Cortisol response and immune-related effects of Atlantic salmon (Salmo salar Linnaeus) subjected to short- and long-term stress

It is generally considered that stress causes decreased immune function in fish. In this study we examined in Atlantic salmon (Salmo salar Linnaeus) the effects of both short- (a single 15s out of water) and long-term (4 weeks of daily handling 15s out of water) stress on plasma cortisol (free and total) and glucose levels, expression of interleukin-1beta (IL-1beta) and survival of head kidney (HK) macrophages under culture with Aeromonas salmonicida. In the short-term study, samples were collected prior to the application of the stressor, and at 1, 3, 6, 12 and 24h post stress. Free and total plasma cortisol levels and the percentage of free cortisol increased significantly in the stressed group at 1 and 3h post stress. Plasma glucose levels were significantly higher than those of control fish at 1, 3 and 6h post stress. Constitutive expression of IL-1beta in macrophages isolated from head kidneys in stressed fish was significantly higher at 1 and 3h post stress. However, lipopolysaccharide (LPS) stimulated expression of IL-1beta in HK macrophages, exhibited significantly higher fold increases in unstressed fish compared to stressed fish. In the long-term study, with the exception of an increase in plasma glucose levels at 1 week, there were no significant differences in stress parameters between groups. There was a significantly higher constitutive IL-1beta expression in macrophages isolated from stressed fish over the first 2 weeks. At weeks 1, 2 and 3 the magnitude of IL-1beta response of isolated HK macrophages to LPS stimulation was reduced in >90% of the stressed fish. At 4 weeks there was no significant difference in inducible IL-1beta expression between the groups. Macrophages isolated from stressed fish also showed significantly decreased survival when exposed to A. salmonicida. This study shows a clear pattern from repeated handling stress, whereby effects on immune cells begin with increased constitutive expression of IL-1beta, followed by decreased stimulation of leucocytes by extracellular antigen, and finally decreased leukocyte survival when exposed to A. salmonicida. The implications of these changes in the immune system will be discussed with respect to the use of classical indicators of stress to predict possible effects on the immune system of fish.     

Transport-associated proteins in Atlantic salmon (Salmo salar)

 The major histocompatibility complex (Mhc) regions of mice, rats, and humans all contain a pair of related genes, TAP1 and TAP2, which encode members of a large superfamily of proteins of similar structure and function. A functional TAP1/TAP2 heterodimer is probably required for efficient presentation of antigens to CD8⁺ T cells. This heterodimer resides in the membrane of the endoplasmic reticulum, and transports peptides from the cytoplasm into the endoplasmic reticulum lumen for binding to Mhc class I molecules. The TAP transporter demonstrates specificity for both peptide sequence and length, and in rats, allelic variation in the sequence of the transporter molecules results in differential ability to transport particular peptides. Here we report two expressed Sasa-TAP2 loci, both of which are polymorphic, as well as an expressed Sasa-TAP1 locus from Atlantic salmon. The Sasa-TAP2A locus has a genomic organization similar to the human TAP2 equivalent. Received: 23 December 1996 / Revised: 26 February 1997     

Cloning and characterization of the executioner caspases 3, 6, 7 and Hsp70 in hyperthermic Atlantic salmon (Salmo salar) embryos

Hyperthermia during embryogenesis has been reported to induce deformities in Atlantic salmon (Salmo salar). To examine the involvement of executioner caspases in hyperthermia-induced cell-death in a poikilotherm vertebrate species, five genes encoding caspase-3,-6, and -7 were cloned from Atlantic salmon, and the expression was studied in thermal stressed salmon embryos. The salmon genome contained two genetically distinct variants of both salmon caspase-3 and caspase-6 that is likely the result of two independent chromosome or genome duplications. Whereas only partial caspase-3A encoding sequences were isolated, the full-length caspase-3B cDNA encodes the inactive proenzyme of 279 amino acids (aa) consisting of an N-terminal prodomain and the large and the small subunit. The salmon caspase-6A and caspase-6B proenzymes include an additional linker region between the two subunits. The deduced salmon caspase-7 consists of only 245 aa and lacks the prodomain and part of the large subunit similar to the predicted caspase-7 of the puffer fish Tetraodon sp.. Increased apoptotic activity as evidenced by cleavage of nuclear DNA was demonstrated in salmon embryos incubated at 18-20 degrees C for 84 h after acclimatization at 8 degrees C. Hyperthermia-induced activation of the executioner caspases was indicated by the increased mRNA levels of caspase-3B, caspase-6A/B and caspase-7 after 54 h heat exposure as quantified by real-time RT-PCR. The 2-2.5 fold increase in the mRNA expression of the heat shock protein Hsp70 gene coincided with the peak mRNA values of the executioner caspases. Whole-mount in situ hybridization of the salmon embryo identified caspase-7 mRNA in the lens exclusively, while caspase-3B and caspase-6A/B were expressed in multiple tissues of exposed and control embryos. Interestingly, cardiac expression of caspase-6A/B was only identified in heat stressed embryos. Altogether, these results shed light on evolutionary aspects of the executioner caspases in vertebrates and their expression in salmon embryos exposed to hyperthermia. In particular, the heat sensitive caspase-6 expression in the embryonic heart is of interest since cardiac malformations are an emergent problem in salmon aquaculture.     

