed in gill. We measured mRNA levels of branchial and hepatic igfbps during smoltification (March, April, and May), desmoltification (July) and following seawater (SW) exposure in March and May. We also characterized parallel changes in a broad suite of osmoregulatory (branchial Na+/K+-ATPase (Nka) activity, Na + /K + /2Cl - cotransporter 1 (nkcc1) and cystic fibrosis transmembrane regulator 1 (cftr1) transcription) and endocrine (plasma Gh and Igf1) parameters. RESULTS: Indicative of smoltification, we observed increased branchial Nka activity, nkcc1 and cftr1 transcription in May. Branchial igfbp6b1 and -6b2 expression increased coincidentally with smoltification. Following a SW challenge in March, igfbp6b1 showed increased expression while igfbp6b2 exhibited diminished expression. igfbp5a,-5b1 and-5b2 mRNA levels did not change during smolting, but each had lower levels following a SW exposure in March. CONCLUSIONS: Salmonids express an especially large suite of igfbps. Our data suggest that dynamic expression of particular igfbps accompanies smoltification and SW challenges; thus, transcriptional control of igfbps may provide a mechanism for the local modulation of Igf activity in salmon gill.