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).

Integrative testis transcriptome analysis reveals differentially expressed miRNAs and their mRNA targets during early puberty in Atlantic salmon.

BACKGROUND: Our understanding of the molecular mechanisms implementing pubertal maturation of the testis in vertebrates is incomplete. This topic is relevant in Atlantic salmon aquaculture, since precocious male puberty negatively impacts animal welfare and growth. We hypothesize that certain miRNAs modulate mRNAs relevant for the initiation of puberty. To explore which miRNAs regulate mRNAs during initiation of puberty in salmon, we performed an integrated transcriptome analysis (miRNA and mRNA-seq) of salmon testis at three stages of development: an immature, long-term quiescent stage, a prepubertal stage just before, and a pubertal stage just after the onset of single cell proliferation activity in the testis. RESULTS: Differentially expressed miRNAs clustered into 5 distinct expression profiles related to the immature, prepubertal and pubertal salmon testis. Potential mRNA targets of these miRNAs were predicted with miRmap and filtered for mRNAs displaying negatively correlated expression patterns. In summary, this analysis revealed miRNAs previously known to be regulated in immature vertebrate testis (miR-101, miR-137, miR-92b, miR-18a, miR-20a), but also miRNAs first reported here as regulated in the testis (miR-new289, miR-30c, miR-724, miR-26b, miR-new271, miR-217, miR-216a, miR-135a, miR-new194 and the novel predicted n268). By KEGG enrichment analysis, progesterone signaling and cell cycle pathway genes were found regulated by these differentially expressed miRNAs. During the transition into puberty we found differential expression of miRNAs previously associated (let7a/b/c), or newly associated (miR-15c, miR-2184, miR-145 and the novel predicted n7a and b) with this stage. KEGG enrichment analysis revealed that mRNAs of the Wnt, Hedgehog and Apelin signaling pathways were potential regulated targets during the transition into puberty. Likewise, several regulated miRNAs in the pubertal stage had earlier been associated (miR-20a, miR-25, miR-181a, miR-202, let7c/d/a, miR-125b, miR-222a/b, miR-190a) or have now been found connected (miR-2188, miR-144, miR-731, miR-8157 and the novel n2) to the initiation of puberty. CONCLUSIONS: This study has - for the first time - linked testis maturation to specific miRNAs and their inversely correlated expressed targets in Atlantic salmon. The study indicates a broad functional conservation of already known miRNAs and associated pathways involved in the transition into puberty in vertebrates. The analysis also reveals miRNAs not previously associated with testis tissue or its maturation, which calls for further functional studies in the testis.

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.

Disturbance of the intestinal mucosal immune system of farmed Atlantic salmon (Salmo salar), in response to long-term hypoxic conditions.

The gastrointestinal (GI) tract has many important biological functions. One is to serve as a barrier between the fish and the external environment. A decreased physical barrier function of the intestine may lead to increased inflow of luminal content and subsequent activation of the intestinal mucosal immune system. This activation is governed by the ability of various compounds to induce cytokine release and immune cell activity, leading to an immune response. In mammals, the impact of stress on the intestinal barrier is well documented and results in increased intestinal permeability and thus increased stimulation of the mucosal immune system. Fish reared in sea cages may at times be exposed to unfavourable environmental conditions leading to chronic stress and disturbed intestinal integrity. This change in permeability may increase the exposure of the mucosal immune system to activating compounds. In the present study, the effect of a prolonged stress on the intestinal mucosal immune system of fish is therefore addressed. Atlantic salmon were exposed to low levels (50%) of dissolved oxygen (DO) for 6-7 weeks in consecutive experiments performed at 8 and 16 °C. Immune parameters were assessed in terms of mRNA expression of the key cytokines, interleukin-1ß (IL-1ß), IL-8, IL-10, interferon-γ (IFNγ) and transforming growth factor-ß (TGFß) as well as the immune regulatory inhibitor of nuclear factor κB (IκB). In the experiment at 8 °C also mucosal neutrophil infiltration was monitored. Subjecting the fish to low DO levels at 8 °C resulted in an increased mucosal neutrophil infiltration together with a down-regulation of IκB. At the higher temperature, 16 °C, low DO levels created decreased expression of the pro-inflammatory cytokine IL-1ß in both intestinal regions as well as an increased expression of IL-10 in the proximal intestine. These results suggest that husbandry conditions in sea cages with DO levels as low as 50% clearly affects the intestinal mucosal immune system and results in a chronic inflammation. Moreover, the effects of low DO levels on the immune factors examined were more pronounced in the 16 °C experiment suggesting additive effects of high temperatures.

