Evidence for a carrier state of infectious hematopoietic necrosis virus in chinook salmon Oncorhynchus tshawytscha.

In British Columbia, Canada, infectious hematopoietic necrosis virus (IHNV) is prevalent in wild sockeye salmon Oncorhynchus nerka and has caused disease in seawater net-pen reared Atlantic salmon Salmo salar. In this study, chinook salmon Oncorhynchus tshawytscha experimentally exposed to an isolate of IHNV found in British Columbia became carriers of the virus. When Atlantic salmon were cohabited with these virus-exposed chinook salmon, IHNV was isolated from the Atlantic salmon. Identification of chinook salmon populations that have been exposed to IHNV may be difficult, as virus isolation was successful only in fish that were concurrently infected with either Renibacterium salmoninarum or Piscirickettisia salmonis. Also, IHNV-specific antibodies were detected in only 2 of the 70 fish experimentally exposed to the virus. Two samples collected from chinook salmon exposed to IHNV while at a salt water net-pen site had a seroprevalence of 19 and 22%; however, the inconsistencies between our laboratory and field data suggest that further research is required before we can rely on serological analysis for identifying potential carrier populations. Because of the difficulty in determining the exposure status of populations of chinook salmon, especially if there is no concurrent disease, it may be prudent not to cohabit Atlantic salmon with chinook salmon on a farm if there is any possibility that the latter have been exposed to the virus.     

Sexual genotype markers absent from small numbers of male New Zealand Oncorhynchus tshawytscha

In New Zealand chinook salmon Oncorhynchus tshawytscha , sexual genotype did not correspond to phenotype in 1·6% of male fish, contrasting starkly with North American chinook populations, where inconsistencies between female genotype and phenotype have been observed at frequencies up to 84%.     

N-Terminal Sequence and Main Characteristics of Atlantic Salmon (Salmo salar) Albumin

Atlantic salmon (Salmo salar) serum albumin was purified from plasma and its N-terminal sequence determined. Atlantic salmon albumin is the predominant plasma protein, negatively charged, at pH 8.6. Albumin was purified to >95% purity which yielded a single band on SDS-PAGE and agarose gel electrophoresis. The molecular weight of the purified albumin was approximately 6,5 kDa. The N-terminal sequence of Atlantic chinook salmon albumin was consistent with that predicted from its previously determined cDNA sequence and was identical to that of salmon (Oncorhynchus tshawytscha) albumin through the first 15 residues. However, the fact that the actual N-terminus was different from that predicted from cDNA sequence indicates that Atlantic salmon albumin, like chinook salmon albumin, lacks a propeptide.     

N‐Terminal Sequence and Main Characteristics of Atlantic Salmon (Salmo salar) Albumin

Abstract

Atlantic salmon (Salmo salar) serum albumin was purified from plasma and its N‐terminal sequence determined. Atlantic salmon albumin is the predominant plasma protein, negatively charged, at pH 8.6. Albumin was purified to >95% purity which yielded a single band on SDS‐PAGE and agarose gel electrophoresis. The molecular weight of the purified albumin was approximately 6,5 kDa. The N‐terminal sequence of Atlantic chinook salmon albumin was consistent with that predicted from its previously determined cDNA sequence and was identical to that of salmon (Oncorhynchus tshawytscha) albumin through the first 15 residues. However, the fact that the actual N‐terminus was different from that predicted from cDNA sequence indicates that Atlantic salmon albumin, like chinook salmon albumin, lacks a propeptide.     

In vitro detection of functional humoral immunocompetence in juvenile chinook salmon (Oncorhynchus tshawytscha) using flow cytometry

