Effects of growth hormone and cortisol administration on plasma insulin-like growth factor binding proteins in juveniles of three subspecies of masu salmon (Oncorhynchus masou).

In this study, we examined the effects of porcine growth hormone (GH) and cortisol on plasma insulin-like growth factor binding proteins (IGFBPs) in juveniles of three subspecies of Oncorhynchus masou (masu, amago, and Biwa salmon). Ligand blotting using digoxigenin-labeled human IGF-I was used to detect and semi-quantify three major circulating IGFBP bands at 41, 28, and 22 kDa, corresponding to IGFBP-2b, -1a, and -1b, respectively. GH increased plasma IGFBP-2b concentration in masu and Biwa salmon but suppressed it in amago salmon. Plasma IGFBP-2b levels were increased by cortisol in the three subspecies. Cortisol induced plasma IGFBP-1a in the three subspecies, whereas GH had a suppressive effect in masu and Biwa salmon. Sham and cortisol injections increased plasma IGFBP-1b levels after 1 day in masu and amago salmon, suggesting that IGFBP-1b is induced following exposure to stressors via cortisol. Increased IGFBP-1b levels were restored to basal levels when co-injected with GH in Biwa salmon, and the same trend was seen in masu and amago salmon. However, the suppressive effect of GH disappeared 2 days after injection in the three subspecies. Despite some differences among subspecies, the findings suggest that cortisol is a primary inducer of plasma IGFBP-1b; however, GH counteracts it in the short term. Therefore, GH has the potential to modulate the degree of increase in circulating IGFBP-1b levels during acute stress.

Intact rather than total circulating insulin-like growth factor binding protein-1a is a negative indicator of growth in masu salmon.

Insulin-like growth factor binding protein (IGFBP)-1a is one of three major circulating forms in salmon and induced under catabolic conditions. However, there is currently no immunoassay available for this form because of a lack of standard and specific antibodies. We developed a time-resolved fluoroimmunoassay (TR-FIA) for salmon IGFBP-1a using recombinant protein for labeling, an assay standard, and production of antiserum. The TR-FIA had a low cross-reactivity (3.6%) with IGFBP-1b, another major form in the circulation. Fasting for 4 wk had no effect on serum immunoreactive (total) IGFBP-1a levels in yearling masu salmon, whereas 6-wk fasting significantly increased it. There was a significant, but weak, negative relationship between serum total IGFBP-1a level and individual growth rate (r2 = 0.12, P = 0.01). We next developed a ligand immuno-functional assay (LIFA) using europium-labeled IGF-I to quantify intact IGFBP-1a. In contrast to total IGFBP-1a, serum intact IGFBP-1a levels increased after 4 wk of fasting, and refeeding for 2 wk restored it to levels similar to those of the fed control. Serum intact IGFBP-1a levels showed a significant negative correlation with individual growth rate (r2 = 0.52, P < 0.001), which was as good as that of IGFBP-1b. Our findings using newly developed TR-FIA and LIFA suggest that regulation of intact IGFBP-1a levels has an important effect on growth in salmon and that intact IGFBP-1a is a negative index of salmon growth.

Molecular cloning of vitellogenin gene promoters and in vitro and in vivo transcription profiles following estradiol-17ß administration in the cutthroat trout.

Transcription of vitellogenin (vtg) genes are initiated when estradiol-17ß (E2)-estrogen receptor (ER) complexes bind estrogen response elements (ERE) located in the gene promoter region. Transcriptional regulation of dual vtg subtypes (major salmonid A-type vtg: vtgAs; minor C-type vtg: vtgC) by E2 was investigated under co-expression of a potential major transcriptional factor, erα1, in cutthroat trout. Two forms of trout vtgAs promoters (1 and 2) and one vtgC promoter were sequenced. These promoters structurally differ based on the number of EREs present. The vtgAs promoter 1 exhibited the highest maximal transcriptional activity by in vitro gene reporter assays. The concentration of E2 that induces 50% of gene reporter activity (half-maximal effective concentrations, EC50) was similar among all vtg promoters and also to the EC50 of E2 administered to induce vtg transcription in vivo. This study revealed a difference in transcriptional properties of multiple vtg promoters for the first time in a salmonid species, providing the basis to understand mechanisms underlying regulation of vitellogenesis via dual vtg gene expression.

Full-Genome Sequencing and Confirmation of the Causative Agent of Erythrocytic Inclusion Body Syndrome in Coho Salmon Identifies a New Type of Piscine Orthoreovirus.