The reproductive cycle of female Ballan wrasse Labrus bergylta in high latitude, temperate waters.

This 2 year study examined the reproductive cycle of wild female Ballan wrasse Labrus bergylta in western Norway as a precursor to captive breeding trials. Light microscopy of ovarian histology was used to stage gonad maturity and enzyme-linked immuno-absorbent assay (ELISA) to measure plasma concentrations of the sex steroids testosterone (T) and 17beta-oestradiol (E(2)). Ovarian recrudescence began in late autumn to early winter with the growth of previtellogenic oocytes and the formation of cortical alveoli. Vitellogenic oocytes developed from January to June and ovaries containing postovulatory follicles (POF) were present between May and June. These POF occurred simultaneously among other late maturity stage oocytes. Plasma steroid concentration and organo-somatic indices increased over winter and spring. Maximal (mean +/-s.e.) values of plasma T (0.95 +/- 0.26 ng ml(-1)), E(2) (1.75 +/- 0.43 ng ml(-1)) and gonado-somatic index (I(G); 10.71 +/- 0.81) occurred in April and May and decreased greatly in July when only postspawned fish with atretic ovaries occurred. Evidence indicates that L. bergylta are group-synchronous multiple spawners with spawning occurring in spring and peaking in May. A short resting period may occur between late summer and autumn when previtellogenic oocytes predominate and steroid levels are minimal.

The effect of hyperoxygenation and reduced flow in fresh water and subsequent infectious pancreatic necrosis virus challenge in sea water, on the intestinal barrier integrity in Atlantic salmon, Salmo salar L.

In high intensive fish production systems, hyperoxygenation and reduced flow are often used to save water and increase the holding capacity. This commonly used husbandry practice has been shown to be stressful to fish and increase mortality after infectious pancreatic necrosis virus (IPNV) challenge, but the cause and effect relationship is not known. Salmonids are particularly sensitive to stress during smoltification and the first weeks after seawater (SW) transfer. This work aimed at investigating the impact of hyperoxygenation combined with reduced flow in fresh water (FW), on the intestinal barrier in FW as well as during later life stages in SW. It further aims at investigating the role of the intestinal barrier during IPNV challenge and possible secondary infections. Hyperoxygenation in FW acted as a stressor as shown by significantly elevated plasma cortisol levels. This stressful husbandry condition tended to increase paracellular permeability (P(app)) as well as translocation of Aeromonas salmonicida in the posterior intestine of Atlantic salmon. After transfer to SW and subsequent IPNV challenge, intestinal permeability, as shown by P(app), and translocation rate of A. salmonicida increased in the anterior intestine, concomitant with further elevation in plasma cortisol levels. In the anterior intestine, four of five fish displayed alterations in intestinal appearance. In two of five fish, IPNV caused massive necrosis with significant loss of cell material and in a further two fish, IPNV caused increased infiltration of lymphocytes into the epithelium and granulocytes in the lamina propria. Hyperoxygenation and reduced flow in the FW stage may serve as stressors with impact mainly during later stages of development. Fish with an early history of hyperoxygenation showed a higher stress response concomitant with a disturbed intestinal barrier function, which may be a cause for the increased susceptibility to IPNV infection and increased susceptibility to secondary infections.

Molecular characterization and quantification of the gonadotropin receptors FSH-R and LH-R from Atlantic cod (Gadus morhua).

In order to elucidate regulatory mechanisms during puberty final oocyte maturation and spawning, full-length sequences coding for the receptors for follicle-stimulating hormone (FSH-R) and luteinizing hormone (LH-R) were isolated from female Atlantic cod (Gadus morhua) by a RACE-PCR based strategy. The nucleotide and amino acid sequences showed high homologies with the corresponding sequences of other fish species but contained some distinct differences. Conserved features important for functionality, such as a long N-terminal extracellular domain (ECD), seven transmembrane domains and a short C-terminal intracellular domain, were identified in both predicted proteins. Partial genomic sequences for these genes were also determined, allowing the design of mRNA-specific quantitative PCR assays. Due to suspected alternative splicing during expression of these genes, additional real-time PCR assays detecting variants containing the membrane-anchoring domain were established. Besides the expected expression of FSH-R and LH-R mRNA in the gonads similarly strong signals for LH-R were also obtained in male gill, and in female and male brain. When relative expression was analysed at different stages of sexual maturation, levels for FSH-R increased moderately during gonadal growth whereas those of LH-R showed a high peak at spawning.

