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.

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).

Water temperature and dietary histidine affect cataract formation in Atlantic salmon (Salmo salar L.) diploid and triploid yearling smolt.

The aim of the present study was to investigate cataract development in diploid (2N) and triploid (3N) Atlantic salmon smolts and post-smolts at two water temperatures (10 and 16 °C) given diets with different histidine supplementation (LH, 10.4 and HH, 13.1 g kg-1 ) before and after seawater transfer. In freshwater, a severe cataract outbreak was recorded in both ploidies reared at 16 °C. The cataract score was significantly higher in triploids compared to diploids, and the severity was lower in both ploidies fed the HH diet. The cataract development at 10 °C was minor. Low gill Na+ , K+ -ATPase activity in fish reared at 16 °C before seawater transfer was followed by osmoregulatory stress with elevated plasma electrolyte concentrations and high mortality in sea water. Both diploids and triploids reared at 10 °C developed cataracts during the seawater period, with higher severities in triploids than diploids and a reduced severity in the fish fed the HH diet. The findings of this study demonstrate the importance of environmental conditions in the husbandry of Atlantic salmon, and particularly triploids, with regard to smoltification and adjusted diets to mitigate cataract development in fresh and sea water.

Immune gene profiles in Atlantic salmon (salmo salar L.) post-smolts infected with SAV3 by bath-challenge show a delayed response and lower levels of gene transcription compared to injected fish.

Salmonid alphavirus (SAV) causes pancreatic disease (PD) in salmonids in Northern Europe which results in large economic losses within the aquaculture industry. In order to better understand the underlying immune mechanisms during a SAV3 infection Atlantic salmon post-smolts were infected by either i.m.-injection or bath immersion and their immune responses compared. Analysis of viral loads showed that by 14 dpi i.m.-injected and bath immersion groups had 95.6% and 100% prevalence respectively and that both groups had developed the severe pathology typical of PD. The immune response was evaluated by using RT-qPCR to measure the transcription of innate immune genes involved in the interferon (IFN) response as well as genes associated with inflammation. Our results showed that IFNa transcription was only weakly upregulated, especially in the bath immersion group. Despite this, high levels of the IFN-stimulated genes (ISGs) such as Mx and viperin were observed. The immune response in the i.m.-injected group as measured by immune gene transcription was generally faster, and more pronounced than the response in the bath immersion group, especially at earlier time-points. The response in the bath immersion group started later as expected and appeared to last longer often exceeding the response in the i.m-injected fish at later time-points. High levels of transcription of many genes indicative of an active innate immune response were present in both groups.

Atlantic salmon (Salmo salar L.) post-smolts challenged two or nine weeks after seawater-transfer show differences in their susceptibility to salmonid alphavirus subtype 3 (SAV3).

BACKGROUND: Pancreas disease (PD), caused by salmonid alphavirus (SAV), is an important disease affecting salmonid aquaculture. It has been speculated that Atlantic salmon post-smolts are more prone to infections in the first few weeks following seawater- transfer. After this period of seawater acclimatization, the post-smolts are more robust and better able to resist infection by pathogens. Here we describe how we established a bath immersion (BI) model for SAV subtype 3 (SAV3) in seawater. We also report how this challenge model was used to study the susceptibility of post-smolts to SAV3 infection in two groups of post-smolts two weeks or nine weeks after seawater - transfer. METHODS: Post-smolts, two weeks (Phase-A) or nine weeks (Phase-B) after seawater- transfer, were infected with SAV3 by BI or intramuscular injection (IM) to evaluate their susceptibility to infection. A RT-qPCR assay targeting the non-structural protein (nsP1) gene was performed to detect SAV3-RNA in blood, heart tissue and electropositive-filtered tank-water. Histopathological changes were examined by light microscope, and the presence of SAV3 antigen in pancreas tissue was confirmed using immuno-histochemistry. RESULTS: Virus shedding from the Phase-B fish injected with SAV3 (IM Phase-B) was markedly lower than that from IM Phase-A fish. A lower percentage of viraemia in Phase-B fish compared with Phase-A fish was also observed. Viral RNA in hearts from IM Phase-A fish was higher than in IM Phase-B fish at all sampling points (p < 0.05) and a similar trend was also seen in the BI groups. Necrosis of exocrine pancreatic cells was observed in all infected groups. Extensive histopathological changes were found in Phase-A fish whereas milder PD-related histopathological lesions were seen in Phase-B fish. The presence of SAV3 in pancreas tissue from all infected groups was also confirmed by immuno-histochemical staining. CONCLUSION: Our results suggest that post-smolts are more susceptible to SAV3 infection two weeks after seawater-transfer than nine weeks after transfer. In addition, the BI challenge model described here offers an alternative SAV3 infection model when better control of the time-of-infection is essential for studying basic immunological mechanisms and disease progression.