Erythrocytic inclusion body syndrome (EIBS) causes mass mortality in farmed salmonid fish, including the coho salmon, Onchorhynchus kisutchi, and chinook salmon, O. tshawytscha. The causative agent of the disease is a virus with an icosahedral virion structure, but this virus has not been characterized at the molecular level. In this study, we sequenced the genome of a virus purified from EIBS-affected coho salmon. The virus has 10 dsRNA genomic segments (L1, L2, L3, M1, M2, M3, S1, S2, S3, and S4), which closely resembles the genomic organization of piscine orthoreovirus (PRV), the causative agent of heart and skeletal inflammation (HSMI) in Atlantic salmon and HSMI-like disease in coho salmon. The genomic segments of the novel virus contain at least 10 open reading frames (ORFs): lambda 1 (λ1), λ2, λ3, mu 1 (µ1), µ2, µNS, sigma 1 (σ1), σ2, σ3, and σNS. An additional ORF encoding a 12.6-kDa protein (homologue of PRV p13) occurs in the same genomic segment as σ3. Phylogenetic analyses based on S1 and λ3 suggest that this novel virus is closely related to PRV, but distinctly different. Therefore, we designated the new virus 'piscine orthoreovirus 2' (PRV-2). Reverse transcription-quantitative real-time PCR revealed a significant increase in PRV-2 RNA in fish blood after the artificial infection of EIBS-naïve fish but not in that of fish that had recovered from EIBS. The degree of anemia in each fish increased as the PRV-2 RNA increased during an epizootic season of EIBS on an inland coho salmon farm. These results indicate that PRV-2 is the probable causative agent of EIBS in coho salmon, and that the host acquires immunity to reinfection with this virus. Further research is required to determine the host range of PRV species and the relationship between EIBS and HSMI in salmonid fish.

Lack of hormonal stimulation prevents the landlocked Biwa salmon (Oncorhynchus masou subspecies) from adapting to seawater.

Landlocking of salmon relaxes selective pressures on hypoosmoregulatory ability (seawater adaptability) and may lead to the abandonment of its physiological system. However, little is known about the mechanism and consequence of the process. Biwa salmon is a strain/subspecies of Oncorhynchus masou that has been landlocked in Lake Biwa for an exceptionally long period (about 500,000 years) and has low ability to adapt to seawater. We compared activity of gill Na(+),K(+)-ATPase (NKA) of Biwa salmon with those of anadromous strains of the same species (masu and amago salmon) during downstream migration periods and after exogenous hormone treatment. Gill NKA activity in anadromous strains increased during their migration periods, while that in Biwa salmon remained low. However, treatments of Biwa salmon with growth hormone (GH) and cortisol increased gill NKA activity. Cortisol treatment also improved the whole body seawater adaptability of Biwa salmon. Receptors for GH and cortisol responded to hormonal treatments, whereas their mRNA levels during downstream migration period were essentially unchanged in Biwa salmon. Circulating levels of cortisol in masu salmon showed a peak during downstream migration period, while no such increase was seen in Biwa salmon. The present results indicate that Biwa salmon can improve its seawater adaptability by exogenous hormonal treatment, and hormone receptors are capable of responding to the signals. However, secretion of the endogenous hormone (cortisol) was not activated during the downstream migration period, which explains, at least in part, their low ability to adapt to seawater.

Increasing versatility of the DNA vaccines through modification of the subcellular location of plasmid-encoded antigen expression in the in vivo transfected cells.

The route of administration of DNA vaccines can play a key role in the magnitude and quality of the immune response triggered after their administration. DNA vaccines containing the gene of the membrane-anchored glycoprotein (gpG) of the fish rhabdoviruses infectious haematopoietic necrosis virus (IHNV) or viral haematopoietic septicaemia virus (VHSV), perhaps the most effective DNA vaccines generated so far, confer maximum protection when injected intramuscularly in contrast to their low efficacy when injected intraperitoneally. In this work, taking as a model the DNA vaccine against VHSV, we focused on developing a more versatile DNA vaccine capable of inducing protective immunity regardless of the administration route used. For that, we designed two alternative constructs to gpG1₋507 (the wild type membrane-anchored gpG of VHSV) encoding either a soluble (gpG1₋462) or a secreted soluble (gpG(LmPle20-462)) form of the VHSV-gpG. In vivo immunisation/challenge assays showed that only gpG(LmPle20-462) (the secreted soluble form) conferred protective immunity against VHSV lethal challenge via both intramuscular and intraperitoneal injection, being this the first description of a fish viral DNA vaccine that confers protection when administered intraperitoneally. Moreover, this new DNA vaccine construct also conferred protection when administered in the presence of an oil adjuvant suggesting that DNA vaccines against rhabdoviruses could be included in the formulation of current multicomponent-intaperitoneally injectable fish vaccines formulated with an oil adjuvant. On the other hand, a strong recruitment of membrane immunoglobulin expressing B cells, mainly membrane IgT, as well as t-bet expressing T cells, at early times post-immunisation, was specifically observed in the fish immunised with the secreted soluble form of the VHSV-gpG protein; this may indicate that the subcellular location of plasmid-encoded antigen expression in the in vivo transfected cells could be an important factor in determining the ways in which DNA vaccines prime the immune response.