A comparative ex vivo and in vivo study of day and night perception in teleosts species using the melatonin rhythm.

The purpose of this study was to determine and compare the light sensitivity of two commercially important, phylogenetically different teleost species in terms of melatonin production. Three series of experiments were performed on both Atlantic salmon and European sea bass. First, a range of light intensities were tested ex vivo on pineal melatonin production in culture during the dark phase. Then, light transmission through the skull was investigated, and finally short-term in vivo light sensitivity trials were performed. Results showed that sea bass pineal gland ex vivo are at least 10 times more sensitive to light than that of the salmon. Light intensity threshold in sea bass appeared to be between 3.8 x 10(-5) and 3.8 x 10(-6) W/m2 in contrast to 3.8 x 10(-4) and 3.8 x 10(-5) W/m2 in salmon. These highlighted species-specific light sensitivities of pineal melatonin production that are likely to be the result of adaptation to particular photic niches. Light transmission results showed that a significantly higher percentage of light penetrates the sea bass pineal window relative to salmon, and confirmed that penetration is directly related to wavelength with higher penetration towards the red end of the visible spectrum. Although results obtained in vivo were comparable, large differences between ex vivo and in vivo were observed in both species. The pineal gland in isolation thus appeared to have different sensitivities as the whole animal, suggesting that retinal and/or deep brain photoreception may contribute, in vivo, to the control of melatonin production.

Three forms of GnRH in the brain and pituitary of the turbot, Scophthalmus maximus: immunological characterization and seasonal variation.

Three forms of GnRH, chicken (c) GnRH-II, salmon (s) and seabream (sb) GnRH, were immunologically characterized in the brain and pituitary of turbot by ELISA. cGnRH-II and sGnRH were detected in the brain, while sbGnRH and sGnRH (but not cGnRH-II) were detected in the pituitary. In females, the levels of cGnRH-II in the turbot brain extracts increased from May to July, concomitant with an increase in oocyte diameter. In the pituitary, sbGnRH was found to be the dominant form, with levels 100-600-fold those of sGnRH. Both sGnRH and sbGnRH in the pituitary showed variation during the spawning season; sbGnRH increased from May to July and correlated with the increase in oocyte diameter, while sGnRH decreased. The overall patterns were the same for male turbot, although levels were generally lower. These findings suggest that sbGnRH could be controlling reproduction in the turbot. However, the seasonal variation in sGnRH indicates a potential physiological role in turbot reproduction. This study gives the first immunological indications that sbGnRH is present in the pituitary of a pleuronectiform fish, and will provide the basis for further studies on the endocrine regulation of reproduction in flatfish.

The interrelation between photoperiod, growth hormone, and sexual maturation of adult Atlantic salmon (Salmo salar).

The plasma profiles of growth hormone (GH) in adult male and female Atlantic salmon were determined in relation to manipulation of the photoperiod and to the development and timing of sexual maturation. Fish were exposed to natural light (NL) or NL + 24L:0D additional light over the netpens from January (ALJ) or March (ALM) to July. Thereafter, these groups were brought indoors, subdivided, and subjected to simulated natural photoperiod (SNP), continuous light (24L), or short day (8L). Assay of salmon GH by RIA in monthly plasma samples revealed that GH levels were generally < 1 ng ml-1 during January to June and were only slightly affected by additional light in January or March. ALJ-24L treatment, and to a lesser extent, ALM-24L treatment, was effective in preventing sexual maturation, and GH levels of immature fish continued to be < or = 1.5 ng ml-1. On the other hand, in sexually maturing fish, GH levels increased to 2-5 ng ml-1 months prior to ovulation. Short-day photoperiod (8L) from July advanced ovulation and spermiation, whereas continuous light from July delayed these processes. The timing of the increase of GH levels was shifted in a parallel manner, indicating a functional relationship between plasma GH levels and the process of sexual maturation.