A flow cytometric (FCM) assay for detection of immunomodulatory effects of environmental factors on the humoral response of chinook salmon (Oncorhynchus tshawytscha) is described and validated. This technique combines exposure of whole animals or leucocyte cultures to immunomodulatory agents/conditions with in vitro mitogenic activation of B-lymphocytes. The proportion of leucocytes undergoing blastogenesis following in vitro stimulation with lipopolysaccharide (LPS) is quantified by FCM analysis of forward and side scatter properties. In addition, binding of a fluorescein isothiocyanate labelled anti-rainbow trout immunoglobulin M monoclonal antibody (anti-RBT SIgM-FITC), quantified by FCM analysis, is used to determine the ability of the lymphoblasts to express surface immunoglobulin M (SIgM).Through a series of calibration steps, it was confirmed that anti-RBT IgM-FITC was specific for B-lymphocyte SIgM in chinook salmon. Binding of anti-RBT IgM-FITC to chinook salmon SIgM positive leucocytes was effectively blocked with salmon serum and an isotype control was established. B-lymphocytes were partially removed from a population of leucocytes through adherence to a nylon wool column, which then demonstrated a consequent reduction in anti-RBT IgM-FITC binding. Using anti-RBT IgM-FITC as a marker, the distribution of resting lymphocytes expressing SIgM in lymphoid tissues of juvenile chinook salmon was described. The mean percentage of SIgM positive cells in spleen, pronephros and blood were found to be 62.1 (+/-2.82), 34.8 (+/-1.86) and 56.7% (+/-4.7) of all viable leucocytes, respectively. In a time-course experiment for optimal in vitro activation of leucocytes for this assay, blastogenesis and up-regulation of SIgM expression of splenic leucocytes were observed through FCM by 4 days post in vitro stimulation with LPS, continued through 7 days, but was no longer visible by 10 days post stimulation. Using this assay, reduced expression of SIgM in splenic and pronephric B-lymphocytes was detected following in vitro exposure to physiologically relevant stress concentrations of cortisol in conjunction with mitogenic stimulation. This technique will be a useful addition to the assays already available in the rapidly growing field of fish immunology.     

Body temperature and seawater adaptation in farmed Atlantic salmon and rainbow trout during prolonged chilling

The core temperature of the rainbow trout Oncorhynchus mykiss (3·5 kg) dropped to 1·0° C during the first 6 h of chilling at 0·5° C, remained stable until 24 h, and dropped significantly to 0·7° C after 39 h. Blood plasma osmolality increased and muscle moisture content decreased gradually with increasing chilling time. After 39 h of chilling, the rainbow trout experienced 40 mosmol l^-1 higher blood plasma osmolality and 2·8% less muscle moisture content compared with initial values. In the Atlantic salmon Salmo salar (5·3 kg), core temperature dropped to 1·3° C and blood plasma osmolality increased significantly during the first 6 h of chilling at 0·5° C, but remained relatively stable throughout the rest of the experimental period. After 39 h of chilling, the salmon experienced 20 mosmol l^-1 higher blood plasma osmolality and 0·5% less muscle moisture content compared with initial values. In rainbow trout muscle moisture content was inversely related to blood plasma osmolality indicating reduced seawater adaptation with increasing hours of chilling. No such relationship was observed in the Atlantic salmon. Hence, changes in plasma osmolality and muscle moisture in the Atlantic salmon do not indicate osmoregulatory failure since the new levels, once established, were maintained throughout the chilling time.     

Experimental and natural host specificity of Loma salmonae (Microsporidia)

The microsporidian Loma salmonae (Putz, Hoffman & Dunbar, 1965) Morrison & Sprague, 1981 has caused significant gill disease in Pacific salmon Oncorhynchus spp. Host specificity of the parasite was examined experimentally by per os challenge of selected salmonids and non-salmonids with infective chinook salmon O. tshawytscha gill material. Pink Oncorhynchus gorbuscha and chum salmon O. keta, brown Salmo trutta and brook trout Salvelinus fontinalis, and chinook salmon (controls) were positive, whereas Atlantic salmon Salmo salar and Arctic char Salvelinus alpinus were negative. In addition, no non-salmonids were susceptible to experimental exposure. Wild Pacific salmon species in British Columbia, Canada, were examined for L. salmonae during their freshwater life history stages (smolts, prespawning, spawning). All stages were infected, although infections in smolts were only detectable using a L. salmonae-specific PCR test. Many previous Loma spp. described from Oncorhychus spp. are likely L. salmonae based on host, parasite morphology, and site of infection.     

Glochidiosis of salmonid fishes. II. Comparison of tissue response of coho and chinook salmon to experimental infection with Margaritifera margaritifera (L.) (Pelecypoda: Margaritanidae)

Coho salmon (Oncorhynchus kisutch) are more resistant to experimental infection with the glochidia of the freshwater mussel (Margaritifera margaritifera) than are chinook salmon (Oncorhynchus tshawytscha). Histological sections made at intervals during the infection showed that coho salmon sloughed the parasites from their gills by 4.5 days postinfection, but the parasites remained encysted in the gills of chinook salmon for 12 weeks, when metamorphosis to juvenile mussels was complete. Coho salmon sloughed the parasites by a well-developed hyperplasia. No such pronounced hyperplastic reaction was seen in the gills of chinook salmon.     

