Hypoxia modulates the transcriptional immunological response in Oncorhynchus kisutch.

Oncorhynchus kisutch is the third most cultivated salmonid species in the Chilean salmon industry and its farming conditions are characterised by high stocking density leading to the generation of high levels of organic matter (food - feces) and decomposition. In addition to the increasingly frequent hypoxic oceanographic events, these inappropriate farming conditions increase the demand for oxygen within the fish farm pen and lead to the appearance of hypoxic events that are harmful to fish.This study aimed to evaluate the stress response (cortisol) and transcription of genes involved in the immune response in head kidney and spleen of Oncorhynchus kisutch subjected to chronic hypoxic stress conditions. The fish were exposed to 100%, 60%, 50%, 35%, and 25% of DO for 28 days, then the blood (plasma), head kidney and spleen were removed. We observed mortality in the 25% DO group at days 15 and 20. Plasma cortisol increased significantly under 35% and 25% DO conditions compared to control. Transcription of Toll-like receptors (TLR1, TLR5M, TLR8, and TLR9) and cytokines (IL-1ß, IL6, IL10, TNF-α) increased in the head kidney only in the 50% DO group, while in spleen there was an increase of these markers in the conditions of 60%, 35%, and 25% DO. As for the markers involved in cell-mediated immunity, CD4-MHCII and CD8-MHCI do not have a clear expression pattern, although there was down-regulation in MHCII transcription in the head kidney, in all the hypoxia conditions evaluated. Finally, IgM transcription was increased in the spleen in all hypoxia conditions, although it wasn't always statistically significant compared to the control. These results indicate that chronic hypoxia induces the stress response, increasing plasma cortisol levels and modulating the transcription of genes involved in the innate and adaptive immune response. The expression patterns were tissue-specific, indicating that the degree of hypoxia differentially affects the transcription of genes involved in the immune response of Oncorhynchus kisutch.

An urban stormwater runoff mortality syndrome in juvenile coho salmon.

Untreated urban runoff poses significant water quality threats to aquatic organisms. In northwestern North America, ongoing development in coastal watersheds is increasing the transport of toxic chemical contaminants to river and stream networks that provide spawning and rearing habitats for several species of Pacific salmon. Adult coho (Oncorhynchus kisutch) are particularly vulnerable to a stormwater-driven mortality syndrome. The phenomenon may prematurely kill more than half of the coho that return each fall to spawn in catchments with a high degree of imperviousness. Here we evaluate the coho mortality syndrome at the juvenile life stage. Freshwater-stage juveniles were exposed to stormwater collected from a high traffic volume urban arterial roadway. Symptoms characteristic of the mortality syndrome were evaluated using digital image analysis, and discrete stages of abnormal behavior were characterized as the syndrome progressed. At a subset of these stages, blood was analyzed for ion homeostasis, hematocrit, pH, glucose, and lactate. Several of these blood chemistry parameters were significantly dysregulated in symptomatic juvenile coho. Affected fish did not recover when transferred to clean water, suggesting a single runoff event to stream habitats could be lethal if resident coho become overtly symptomatic. Among coho life stages, our findings indicate the urban runoff mortality syndrome is not unique to adult spawners. Therefore, the consequences for wild coho populations in developing watersheds are likely to be greater than previously anticipated.

Utilization of an endocrine growth index, insulin-like growth factor binding protein (IGFBP)-1b, for postsmolt coho salmon in the Strait of Georgia, British Columbia, Canada.