The efficacy of field techniques for obtaining and storing blood samples from fishes.

Prompted by the dramatic increase in the use of blood analyses in fisheries research and monitoring, this study investigated the efficacy of common field techniques for sampling and storing blood from fishes. Three questions were addressed: (1) Do blood samples taken via rapid caudal puncture (the 'grab-and-stab' technique) yield similar results for live v. sacrificed groups of fishes? (2) Do rapidly obtained caudal blood samples accurately represent blood properties of fishes prior to capture? (3) Does storage of whole blood in an ice slurry for a working day (8·5 h) modify the properties of the plasma? It was shown that haematocrit, plasma ions, metabolites, stress hormones and sex hormones of caudal blood samples were statistically similar when taken from live v. recently sacrificed groups of adult coho salmon Oncorhynchus kisutch. Moreover, this study confirmed by using paired blood samples from cannulated O. kisutch that blood acquired through the caudal puncture technique (mean ±s.e. 142 ± 26 s after capture) was representative of fish prior to capture. Long-term (8·5 h) cold storage of sockeye salmon Oncorhynchus nerka whole blood caused significant decreases in plasma potassium and chloride, and a significant increase in plasma glucose. Previous research has suggested that these changes largely result from net movements of ions and molecules between the plasma and erythrocytes, movements that can occur within minutes of storage. Thus, blood samples from fishes should be centrifuged as quickly as practicable in the field for separation of plasma and erythrocytes to prevent potentially misleading data.

Effects of external and surgically implanted dummy radio transmitters on mortality, swimming performance and physiological status of juvenile masu salmon Oncorhynchus masou.

The effects of external and surgically implanted radio transmitters on juvenile masu salmon Oncorhynchus masou were examined. External attachment of transmitters significantly decreased the survival of fish and caused a decreased critical swimming speed compared with surgical implantation. Although plasma cortisol, plasma glucose and haematocrit values did not differ significantly among groups, it appeared that the most suitable transmitter attachment method for juvenile O. masou may be surgical implantation.

Effects of growth hormone and cortisol on the downstream migratory behavior in masu salmon, Oncorhynchus masou.

The effects of ovine growth hormone (oGH) and cortisol on downstream migratory behavior in yearling (1(+)) smolts and underyearling (0(+)) parr of masu salmon, Oncorhynchus masou, were examined during the downstream migratory period in spring using artificial raceways. In May, each of 22 1(+) smolts and 0(+) parr were implanted with cholesterol pellets containing 250 microg of oGH and/or 2 mg of cortisol. Their downstream migratory behavior was subsequently observed in artificial raceways, along with control groups 4-23 days after implantation. In 1(+) smolts, the frequency of downstream migratory behavior was 23%, 18%, 72%, and 82% in the control, oGH, cortisol, and oGH+cortisol-treated groups, respectively. The frequency was significantly higher in the cortisol and oGH+cortisol-treated groups than in the control and oGH-treated groups. In 0(+) parr, the frequency of downstream migratory behavior in the cortisol (82%) and cortisol+oGH-treated (90%) groups was significantly higher than in the control (18%) and oGH-treated (0%) groups. These results indicate that cortisol is an important endocrine factor inducing downstream migratory behavior in both 1(+) smolt and 0(+) parr of masu salmon.

Effects of melatonin feeding on smoltification in masu salmon (Oncorhynchus masou).

Influences of photoperiod on plasma melatonin profiles and effects of melatonin administration on long-day-induced smoltification in masu salmon (Oncorhynchus masou) were investigated in order to reveal the roles of melatonin in the regulation of smoltification in salmonids. Under light-dark (LD) cycles, plasma melatonin levels exhibited daily variation, with higher values during the dark phase than during the light phase. The duration of nocturnal elevation under short photoperiod (LD 8:16) was longer than that under long photoperiod (LD 16:8). Melatonin feeding (0.01, 0.1 and 1 mg/kg body weight) elevated plasma levels of melatonin in a dose-dependent manner for at least 7 h but not for 24 h. When masu salmon reared under short photoperiod were exposed to long photoperiod (LD 16:8) and fed melatonin (1 mg/kg body weight) 7 hours before the onset of darkness, a significantly smaller proportion of smolts appeared in the melatonin-fed group after 32 days than in the control group. However, after 59 days of the treatment, there was no difference in the proportion of smolts between the control and melatonin-treated groups. Thus, melatonin feeding mimicked the effects of short photoperiod, which delays but does not completely suppress smoltification. These results indicate that the day length is transduced into changes in the duration of nocturnal elevation in plasma melatonin levels, and that artificial modification of the plasma melatonin pattern possibly delays the physiological processes of smoltification induced by long-day photoperiodic treatment.