Long-term hypo-osmoregulatory capacity in downstream migrating Atlantic salmon Salmo salar L. smolts.

The duration of hypo-osmoregulatory capacity in downstream migrating Atlantic salmon Salmo salar L smolts previously stocked as startfed young-of-the year (YOY) parr was tested in the River Dalåa from mid-May to late-June 1999. Hypo-osmoregulatory capacity, measured as plasma osmolality and chloride, was assessed after seawater (SW) challenge tests (168 h, salinity = 35). All S. salar exhibited sufficient hypo-osmoregulatory capacity at the initiation of downstream migration in mid-May. Migrating S. salar smolts caught in mid-May and retained in fresh water displayed no signs of de-smoltification as they maintained hypo-osmoregulatory capacity through June. This indicates a physiological smolt window that lasts a minimum of 6 weeks (330 degree days; D°) for hatchery-produced S. salar smolts stocked as YOY parr. Based on the observed river migration speeds, it can be assumed that the S. salar smolts entered SW 2-4 weeks after initiation of migration in the upper parts of the River Dalåa. Hence, based on smolt migration and SW tolerance, it is suggested that stocking of YOY parr is a viable enhancement strategy in the River Dalåa.

Development of intestinal ion-transporting mechanisms during smoltification and seawater acclimation in Atlantic salmon Salmo salar.

This study investigated the expression of ion transporters involved in intestinal fluid absorption and presents evidence for developmental changes in abundance and tissue distribution of these transporters during smoltification and seawater (SW) acclimation of Atlantic salmon Salmo salar. Emphasis was placed on Na(+) , K(+) -ATPase (NKA) and Na(+) , K(+) , Cl(-) co-transporter (NKCC) isoforms, at both transcriptional and protein levels, together with transcription of chloride channel genes. The nka α1c was the dominant isoform at the transcript level in both proximal and distal intestines; also, it was the most abundant isoform expressed in the basolateral membrane of enterocytes in the proximal intestine. This isoform was also abundantly expressed in the distal intestine in the lower part of the mucosal folds. The protein expression of intestinal Nkaα1c increased during smoltification. Immunostaining was localized to the basal membrane of the enterocytes in freshwater (FW) fish, and re-distributed to a lateral position after SW entry. Two other Nka isoforms, α1a and α1b, were expressed in the intestine but were not regulated to the same extent during smoltification and subsequent SW transfer. Their localization in the intestinal wall indicates a house-keeping function in excitatory tissues. The absorptive form of the NKCC-like isoform (sub-apically located NKCC2 and/or Na(+) , Cl(-) co-transporter) increased during smoltification and further after SW transfer. The cellular distribution changed from a diffuse expression in the sub-apical regions during smoltification to clustering of the transporters closer to the apical membrane after entry to SW. Furthermore, transcript abundance indicates that the mechanisms necessary for exit of chloride ions across the basolateral membrane and into the lateral intercellular space are present in the form of one or more of three different chloride channels: cystic fibrosis transmembrane conductance regulator I and II and chloride channel 3.

Osmoregulation in Atlantic salmon Salmo salar smolts transferred to seawater at different temperatures.

In order to investigate how changes in gill Na(+) , K(+) -ATPase (NKA) α1a and α1b subunits, Na(+) , K(+) , 2Cl(-) co-transporter (NKCC1) and the apical cystic fibrosis trans-membrane conductance regulator-I (CFTR-I) transcripts in wild strain of Atlantic salmon, Salmo salar, smolts are affected by temperature during spring, hatchery-reared parr (mean ± s.e. fork length = 14·1 ± 0·5; mean ± s.e. body mass = 28·5 ± 4·5 g) originating from broodstock from the Vosso river (western Norway) were acclimated to three temperature regimes (4·1, 8·1 and 12·9° C) in May and reared under gradually increasing salinity between May and June. Changes in plasma Na(+) , haematocrit (Hct) and PCO2 were monitored in order to assess and compare key physiological changes with the transcriptional changes in key ion transporters. The temperatures reflect the natural temperature range in the River Vosso during late spring. Overall, higher gill NKA α1b mRNA levels, gill NKCC1a levels and CFTR-I levels were observed in the 4·1° C group compared to the 11·9° C group. This coincided with a 2-3 week period with decreased Hct and PCO2 and may indicate a critical window when smolts suffer from reduced physical performance during migration. Further research is needed to confirm the potential interaction between ecological and physiological conditions on mortality of hatchery-reared smolts from River Vosso during their natural migration.