Monitoring the growth of salmon during their early marine phase provides insights into prey availability, and growth rates may be linked to risks of size-dependent mortality. However, the measurement of growth rate is challenging for free-living salmon in the ocean. Insulin-like growth factor (IGF)-I is a growth-promoting hormone that is emerging as a useful index of growth in salmon. In addition, laboratory-based studies using coho salmon have shown that one of circulating IGF-binding proteins (IGFBPs), IGFBP-1b, is induced by fasting and thus could be used as an inverse index of growth and/or catabolic state in salmon. However, few studies have measured plasma levels of IGFBP-1b in salmon in the wild. We measured plasma IGFBP-1b levels for postsmolt coho salmon collected in the Strait of Georgia and surrounding waters, British Columbia, Canada, and compared regional differences in IGFBP-1b to ecological information such as seawater temperature and stomach fullness. Plasma IGFBP-1b levels were the highest in fish from Eastern Johnstone Strait and relatively high in Queen Charlotte Strait and Western Johnstone Strait, which was in good agreement with the poor ocean conditions for salmon hypothesized to occur in that region. The molar ratio of plasma IGF-I to IGFBP-1b, a theoretical parameter of IGF-I availability to the receptor, discriminated differences among regions better than IGF-I or IGFBP-1b alone. Our data suggest that plasma IGFBP-1b reflects catabolic status in postsmolt coho salmon, as highlighted in fish in Eastern Johnston Strait, and is a useful tool to monitor negative aspects of salmon growth in the ocean.

The pituitary-thyroid axis during the parr-smolt transformation of Coho salmon, Oncorhynchus kisutch: quantification of TSH ß mRNA, TSH, and thyroid hormones.

The objective of this investigation was to quantify pituitary thyroid stimulating hormone (TSH) ß mRNA, pituitary and plasma TSH and plasma thyroid hormone levels during the parr-smolt transformation of Coho salmon that occurs in spring from February to May. The status of the pituitary-thyroid axis was assessed using an RNase protection assay for pituitary TSH ß mRNA and radioimmunoassays for salmon pituitary and plasma TSH and thyroid hormones. TSH ß mRNA was highest during late winter (February) (4.9 pg/µg DNA) and gradually declined during spring (2.3 pg/µg DNA). In contrast, pituitary and plasma TSH levels showed a small, but statistically non-significant change during smoltification. Despite minimal change in plasma TSH levels, characteristically large increases in plasma T4 (January-3.3 ng/ml to April-10.2 ng/ml) and significant, but modest increases in plasma T3 (February-2.4 ng/ml to April-5.8 ng/ml) were observed. Regression analysis showed a significant positive relationship between plasma T4 and T3 and negative relationship between plasma T3 and pituitary TSH ß mRNA. However, all other relations were not significant. These data suggest a significant role for peripheral regulation (i.e. T4-T3 conversion, change in tissue sensitivity, hormone degradation rate) as well as evidence of central regulation via negative feedback at the level of the pituitary gland in regulation of thyroid activity in salmon. Furthermore, the increased thyroid sensitivity to TSH (shown previously), in the face of relatively constant plasma TSH levels, may be the major factor responsible for the increased thyroid activity observed during smoltification.

Physiological responses of free-swimming adult coho salmon to simulated predator and fisheries encounters.

The responses of free-swimming adult coho salmon (Oncorhynchus kisutch) to simulated predator and fisheries encounters were assessed by monitoring heart rate (f(H)) with implanted data loggers and periodically taking caudal blood samples. A 10- or 30-min corralling treatment was conducted to simulate conspecifics being cornered by a predator or corralled by fisheries gear without physical contact. Corralling rapidly doubled f(H) from ∼31 beats min(-1) to a maximum of ∼60 beats min(-1), regardless of the duration of the corralling. However, recovery of f(H) to precorralling levels was significantly faster after the 10-min corralling (7.6 h) than after the 30-min corralling (11.5 h). An exhaustive-exercise treatment (chasing for 3 min, with physical contact) to simulate a predator chasing a fish to exhaustion or a fish becoming exhausted after encountering fisheries gear resulted in increased f(H) (to 60 beats min(-1)), plasma lactate, glucose, sodium, osmolality, and cortisol (males only) and a significant decrease in mean corpuscular hemoglobin concentration. Recovery of f(H) and most blood variables was complete about 16 h after exhaustive exercise and handling. The results illustrate a clear relationship between the intensity of exercise and the duration required for recovery of f(H). Changes in f(H) were significantly correlated with those in plasma lactate, chloride, and sodium at 1 h after the exercise treatment protocols. Thus, measurements of f(H) may provide an accurate indication of the general physiological response of salmonids to exhaustive exercise in the natural environment.