Identification of the sex chromosome pair in coho salmon (Oncorhynchus kisutch): lack of conservation of the sex linkage group with chinook salmon (Oncorhynchus tshawytscha)

Fluorescence in situ hybridization (FISH) using a probe to the male-specific GH-Y (growth hormone pseudogene) was used to identify the Y chromosome in coho salmon (Oncorhynchus kisutch). The sex chromosome pair is morphologically similar to chinook salmon (Oncorhynchus tshawytscha) with the GH-Y localized to the small short arm of the largest subtelocentric chromosome pair. FISH experiments with probes containing sex-linked genes in rainbow trout (Oncorhynchus mykiss) (SCAR163) and chinook salmon (Omy7INRA) showed that the coho sex linkage group is different from chinook and rainbow trout and this was confirmed by segregation analysis for the Omy7INRA locus. The telomeric location of the SEX locus, the presence of shared male-specific markers in coho and chinook salmon, and the lack of conservation of sex-linkage groups suggest that transposition of a small male-specific region may have occurred repeatedly in salmonid fishes of the genus Oncorhynchus.     

Physiological impact of sea lice on swimming performance of Atlantic salmon

Atlantic salmon Salmo salar were infected with two levels of sea lice Lepeophtheirus salmonis (0·13 ± 0·02 and 0·02 ± 0·00 sea lice g⁻¹). Once sea lice became adults, the ventral aorta of each fish was fitted with a Doppler cuff to measure cardiac output ( ̇ ), heart rate ( f _H) and stroke volume ( V _S) during swimming. Critical swimming speeds ( U _crit) of fish with higher sea lice numbers [2·1 ± 0·1 BL (body lengths) s⁻¹] were significantly lower ( P  < 0·05) than fish with lower numbers (2·4 ± 0·1 BL s⁻¹) and controls (sham infected, 2·6 ± 0·1 BL s⁻¹). After swimming, chloride levels in fish with higher sea lice numbers (184·4 ± 11·3 mmol l⁻¹) increased significantly (54%) from levels at rest and were significantly higher than fish with fewer lice (142·0 ± 3·7 mmol l⁻¹) or control fish (159·5 ± 3·5 mmol l⁻¹). The f _H of fish with more lice was 9% slower than the other two groups at U _crit. This decrease resulted in ̇ not increasing from resting levels. Sublethal infection by sea lice compromised the overall fitness of Atlantic salmon. The level of sea lice infection used in the present study was lower than has previously been reported to be detrimental to wild Atlantic salmon.     

Genetic variation in mortality of chinook salmon during a bloom of the marine alga Heterosigma akashiwo

Mortality in a netpen-reared population of 3-year-old chinook salmon Oncorhynchus tshawytscha during an extensive 1997 bloom of the alga Heterosigma akashiwo in Puget Sound, Washington, was low (7·2%), and corresponded to a reduction in variance effective population size of 9·4%. Under a liability threshold model, the heritability of mortality (± S.E.), based on paternal half-sibs, was estimated at 0·15 ± 0·04. No significant genetic variation was detected for date of death. Despite the low overall mortality, the consequences for variation in family size underscore the importance of maximizing genetic variation in cultured fish populations later released to the wild as a precaution against mortality and losses of genetic variation over the life cycle. The pattern of family variation in response to this algal bloom provides evidence for potentially selective mortality of anadromous salmonids in the marine environment during natural perturbations.     

Functional response of juvenile pink and chum salmon: effects of consumer size and two types of zooplankton prey

Feeding rate experiments were conducted for pink salmon Oncorhynchus gorbuscha fry [mean fork length ( L _F) 39 mm], juveniles (103–104 mm L _F) and juvenile chum salmon Oncorhynchus keta (106–107 mm L _F). Fishes were presented with small copepod ( Tisbi sp.) or larger mysid shrimp ( Mysidopsis bahia ) prey at varying densities ranging from 1 to 235 prey l⁻¹ in feeding rate experiments conducted at water temperatures ranging from 10·5 to 12·0° C under high light levels and low turbidity conditions. Juvenile pink and chum salmon demonstrated a type II functional response to mysid and copepod prey. Mysid prey was readily selected by both species whereas the smaller bodied copepod prey was not. When offered copepods, pink salmon fry fed at a higher maximum consumption rate (2·5 copepods min⁻¹) than larger juvenile pink salmon (0·4 copepods min⁻¹), whereas larger juvenile chum salmon exhibited the highest feeding rate (3·8 copepods min⁻¹). When feeding on mysids, the maximum feeding rate for larger juvenile pink (12·3 mysids min⁻¹) and chum (11·5 mysids min⁻¹) salmon were similar in magnitude, and higher than feeding rates on copepods. Functional response models parameterized for specific sizes of juvenile salmon and zooplankton prey provide an important tool for linking feeding rates to ambient foraging conditions in marine environments, and can enable mechanistic predictions for how feeding and growth should respond to spatial-temporal variability in biological and physical conditions during early marine life stages.     