A lymphosarcoma in an Atlantic salmon (Salmo salar)

A lymphosarcoma that appeared to be of thymic origin and of lymphoblastic type was found in a 3.5-yr-old Atlantic salmon (Salmo salar). The fish was from a population of 60 broodfish maintained at a research fish laboratory. A large tumor mass was found under the left operculum. Small tumor nodules were found on the swim bladder and in the abdominal adipose tissue. The location of this neoplasm differed from those of previously described tumors in this fish species.     

The two myostatin genes of Atlantic salmon (Salmo salar) are expressed in a variety of tissues.

Two myostatin isoforms were identified in Atlantic salmon (Salmo salar) by RT-PCR, and genomic sequences encoding this negative muscle growth factor were for the first time isolated from a nonmammalian species. Salmon myostatin isoform I is transcribed in white skeletal muscle as a 2346-nucleotide mRNA species that encodes a precursor protein of 373 amino acids. Salmon myostatin I shows 93% sequence identity with isoform II which was isolated from white muscle as a partial cDNA sequence of 1409 nucleotides. In contrast to the restricted gene expression of myostatin in mammals, salmon myostatin I and II mRNAs were identified by RT-PCR in multiple tissues, including white muscle, intestine, brain, gills, tongue and eye. In addition, isoform I mRNA was found in red skeletal muscle, heart, spleen, and ovarian tissue. Using polyclonal antibodies against both isoforms, a 55-kDa precursor protein was detected by Western blot analysis in the red and white skeletal muscle, heart, intestine, and brain. Immunoreactive peptides of 35-40 kDa were identified in the gills, tongue, spleen, and head kidney, while the 25-kDa mature myostatin was found in the eye and serum, and in vitro expressed in rabbit reticulocyte lysate. Salmon myostatin was immunohistochemically localized in the sarcoplasma of red and white muscle fibres, in intestinal epithelial cells, at the basis of the branchial primary lamellae, and in odontoblasts and ameloblasts of the tongue teeth. The results indicate that the role of fish myostatin may not be restricted to muscle growth regulation, but may have additional functions similar to the growth/differentiation factor-11 in mammals.     

Tolerance and resistance to thermal stress in juvenile Atlantic salmon, Salmo salar

SUMMARY. 1. The chief objective was to construct a thermal tolerance polygon for juvenile Atlantic salmon, Salmo salar L., using fish from four groups and two populations: two age groups from one population (0+, 1+ parr from River Leven), two size groups from the other population (slow and Fast growing 1+ parr from River Lune). 2. Fish were acclimated to constant temperatures of 5, 10, 15, 20, 25 and 27°C; then the temperature was raised or lowered at 1°C h^?1 to determine the upper and lower limits for feeding and survival over 10 min, 100 min, 1000 min and 7 days. As they were not significantly different between the four groups of fish, values at each acclimation temperature were pooled to provide arithmetic means (with SE) for the thermal tolerance polygon. 3. Incipient lethal levels (survival over 7 days) defined a tolerance zone within which salmon lived for a considerable time; upper mean incipient values increased with increasing acclimation temperature to reach a maximum of 27.8±0.2°C, lower mean incipient values were below 0°C and were therefore undetermined at acclimation temperatures <20°C but increased at higher acclimation temperatures to 2.2±0.4°C. Resistance to thermal stress outside the tolerance zone was a function of time; the ultimate lethal level (survival for 10 min) increased with acclimation temperature to a maximum of 33°C whilst the minimum value remained close to 0°C. Temperature limits for feeding increased slightly with acclimation temperature to upper and lower mean values of 22.5±0.3°C and 7.0±0.3°C. 4. In spite of different methodologies, values in the present investigation are similar to those obtained in previous, less comprehensive studies in the laboratory. They also agree with field observations on the temperature limits for feeding and survival. Thermal tolerance polygons are now available for eight species of salmonids and show that the highest temperature limits for feeding and survival are those recorded for juvenile Atlantic salmon.     