Postprandial changes in plasma growth hormone, insulin, insulin-like growth factor (IGF)-I, and IGF-binding proteins in coho salmon fasted for varying periods.

We examined postprandial changes in circulating growth hormone (GH), insulin, insulin-like growth factor (IGF)-I, and IGF-binding proteins (IGFBPs) in yearling coho salmon under different feeding regimes. Fish were initially fasted for 1 day, 1 wk, or 3 wk. Fasted fish were then fed, and blood was collected at 4-h intervals over 26 h. After the various periods of fasting, basal levels of insulin were relatively constant, whereas those of IGF-I, IGFBPs and GH changed in proportion to the duration of the fast. A single meal caused a rapid, large increase in the circulating insulin levels, but the degree of the increase was influenced by the fasting period. IGF-I showed a moderate increase 2 h after the meal but only in the regularly fed fish. Plasma levels of 41-kDa IGFBP were increased in all groups within 6 h after the single meal. The fasting period did not influence the response of 41-kDa IGFBP to the meal. IGFBP-1 and GH decreased after the meal to the same extent among groups regardless of the fasting period. The present study shows that insulin and IGF-I respond differently to long (weeks)- and short (hours)-term nutritional changes in salmon; insulin maintains its basal level but changes acutely in response to food intake, whereas IGF-I adjusts its basal levels to the long-term nutritional status and is less responsive to acute nutritional input. IGFBPs maintain their sensitivity to food intake, even after prolonged fasting, suggesting their critical role in the nutritional regulation of salmon growth.

Characterization and measurement of the plasma alpha- and beta-sex hormone-binding globulin paralogs in salmon.

When the biochemical characteristics of coho salmon SHBG (csSHBG) plasma were examined, two different steroid-binding profiles were obtained corresponding to recombinant csSHBG-alpha and csSHBG-beta. These SHBG paralogs share only 24% sequence identity, and this explains their unique steroid-binding properties. Both proteins bind testosterone, but csSHBG-alpha also binds androstenedione (Kd = 2.8 nm) and ethinylestradiol with high affinity, whereas csSHBG-beta binds estradiol (Kd = 0.8 nm) preferentially. When analyzed by gel filtration, csSHBG-alpha displays the properties of a 153-kDa homodimer, whereas csSHBG-beta elutes as a 68-kDa monomer. The unique steroid-binding properties of csSHBG-alpha and csSHBG-beta allowed us to develop an assay for their measurements in immature (pre-smolt) and mature coho salmon blood. Plasma csSHBG-alpha levels were 3- to 4-fold higher than those of csSHBG-beta irrespective of developmental stage or sex and correlate with each other. The major site of csSHBG-alpha expression in pre-smolts and mature fish is the liver, but low levels of csSHBG-alpha mRNA are present in stomach/intestine of mature fish. In pre-smolts, high levels of csSHBG-beta mRNA are present in gills and ovary, whereas csSHBG-beta mRNA is most abundant in muscle and stomach/intestine of mature fish. Based on the differences in csSHBG-alpha and csSHBG-beta plasma levels and their tissue expression profiles, we conclude that gills and/or muscle contribute mainly to plasma SHBG-beta in coho salmon. The assays we have developed will enable studies of how SHBG-alpha/SHBG-beta biosynthesis is regulated throughout the salmonid life cycle and how they influence steroid hormone action in these fish.

Endocrine effects of growth hormone overexpression in transgenic coho salmon.