Radio-transmitted electromyogram signals as indicators of physical activity in Atlantic salmon

Surgical methods developed to implant EMG (electromyogram) transmitters in Atlantic salmon Salmo salar were tested to calibrate electromyograms from axial red musculature to swimming speed in a swim speed chamber, and to compare electromyograms of fish from two stocks (Lone and Imsa). Ten Lone and eight Imsa salmon were equipped with internal EMG transmitters. Surgical procedures were acceptable, with 100% survival of all implanted fish during the study. It was possible to calibrate EMG pulse intervals to swimming speed in 14 of the 18 salmon run in the swim speed chamber ( r²= 0·35-0·76 for individuals, 0·63 for pooled data). Individuals differed in their EMG resting levels (EMGs recorded at 0·5 ms⁻¹), and so higher correlations were obtained between swimming speed and an activity index (EMG pulse intervals at different speeds/EMG resting levels) (pooled data, r² =0·75). The linear relationship between swimming speed and EMG pulse intervals differed significantly between the two stocks ( P <0·05). This successful calibration of EMGs to swimming speed opens the possibility of calibrating EMGs to oxygen consumption and the measurement of the metabolic costs of activity in field experiments.     

Juvenile sockeye salmon distribution, size, condition and diet during years with warm and cool spring sea temperatures along the eastern Bering Sea shelf

Interannual variations in distribution, size, indices of feeding and condition of juvenile Bristol Bay sockeye salmon Oncorhynchus nerka collected in August to September (2000–2003) during Bering–Aleutian Salmon International Surveys were examined to test possible mechanisms influencing their early marine growth and survival. Juvenile sockeye salmon were mainly distributed within the southern region of the eastern Bering Sea, south of 57°0' N during 2000 and 2001 and farther offshore, south of 58°0' N during 2002 and 2003. In general, juvenile sockeye salmon were significantly larger ( P < 0·05) and had significantly higher indices of condition ( P < 0·05) during 2002 and 2003 than during 2000 and 2001. The feeding index was generally higher for age 1.0 year sockeye salmon than age 2.0 year during all years. Among-year comparisons suggested that Pacific sand lance Ammodytes hexapterus were important components of the juvenile sockeye salmon diet during 2000 and 2001 (20 to 50% of the mean wet mass) and age 0 year walleye pollock Theragra chalcogramma were important components during 2002 and 2003 (50 to 60% of the mean wet mass). Warmer sea temperatures during spring and summer of 2002 and 2003 probably increased productivity on the eastern Bering Sea shelf, enhancing juvenile sockeye salmon growth.     

Isolation and characterization of a trypsin fraction from the pyloric ceca of chinook salmon (Oncorhynchus tshawytscha)

A trypsin fraction was isolated from the pyloric ceca of New Zealand farmed chinook salmon (Oncorhynchus tshawytscha) by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The chinook salmon enzyme hydrolyzed the trypsin-specific synthetic substrate benzoyl-dl-arginine-p-nitroanilide (dl-BAPNA), and was inhibited by the general serine protease inhibitor phenyl methyl sulfonyl fluoride (PMSF), and also by the specific trypsin inhibitors — soybean trypsin inhibitor (SBTI) and benzamidine. The enzyme was active over a broad pH range (from 7.5 to at least pH 10.0) at 25 °C and was stable from pH 4.0 to pH 10.0 when incubated at 20 °C, with a maximum at pH 8.0. The optimum temperature for the hydrolysis of dl-BAPNA by the chinook salmon enzyme was 60 °C, however, the enzyme was unstable at temperatures above 40 °C. The molecular mass of the chinook salmon trypsin was estimated as 28 kDa by SDS–PAGE.     