Predictions of response to selection caused by angling in a wild population of Atlantic salmon (Salmo salar)

1. Recreational angling activities in wild populations of Atlantic salmon may induce a selection pressure towards a reduction in body size and length if the angling season coincides with the return of the largest sea age fish class. 2. Using estimates of heritability for growth traits and estimates of the selection pressure from angling operating on growth, we predicted the response to selection expected to occur in a wild population of Atlantic salmon. 3. The dataset used here comprised individuals from two consecutive generations (parents and offspring) from the River Bidasoa (NW Spain). Offspring were assigned to parents using six highly polymorphic microsatellite loci. Use of restricted maximum likelihood methodology and the animal model allowed us to estimate the heritability for body length and body weight as well as their genetic correlation. 4. Estimated heritabilities (0.32 ± 0.12 for length and 0.32 ± 0.11 for weight) and selection pressure caused by angling were used to obtain predictions of response to selection because of angling. Our results suggested a decline of 1.9 mm in body length and 103.3 g in body weight per generation because of angling pressure. 5. The results derived from this study suggest that the angling season should be annually delayed in order to avoid selective angling of the multi‐year class and further reductions in body weight and length.     

The effects of Lepeophtheirus salmonis infections on the stress response and immunological status of Atlantic salmon (Salmo salar)

This study was conducted to determine the effects of a high level of infection of the parasitic copepod L. salmonis on the stress response and immunological status of Atlantic salmon. An initial low-level initial infection was carried out 14d prior to a second infection in which twice as many parasites were introduced. Plasma cortisol and prostaglandin E(2) (PGE(2)) levels were monitored concurrent to the expression of six immune-related genes over five sample times (9, 21, 26, 33 and 40days post initial infection, dpii). The mean lice counts on the infected fish increased significantly from the first infection (16.3+/-1.89 at 9dpii) to the second (142.8+/-12.8 at 26dpii). Plasma cortisol levels increased significantly at 26, 33 and 40dpii in infected fish compared to controls. Plasma PGE(2) levels were significantly higher in infected fish at 9, 33 and 40dpii, when compared to controls. At 9dpii, expression of interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNFalpha)-like cytokine, major histocompatibility class II (MH II), transforming growth factor-beta (TGFbeta)-like cytokine and cyclooxygenase-2 genes were increased in infected fish compared to controls. The expression of most of these genes returned to control levels at 21dpii when the highest expression of the MH class I gene was observed in infected fish (significantly higher than controls). Major histocompatibility class I gene expression remained higher in infected fish at 26 and 33dpii compared to controls and this was observed for the TNFalpha-like gene. By 33dpii, MH class II and TGFbeta-like genes had higher expression in infected fish compared to controls. Interleukin-1beta and TNFalpha-like gene were the only genes that showed significantly higher expression in infected fish compared to controls at 40dpii, while MH class I gene expression was significantly depressed in infected fish at this time. The expression of nearly all immune-related genes studied here increased following initial infection with L. salmonis, however, immunological stimulation did not reduce parasite numbers or protect against re-infection.     

Histone H1: an antimicrobial protein of Atlantic salmon (Salmo salar)

Antimicrobial activity was detected in acid extracts of liver, intestine, and stomach of healthy Atlantic salmon (Salmo salar). An antimicrobial protein was isolated from salmon liver using acid extraction followed by ammonium sulfate precipitation, large-scale gel filtration chromatography, reverse-phase HPLC, and size exclusion HPLC. The salmon antimicrobial (SAM) protein was found to have a molecular mass of 20,734 Da by MALDI TOF mass spectrometry. Peptide mass fingerprinting and partial sequencing by tandem nanoelectrospray mass spectrometry identified the protein as histone H1. The protein had a minimal inhibitory concentration of 31 microg/mL against E. coli D31 in a plate clearing assay. The effect of the SAM protein on bacterial morphology was indistinguishable from that of (Ala-(8,13,18))-magainin II, as shown by scanning electron microscopy, which suggests that the protein disrupts E. coli membranes in a manner similar to that of most antimicrobial peptides. This protein may act as an antimicrobial in vivo through active secretion or by release from cells during infection-related apoptosis.     