Non-transgenic (wild-type) coho salmon (Oncorhynchus kisutch), growth hormone (GH) transgenic salmon (with highly elevated growth rates), and GH transgenic salmon pair fed a non-transgenic ration level (and thus growing at the non-transgenic rate) were examined for plasma hormone concentrations, and liver, muscle, hypothalamus, telencephalon, and pituitary mRNA levels. GH transgenic salmon exhibited increased plasma GH levels, and enhanced liver, muscle and hypothalamic GH mRNA levels. Insulin-like growth factor-I (IGF-I) in plasma, and growth hormone receptor (GHR) and IGF-I mRNA levels in liver and muscle, were higher in fully fed transgenic than non-transgenic fish. GHR mRNA levels in transgenic fish were unaffected by ration-restriction, whereas plasma GH was increased and plasma IGF-I and liver IGF-I mRNA were decreased to wild-type levels. These data reveal that strong nutritional modulation of IGF-I production remains even in the presence of constitutive ectopic GH expression in these transgenic fish. Liver GHR membrane protein levels were not different from controls, whereas, in muscle, GHR levels were elevated approximately 5-fold in transgenic fish. Paracrine stimulation of IGF-I by ectopic GH production in non-pituitary tissues is suggested by increased basal cartilage sulphation observed in the transgenic salmon. Levels of mRNA for growth hormone-releasing hormone (GHRH) and cholecystokinin (CCK) did not differ between groups. Despite its role in appetite stimulation, neuropeptide Y (NPY) mRNA was not found to be elevated in transgenic groups.

Measurement of circulating salmon IGF binding protein-1: assay development, response to feeding ration and temperature, and relation to growth parameters.

Fish plasma/serum contains multiple IGF binding proteins (IGFBPs), although their identity and physiological regulation are poorly understood. In salmon plasma, at least three IGFBPs with molecular masses of 22, 28 and 41 kDa are detected by Western ligand blotting. The 22 kDa IGFBP has recently been identified as a homolog of mammalian IGFBP-1. In the present study, an RIA for salmon IGFBP-1 was established and regulation of IGFBP-1 by food intake and temperature, and changes in IGFBP-1 during smoltification, were examined. Purified IGFBP-1 from serum was used for as a standard, for tracer preparation and for antiserum production. Cross-linking (125)I-labelled IGFBP-1 with salmon IGF-I eliminated interference by IGFs. The RIA had little cross-reactivity with salmon 28 and 41 kDa IGFBPs (< 0.5%) and measured IGFBP-1 levels as low as 0.1 ng/ml. Fasted fish had significantly higher IGFBP-1 levels than fed fish (21.6 +/- 4.6 vs 3.0 +/- 2.2 ng/ml). Plasma IGFBP-1 was measured in individually tagged 1-year-old coho salmon reared for 10 weeks under four different feeding regimes as follows: high constant (2% body weight/day), medium constant (1% body weight/day), high variable (2% to 0.5% body weight/day) and medium variable (1% to 0.5% body weight/day). Fish fed with the high ration had lower IGFBP-1 levels than those fed with the medium ration. Circulating IGFBP-1 increased following a drop in feeding ration to 0.5% and returned to the basal levels when feeding ration was increased. Another group of coho salmon were reared for 9 weeks under different water temperatures (11 or 7 degrees C) and feeding rations (1.75, 1 or 0.5% body weight/day). Circulating IGFBP-1 levels were separated by temperature during the first 4 weeks; a combined effect of temperature and feeding ration was seen in week 7; only feeding ration influenced IGFBP-1 level thereafter. These results indicate that IGFBP-1 is responsive to moderate nutritional and temperature changes. There was a clear trend that circulating IGFBP-1 levels were negatively correlated with body weight, condition factor (body weight/body length(3) x 100), growth rates and circulating 41 kDa IGFBP levels but not IGF-I levels. During parr-smolt transformation of coho salmon, IGFBP-1 levels showed a transient peak in late April, which was opposite to the changes in condition factor. Together, these findings suggest that salmon IGFBP-1 is inhibitory to IGF action. In addition, IGFBP-1 responds to moderate changes in dietary ration and temperature, and shows a significant negative relationship to condition factor.

Comparison of extraction methods and assay validation for salmon insulin-like growth factor-I using commercially available components.