Plasma growth hormone-binding protein levels in Atlantic salmon Salmo salar during smoltification and seawater transfer.

Specific growth hormone (GH)-binding protein (Ghbp) was purified from Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss plasma with immunoprecipitation and characterized in cross-linking studies using autoradiography and western blots. The size of the Ghbp was estimated to be c. 53 kDa. A radioimmunoassay was established to measure Ghbp in salmonids, using antibodies specific against the extracellular segment of the S. salar growth hormone receptor 1 (grh1; GenBank AY462105). Plasma Ghbp levels were measured in S. salar smolts in fresh water and after transfer to seawater (SW; experiments 1 and 2), and in post-smolts kept at different salinities (0, 12, 22 and 34) for 3 months (experiment 3). A transient increase in plasma Ghbp, which lasted for 1 month or less, was noted in smolts after transfer to SW. Concomitantly, plasma GH and gill Na(+) -K(+) -ATPase activity increased during smoltification (in experiment 2). No difference in plasma Ghbp was evident between post-smolts kept at different salinities, although the fish kept at salinity 34 had higher plasma GH than the group kept at salinity 22 and higher hepatic ghr1 expression than post-smolts kept at salinity 12. This suggests that plasma Ghbp regulation may respond to salinity changes in the short term. The lack of correlation between Ghbp, plasma GH and hepatic ghr1 expression in the long-term post-smolt experiment indicates that Ghbp levels may be regulated independently of other components of the endocrine GH system in salmonids.

Development of seawater tolerance and subsequent downstream migration in wild and stocked young-of-the-year derived Atlantic salmon Salmo salar smolts.

This study investigated the development of hypo-osmoregulatory capacity and timing of downstream migration in wild Atlantic salmon Salmo salar smolts from the River Stjørdalselva and stocked young-of-the-year (YOY), derived S. salar smolts from the tributary River Dalåa. Both wild and stocked S. salar smolts developed seawater (SW) tolerance in early May, persisting through June, measured as their ability to regulate plasma osmolality and chloride following 24 h SW (salinity = 35) exposure. Although the majority of downstream migration among the stocked S. salar smolts occurred later than observed in their wild counterparts, the development of SW tolerance occurred concurrently. The wild S. salar from Stjørdalselva and stocked YOY smolts from the River Dalåa started to migrate on the same cumulative day-degrees (D°). The study revealed no downstream migration before development of SW tolerance. This emphasizes the importance of incorporating physiological status when studying environmental triggers for downstream migration of S. salar smolts. Overall, these findings suggest that the onset of smolt migration in stocked S. salar smolts was within the smolt window from an osmoregulatory point of view.

Aluminum exposure impacts brain plasticity and behavior in Atlantic salmon (Salmo salar).

Aluminum (Al) toxicity occurs frequently in natural aquatic ecosystems as a result of acid deposition and natural weathering processes. Detrimental effects of Al toxicity on aquatic organisms are well known and can have consequences for survival. Fish exposed to Al in low pH waters will experience physiological and neuroendocrine changes that disrupt homeostasis and alter behavior. To investigate the effects of Al exposure on both the brain and behavior, Atlantic salmon (Salmo salar) kept in water treated with Al (pH 5.7, 0.37±0.04 µmol 1(-1) Al) for 2 weeks were compared with fish kept in under control conditions (pH 6.7, <0.04 µmol 1(-1) Al). Fish exposed to Al and acidic conditions had increased Al accumulation in the gills and decreased gill Na(+), K(+)-ATPase activity, which impaired osmoregulatory capacity and caused physiological stress, indicated by elevated plasma cortisol and glucose levels. Here we show for the first time that exposure to Al in acidic conditions also impaired learning performance in a maze task. Al toxicity also reduced the expression of NeuroD1 transcript levels in the forebrain of exposed fish. As in mammals, these data show that exposure to chronic stress, such as acidified Al, can reduce neural plasticity during behavioral challenges in salmon, and may impair the ability to cope with new environments.

Seawater tolerance and post-smolt migration of wild Atlantic salmon Salmo salar × brown trout S. trutta hybrid smolts.