Cloning of the Atlantic salmon (Salmo salar) IL-1 receptor associated protein

In mammals, the pro-inflammatory cytokine interleukin-1 signals through a receptor complex containing a type I interleukin-1 receptor (IL-1RI) and a receptor associated protein (IL-1RAcP). Previously, we have described a cDNA from Atlantic salmon encoding a molecule with homology to the mammalian IL-RI. This molecule was named IL-1 receptor like protein (IL-1RLP) in the absence of functional data to support its proposed role as the salmon IL-1RI. Here, we describe the cloning and characterisation of a cDNA encoding salmon IL-1RAcP. Like other members of the IL-1R family, the salmon IL-1RAcP encodes three extracellular immunoglobulin-like domains and a cytoplasmic Toll/Interleukin-1 receptor (TIR) domain involved in signalling. Specific binding of salmon IL-1RAcP to IL-1RLP was shown by co-immunoprecipitation studies.     

Purification and characterization of Atlantic salmon (Salmo salar) fibrinogen

This study describes a purification protocol of salmon fibrinogen that gives a consumable and highly clottable fibrinogen. Some characteristics of salmon and human fibrinogen are compared. Fibrinogen was purified from barium sulphate adsorbed plasma of Atlantic salmon, using two steps of 25% ammonium sulphate precipitation followed by ultrafiltration. The clottability of the purified salmon fibrinogen was 91%. The Aalpha chains of salmon fibrinogen were heterogeneous with a molecular mass of 90-110 kDa, compared to approximately 67 kDa of human fibrinogen Aalpha chains. The Bbeta and gamma chains of salmon and human fibrinogen had molecular masses of approximately 55 and 50 kDa, respectively. Western blotting revealed that polyclonal rabbit anti-human fibrinogen antibodies had affinity for the gamma chains of salmon fibrinogen, making it possible to study factor XIII activity in purified salmon fibrinogen. Cross-linking of either gamma-gamma or gamma-alpha chains was not detected upon incubation of the purified fibrinogen with thrombin and calcium alone, but was detected when clotting was performed in plasma indicating absence of factor XIII activity in the purified product.     

Experimentally induced marine flexibacteriosis in Atlantic salmon smolts Salmo salar. I. Pathogenicity

Tenacibaculum maritimum causes marine flexibacteriosis in many cultured fish species, including Atlantic salmon Salmo salar in Tasmania, Australia. Several aspects of the pathogenicity of this bacterium were investigated in naive Atlantic salmon smolts using different isolates, growth conditions and doses to produce a model of infection. We found that T. maritimum is pathogenic to Atlantic salmon using either marine Shieh's or marine Ordal's culture medium. The use of aeration in broth culture produced a dose effect in challenge due to a 'clumping' of the bacteria during culture. The virulence of a strain appears to be connected with this 'clumping'; the more adherent the cells, the more pathogenic the strain. Differences in virulence between 3 strains was apparent, with 1 of the strains (89/4747) being non-pathogenic and unable to produce disease in the host. The 2 other strains (89/4762, 00/3280) were highly virulent, resulting in 100% mortalities within 3 d. A reproducible model of infection has been established in the present study using strain 89/4762. Results from the present study provide a better insight into the nature of the disease.     

Swimbladder Leiomyosarcoma in Atlantic salmon (Salmo salar) in North America

Leiomyosarcoma with associated retrovirus were found in North America for the first time in adult Atlantic salmon (Salmo salar) held in a quarantine facility at the North Attleboro National Fish Hatchery (NANFH), Massachusetts, USA. The fish had been collected as age 1-2 yr animals from the Pleasant River, Maine, and were to be used as brood stock in a population augmentation program for that river. Neoplastic disease was observed at NANFH initially in older (age 4 yr) fish, followed by age 3 yr fish. Disease was not observed in age 2 yr fish. The mortality pattern was chronic.     

Molecular characterisation of a putative Atlantic salmon (Salmo salar) odorant receptor

The olfactory system of fish is extremely important as it is able to recognise and distinguish a vast array of odorous molecules that are involved in behaviours paramount to survival. This is achieved by the activation of a diverse multigene family of G-protein coupled receptors through odorous ligand binding. Using molecular techniques, the nucleotide sequence of the cDNA coding for an Atlantic salmon (Salmo salar) odorant receptor (ASOR1) has been determined. The full-length cDNA (1260 nt) encodes a protein of 320 amino acid residues, including one potential N-linked glycosylation site, within the short extracellular amino terminal of the receptor. Hydrophobicity analysis revealed seven hydrophobic regions within the amino acid sequence, corresponding to possible positions of the transmembrane domains characteristic of the G-protein coupled receptor superfamily. Several conserved motifs unique to odorant receptors were also present. Through characterisation of this receptor, we hope to increase the understanding of the mechanisms underlying olfaction in salmonid species.     

Spawning behaviour of a farmed escaped female Atlantic salmon (Salmo salar)

Excavation of stranded redds revealed differences in spawning behaviour between farmed and wild Atlantic salmon. The redd of a farmed fish contained more egg pockets (nine v . average of two) and fewer eggs per pocket (averages: 459 v . 707). No other pocket measures differed.     