Insulin-like growth factor-binding proteins (IGFBPs) may interfere with accurate measurement of plasma IGFs in radioimmunoassay (RIA). Although several simplified extraction methods for IGFs have been developed, these methods are not always validated for differing physiological states, developmental stages, and animal species. For teleost fish, neither the necessity of plasma extraction nor the validity of extraction methods for IGF RIA is widely established. We systematically examined the validity of acid-ethanol (AE) extraction, AE extraction followed by cryoprecipitation (AEC extraction), and SP-Sephadex extraction in RIA for salmon IGF-I using commercially available components (GroPep Pty Ltd). Displacement curves of plasma extracted by AE, AEC, and SP-Sephadex were parallel to those of the standard. Measured IGF-I levels in plasma from several developmental stages and under different physiological and experimental conditions were significantly increased by the extractions and comparable to those after acid-size exclusion chromatography (SEC). On Western ligand blotting using digoxigenin-labeled human IGF-I, the intensity of IGFBP bands remaining in plasma were reduced after extraction, although some IGFBPs remained. However, these residual IGFBPs did not interfere measurably with the RIA based on quantitative comparison of IGF-I levels with acid-SEC. We conclude that with this RIA extraction is necessary for measurement of salmon IGF-I in plasma since measured values were routinely lower in unextracted samples, and AE, AEC, and SP-Sephadex extractions are applicable to the IGF-I RIA using the commercially available components. Using the validated RIA for IGF-I, plasma IGF-I levels in nonmaturing and precociously maturing chinook salmon in spring were measured after AE extraction. During spring, nonmaturing and maturing fish fed and grew well, and plasma IGF-I level was significantly correlated with body weight in both fish. This result indicates that circulating IGF-I plays a key role in controlling growth in precociously maturing chinook salmon in spring as in nonmaturing fish.

Free and protein-bound insulin-like growth factor-I (IGF-I) and IGF-binding proteins in plasma of coho salmon, Oncorhynchus kisutch.

Total and free insulin-like growth factor-I (IGF-I) levels were quantified in plasma from growth hormone (GH)-treated and fasted coho salmon. Total IGF-I was measured by radioimmunoassay after acid-ethanol extraction and free IGF-I was separated from protein-bound IGF-I using ultrafiltration by centrifugation. Total and free IGF-I increased in plasma after GH treatment and decreased after fasting. The level of free IGF-I, however, was maintained at approximately 0.3% in both experiments. Unsaturated binding activity in plasma for IGF-I was assessed by incubation with (125)I-recombinant salmon IGF-I ((125)I-sIGF-I). Although there was no difference in binding activity between GH-treated and control fish, fasted fish showed higher binding activity than did fed fish, suggesting induction of unsaturated binding protein by fasting. IGF-binding protein (IGFBP) bands were observed in plasma of coho salmon by Western ligand blotting using (125)I-sIGF-I. A low-molecular-weight (22 kDa) band was clear in fasted fish but not detectable in fed fish. The IGFBP band, which has molecular weight similar to that of human IGFBP-3 (41 kDa), was more intense in GH-treated fish than in controls. The molecular distribution of IGF-I in plasma was examined by gel filtration under neutral conditions. Most IGF-I was eluted around 40 kDa. This result suggests that the major form of bound IGF-I in the circulation of coho salmon may be in a 40-kDa binary complex rather than in a 150-kDa ternary complex, as in mammals.

Calcium is an equipotent stimulator of stanniocalcin secretion in freshwater and seawater salmon.

Stanniocalcin (STC) is a calcium- and phosphate-regulating hormone produced by the corpuscles of Stannius in fishes. A rise in ion calcium (Ca2+) levels is the principal stimulus for secretion, and the hormone acts on the gills, gut, and kidneys to restore normocalcemia. The STC-producing cells in marine fishes are metabolically more active and secrete more hormone than those in freshwater fishes, which has been attributed to the higher calcium content of seawater placing a greater burden on the organ systems governing Ca2+ homeostasis. In this study we have addressed the question of whether or not the STC cells in marine fishes are more sensitive to Ca2+, by comparing the secretagogic effects of Ca2+ in freshwater- and seawater-adapted coho salmon. The results showed that the STC cells were equally Ca(2+)-sensitive in the two groups. Therefore, in spite of the fact that the STC cells are more active in marine fishes this requires no apparent adjustment in cellular sensitivity to calcium.