High levels of hybridization between Atlantic salmon Salmo salar and brown trout Salmo trutta have been reported in the River Driva. This study presents the underlying mechanisms of development of seawater (SW) tolerance and marine migration pattern for S. salar×S. trutta hybrids. Migrating S. salar×S. trutta hybrid smolts caught in the River Driva, Norway (a river containing Gyrodactylus salaris), displayed freshwater (FW) gill Na(+), K(+) -ATPase (NKA) activity levels of 11·8 µmol ADP mg protein h(-1), which were equal to or higher than activity levels observed in S. salar and S. trutta smolts. Following 4 days of SW exposure (salinity 32·3), enzyme activity remained high and plasma ion levels were maintained within the normal physiological range observed in S. salar smolts, indicating no signs of ion perturbations in S. salar×S. trutta hybrids. SW exposure induced an increase in NKA α1b-subunit mRNA levels with a concurrent decrease in α1a levels. Salmo salar×S. trutta post-smolts migrated rapidly through the fjord system, with increasing speed with distance from the river, as is often seen in S. salar smolts. The present findings suggest that S. salar×S. trutta smolts, as judged by the activity and transcription of the NKA system, regulation of plasma ion levels and migration speed more closely resemble S. salar than S. trutta.

Corticotropin-releasing factor neurogenesis during midlife development in salmon: genetic, environmental and thyroid hormone regulation.

Salmon parr-smolt transformation (smoltification) is a mid-life transitional stage between life in freshwater and seawater that entails a wide range of neural, endocrine and physiological modifications. In salmon, the neuroendocrine corticotropin-releasing factor (CRF) system regulates pituitary adrenocorticotrophic hormone and thyrotrophin release. Four experimental groups of Atlantic salmon, Salmo salar, were used to investigated CRF neurogenesis and its regulation during smoltification. We compared: (i) developmental stages (parr and early-smolt) in anadromous controls; (ii) a developmentally arrested model: anadromous reared under continuous light (LL) with anadromous controls; (iii) a natural hypoendocrine/incomplete smolt development salmon model (landlocked) with anadromous controls; and (iv) landlocked treated with thyroxine to anadromous control smolt levels. CRF neurogenesis between groups was studied with bromodeoxyuradine (BrdU) incorporation followed by double-labelling CRF and BrdU immunhistochemistry. The rate of CRF neurogenesis in the preoptic area (POA) increased from parr to early-smolts in anadromous salmon. By contrast, neurogenesis was inhibited in the LL group and reduced in the landlocked salmon. The administration of thyroxine in landlocked salmon to match anadromous levels increased the rate of CRF neurogenesis to anadromous levels. In conclusion, newly-formed CRF cells in the POA during smoltification are associated with increased retinal innervation to the POA and endocrine responsiveness to increased photoperiod. Both genetic and environmental factors influence the degree of salmon brain development. Thyroid hormones increase CRF neurogenesis during this critical period of development in salmon. We hypothesise that a positive-feedback of thyroid hormones on CRF neurogenesis may be an important event in reaching the developmental climax during critical periods.

Branchial expression patterns of claudin isoforms in Atlantic salmon during seawater acclimation and smoltification.

In euryhaline teleosts, permeability changes in gill epithelia are essential during acclimation to changed salinity. This study examined expression patterns of branchial tight junction proteins called claudins, which are important determinants of ion selectivity and general permeability in epithelia. We identified Atlantic salmon genes belonging to the claudin family by screening expressed sequence tag libraries available at NCBI, and classification was performed with the aid of maximum likelihood analysis. In gill libraries, five isoforms (10e, 27a, 28a, 28b, and 30) were present, and quantitative PCR analysis confirmed tissue-specific expression in gill when compared with kidney, intestine, heart, muscle, brain, and liver. Expression patterns during acclimation of freshwater salmon to seawater (SW) and during the smoltification process were examined. Acclimation to SW reduced the expression of claudin 27a and claudin 30 but had no overall effect on claudin 28a and claudin 28b. In contrast, SW induced a fourfold increase in expression of claudin 10e. In accord, a peak in branchial claudin 10e was observed during smoltification in May, coinciding with optimal SW tolerance. Smoltification induced no significant changes in expression of the other isoforms. This study demonstrates the expression of an array of salmon claudin isoforms and shows that SW acclimation involves inverse regulation, in the gill, of claudin 10e vs. claudin 27a and 30. It is possible that claudin 10e is an important component of cation selective channels, whereas reduction in claudin 27a and 30 may change permeability conditions in favor of the ion secretory mode of the SW gill.