Oxygen consumption, ammonia excretion and protein use in response to thermal changes in juvenile Atlantic salmon Salmo salar

Experiments were designed to examine the effects of various temperature challenges on oxygen consumption and ammonia excretion rates and protein utilization in juvenile Atlantic salmon Salmo salar . Fish acclimated to 15° C were acutely and abruptly exposed to either 20 or 25° C for a period of 3 h. To simulate a more environmentally relevant temperature challenge, a third group of fish was exposed to a gradual increase in temperature from 15 to 20° C over a period of 3 h ( c. 1·7° C h⁻¹). Oxygen consumption and ammonia excretion rates were monitored before, during and after the temperature shift. From the ammonia excretion and oxygen consumption rates, protein utilization rates were calculated. Acute temperature changes (15–20° C or 15–25° C) caused large and immediate increases in the oxygen consumption rates. When the temperature was gradually changed ( i.e. 1·7° C h⁻¹), however, the rates of oxygen consumption and ammonia excretion were only marginally altered. When fish were exposed to warmer temperatures ( i.e. 15–20° C or 15–25° C) protein use generally remained at pre-exposure (15° C) levels. A rapid transfer back to 15° C (20–15° C or 25–15° C) generally increased protein use in S. salar . These results indicate that both the magnitude and the rate of temperature change are important in describing the physiological response in juvenile salmonids.     

Mortality in Atlantic salmon (Salmo salar) associated with trichodinid ciliates

A protozoan infection (Trichodina truttae) was identified in captive Atlantic salmon (Salmo salar) kelts that died in spring of 1988 and 1989. Fish with intense infections showed signs of listlessness, erratic swimming and inappetence. The infection induced excessive mucus secretion, epithelial sloughing and lesions that probably permitted entry of opportunistic bacteria which eventually caused ulcers and death. A seawater bath for 30 min each week for 4 wk effectively controlled the parasite.     

Catabolism of circulating collagen in the Atlantic salmon (Salmo salar)

The catabolic fate of circulating collagen (Col) in the Atlantic salmon (Salmo salar) was studied. Serum t_1/2 and organ distribution of circulating Col in salmon were determined using Col conjugated with ^l25I-tyramine cellobiose (¹²⁵I-TC), a low molecular weight adduct which is trapped intralysosomally at the site of uptake. Intravenously administered ^l25I-tyramine cellobioselabelled Col type I was prepared either from salmon skin (sCol) or rat skin (rCol). Biphasic clearance kinetics of ^l25I-TC-sCol in salmon were apparent, with 78% being removed from the circulation in an initial rapid α-phase (t_1/2(α) = 2.4 min), and 22% being removed more slowly in a terminalβ-phase (t]2(β) = 25.8 min). Serum half life of ¹²⁵I-TC-rCol was found to be 5.4 min (in this type of experiment the number of data points allow the determination of only a monophasic decay slope). Approximately 90% of recovered radioactivity was found in the kidney of the fish. In comparative experiments, 74% of administered ¹²⁵I-TC-sCol was cleared from the circulation of rats during an initial rapid α-phase with t_l/2(α) = 0.8 min, and 26% was eliminated in the terminal β-phase with t_1/2(β) = 7.2 min ^l25I-sCol was endocytosed and degraded in pure cultures of ral liver endothelial cells, which are the main site of clearance of circulating Col in the rat. Moreover, Col from the two species competed for the same receptor on cultured rat liver endothelial cells, Intravenous administration of tetramethyl rhodamine isothiocyanate-labelled sCol (TRITC-sCol) in salmon, and subsequent examination of sections of kidney in the fluorescence microscope, revealed that the fluorochrome was accumulated exclusively in discrete vesicles of sinusoidal lining cells. Analyses of kidney tissue 24h after intravenous administration of a mixture of fluorescein isothiocyanate-labelled latex beads and TRITC-sCol revealed no codistribution of the two fluorochromes, suggesting that the injected Col was taken up in cells different from macrophages. Purified pronephros macrophages prepared after simultaneous injections of stained beads and Col contained only fluorescein-labelled latex particles. Interestingly, the cells which had accumulated TRITC-sCol appeared to be equally distributed in both pronephros and the part of the kidney containing tubuli. We conclude that Col which gains access to the circulation of the Atlantic salmon is cleared mainly by uptake into sinusoidal lining cells of the kidney. These cells are distinct from phagocytosing macrophages, and morphologically similar to the highly specialized scavenging endothelial cells of mammalian liver sinusoids.