Development of a homologous radioimmunoassay for coho salmon insulin-like growth factor-I.

A specific homologous radioimmunoassay (RIA) for measurement of insulin-like growth factor-I (IGF-I) in plasma of salmonid and a few non-salmonid fish species was developed using recombinant coho salmon IGF-I (rsIGF-I) as tracer and standard, and antiserum against this peptide raised in rabbits. The minimum detection level of IGF-I was 1.5 ng/ml and linearity was obtained in a range from 1.5 to 23 ng/ml. No cross-reaction was detected in the salmon IGF-I RIA with mammalian growth factors, salmon pituitary hormones, salmon or mammalian insulin, or any peptide in rat plasma. Although salmon IGF-I has high sequence similarity to mammalian IGF-I, it did not cross-react with anti-human IGF-I serum in human RIA and serial dilutions of plasma from salmon were not parallel to the human IGF-I standards in this assay system. In contrast, dilution curves for plasma of salmonids, such as coho (Oncorhynchus kisutch), Atlantic (Salmo salar), and sockeye (O. nerka) salmon, rainbow trout (O. mykiss), some other teleost fish, such as tilapia (Oreochromis mossabmica), carp (Cyprus carpio), eel (Anguilla rostrata), Atlantic halibut (Hippoglossus hippoglossus), and agnathan, the sea lamprey (Petromyzon marinus), assessed in salmon IGF-I RIA were parallel to the rsIGF-I standards. Acid-ethanol extraction of plasma samples altered the molecular weight, but not the quantity, of immunoreactive IGF-I, implying that IGF-I binding proteins in salmon plasma do not affect the performance of the salmon IGF-I RIA. Gel filtration of nonacidified plasma on a Sephadex G-75 superfine column produced two immunoreactive IGF-I peaks of molecular weights of approximately > 70 k and 7 kDa, whereas acidification of plasma increased the relative amount of the 7-kDa peak (IGF-I) and the > 70-kDa peak disappeared. The recoveries of rsIGF-I added to extracted or nonextracted plasma were 97.4 and 94.9%, respectively. Inter- and intraassay coefficients of variation were 3.6 and 3.3%, respectively. Plasma IGF-I levels in coho salmon smolts were 117.4 +/- 19.1 ng/ml as compared to IGF-I levels in parr (45.3 +/- 2.5 ng/ml) or in adult fish (45.2 +/- 5.4 ng/ml) measured in the same assay. Injection of salmon growth hormone, but not prolactin or somatolactin, caused a significant and dose-dependent elevation of plasma IGF-I levels, while either fasting or injection of streptozotocin led to a significant decline in systemic IGF-I.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunocytochemical study of the somatolactin cells in the pituitary of Pacific salmon, Oncorhynchus nerka, and O keta at some stages of the reproductive cycle.

Cells of the intermediate lobe of salmonids, homologous to the PAS-positive cells of other teleost species, cannot be differentiated by normal staining techniques, but can be immunostained with an antiserum against cod somatolactin (SL). Immunocytochemical techniques were applied to pituitary sections of two Pacific salmon, Oncorhynchus nerka and O. keta. Samples of immature or early maturing fish were collected in the Pacific Ocean and from mature spawning fish from hatcheries near Seattle and Willard (Washington). SL cells were rather small and moderately immunoreactive in immature fish. They were slightly enlarged in the early stages of gonadal development and more often contacted the basal lamina through processes with terminal swellings, suggesting granule release into perivascular spaces. In spawning fish, cells were enlarged and frequently more granulated, showing a wide contact with the basal lamina and a proximodistal transport of granules. In addition, large and more or less degranulated cells were noted, also indicating an active release of SL granules. Spawning females tended to have more SL cells than equivalent males. The gradual stimulation of SL synthesis and release during sexual maturation suggests that SL may be involved in the control of some steps of reproduction as previously shown by the increase in SL plasma levels in maturing coho salmon.