Annual changes in serum levels of two choriogenins and vitellogenin in masu salmon, Oncorhynchus masou.

Annual changes in serum levels of two chorion precursors, choriogenin H (Chg H) and choriogenin L (Chg L), vitellogenin (Vg) and estradiol-17beta (E2) were quantified in masu salmon, Oncorhynchus masou, using specific immunoassays. Serum Chg levels were higher than Vg during the previtellogenic growth phase when circulating E2 levels were low ( approximately 0.1 ng/mL), suggesting higher sensitivity of Chg to E2. When oocyte growth shifted to the vitellogenic phase, Vg levels increased and became the most abundant in serum coincident with elevations of E2 and GSI. Chg H, Chg L and Vg peaked 1 month prior to ovulation at 0.61+/-0.08, 0.98+/-0.18 and 10.93+/-3.24 mg/mL, respectively. These results suggest that chorion formation by Chgs occurs prior to vitellogenesis and that the sensitivity of Chgs to low circulating E2 is closely related to the sequential events of oocyte growth.

Purification of serum precursor proteins to vitelline envelope (choriogenins) in masu salmon, Oncorhynchus masou.

Three vitelline envelope-related proteins (VERPs), very-high-molecular-weight VERP (vhVERP), high-molecular-weight VERP (hVERP) and low-molecular-weight VERP (lVERP) were purified from female masu salmon serum. The apparent molecular weights of vhVERP, hVERP and lVERP, in their native state, were 520, 88 and 54 kDa, respectively, by gel-filtration chromatography. Very-high-molecular-weight VERP comprises two subunits, corresponding to 175 and 126 kDa. On SDS-PAGE, hVERP and lVERP migrate at 53 and 47 kDa, respectively. Amino acid analysis of vhVERP and hVERP showed that they share a high content of glutamic acid and proline. By contrast, lVERP is rich in glutamic acid and asparatic acid. These features are in good agreement with the amino acid composition of the vitelline envelope. Immuno-biochemical analysis suggested that vhVERP is derived from hVERP by polymerization and/or aggregation. Antibodies against hVERP and lVERP specifically immunostained the vitelline envelope and liver of female masu salmon. In addition, both hVERP and lVERP were induced in the serum of estrogen-treated male fish. Taken together, it is suggested that hVERP and lVERP are homologous molecules with choriogenin H and choriogenin L in medaka, respectively. These results indicate that hVERP and lVERP are precursor proteins to the vitelline envelope (choriogenins) in masu salmon.

Cloning and expression analysis of rainbow trout Oncorhynchus mykiss tumour necrosis factor-alpha.

A rainbow trout (Oncorhynchus mykiss) gene for tumor necrosis factor (TNF) has been cloned and sequenced. The cDNA contains an open reading frame of 738 nucleotides that translate into a 246 amino-acid putative peptide, with a 5' untranslated region (UTR) of 140 bp and a 3' UTR of 506 bp. Two potential N-linked glycosylation sites exist in the translation. The genomic sequence measures 2007 bp and contains three introns that intercept four coding exons. Expression studies using RT-PCR have shown that the trout TNF gene is constitutively expressed in the gill and kidney of unstimulated fish. Trout TNF expression could be up-regulated by stimulation of isolated head kidney leucocytes with lipopolysaccharide (LPS). Similarly, stimulation of a trout macrophage cell line (RTS11) with LPS resulted in an increased transcript level, as did incubation with recombinant trout interleukin (IL)-1 beta. The optimal timing for induction of TNF expression in trout macrophages was determined using recombinant trout IL-1 beta, where a clear induction was apparent by 2 h and peaked at 4 h. Evidence that this TNF gene is equivalent to mammalian TNF-alpha is discussed.

Physiological levels of testosterone kill salmonid leukocytes in vitro.

Adult spring chinook salmon (Oncorhynchus tshawytscha) elaborate high plasma concentrations of testosterone during sexual maturation, and these levels of testosterone have been shown to reduce the salmonid immune response in vitro. Our search for the mechanism of testosterone's immunosuppressive action has led to the characterization of an androgen receptor in salmonid leukocytes. In the present study we examined the specific effects that testosterone had on salmonid leukocytes. Direct counts of viable leukocytes after incubation with and without physiological levels of testosterone demonstrate a significant loss of leukocytes in cultures exposed to testosterone. At least 5 days of contact with testosterone was required to produce significant immunosuppression and addition of a "conditioned media" (supernatant from proliferating lymphocytes not exposed to testosterone) did not reverse the immunosuppressive effects of testosterone. These data lead us to conclude that testosterone may exert its immunosuppressive effects by direct action on salmonid leukocytes, through the androgen receptor described, and that this action leads to the death of a significant number of these leukocytes.