An epizootic in chinook salmon (Oncorhynchus tshawytscha) caused by a sorbitol-positive serovar 2 strain of Yersinia ruckeri

Enteric redmouth disease is described in chinook salmon (Oncorhynchus tshawytscha) at a state hatchery in Sand Ridge, Illinois. Biochemical, isoenzyme, and serological data indicated that the epizootic was caused by a sorbitol-fermenting Serovar 2 strain of Yersinia ruckeri. In laboratory experiments the isolate was pathogenic for both brook trout (Salvelinus fontinalis) and Atlantic salmon (Salmo salar).     

Innate and enhanced predator recognition in hatchery-reared chinook salmon

We used a laboratory behaviour assay to investigate how innate predator recognition, handling stress, retention time, and number of conditioning events might affect chemically mediated anti-predator conditioning for hatchery-reared chinook salmon, Oncorhynchus tshawytscha. Juvenile chinook salmon with no prior exposure to predatory stimuli exhibited innate fright responses to northern pikeminnow, Ptychocheilis oregonensis, odour, regardless of whether the salmon came from a population that exists in sympatry or allopatry with northern pikeminnows. Juvenile chinook salmon exhibited enhanced predator recognition following a single conditioning event with conspecific extract and northern pikeminnow odour. Handling similar to what hatchery salmon might experience prior to release did not substantially reduce the conditioned response. When we conditioned juvenile chinook salmon in hatchery rearing vessels, fish from tanks treated once exhibited a conditioned response to northern pikeminnow odour in aquaria, but only for one behaviour (feeding response), and fish treated twice did not respond. The results suggest that enhanced recognition of predator stimuli occurs quickly, but may be to some extent context-specific, which may limit conditioned fright responses after release into the natural environment.     

Flow cytometry assays of respiratory burst in Atlantic salmon (Salmo salar L.) and in Atlantic cod (Gadus morhua L.) leucocytes

The oxidation of dihydrorhodamine 123 (DHR) to the fluorescent rhodamine 123 (RHO) was detected using flow cytometry. This assay for detection of respiratory burst activity was established in peripheral blood leucocytes (PBL) and head kidney leucocytes (HKL) of Atlantic salmon and Atlantic cod. The leucocytes were stimulated by phorbol 12-myristate 13-acetate (PMA). For cod cells 10 times lower concentration of PMA had to be used compared to salmon cells, as higher concentrations were toxic and resulted in considerable cell death. The cells found to be RHO-positive were monocytes/macrophages and neutrophils based on the scatter dot plots, but for salmon also some small cells were found to have high fluorescence intensity both in the flow cytometry analyses and by fluorescence microscopy of cytospin preparations. The nature of these cells is not known. For cod leucocytes, such cells were not obvious. The instrument settings are a bit more demanding for cod, as cod cells die more easily compared to salmon cells. In both assays the limit between negative and positive cells has to be carefully considered. The presented flow cytometry protocols for measurements of respiratory burst in salmon and cod leucocytes can be applied in various studies where respiratory burst functions are involved, such as to verify if it is activated or suppressed in connection with infections and immunostimulation.     

Female mate choice and spawning behaviour of Chinook salmon under experimental conditions

After spawning their first nest, female chinock salmon Oncorhynchus tshawytscha paired with small males ( c. 46% of female weight) spent an average of 16·2 h between spawning of successive nests compared with 9·6 h for females paired with large males ( c. 112% of female weight) ( P< 0·05). Neither frequencies of female nest construction behaviours (digging and probing) nor male courtship behaviour (crossovers and quivers) differed between large- and small-male pairs. Male quivering frequencies were correlated significantly with female digging and probing frequencies, whereas the crossover frequencies were not. It is suggested that delayed spawning by females in the presence of relatively small males is a primary mechanism by which females in the genus Oncorhynchus exhibit mate choice.     

Temporal repeatability of relative standard metabolic rate in juvenile Atlantic salmon and its relation to life history variation

There was a strong correlation between the relative standard metabolic rate (rSMR) values of individual Atlantic salmon Salmo salar L. measured 5 and 22 weeks after first feeding in June and October respectively (Pearson's r =0·68, 26 d.f., P <0·001). However, this is a conservative estimate of repeatability as two separate regressions were used to calculate SMR in October due to the separation of the population into an upper modal group made up of early migrants and a lower modal group comprised of delayed migrants. SMR values in June and October were similar (paired t test, t =-0·85, 27 d.f., P >0·05) when expressed as the percentage deviation from those predicted for a fish of that size based on the body mass/SMR relations in June or October indicating that relative standard metabolic rates were stable over time when food was not limiting. rSMR status was maintained in 19 of the 28 fish (i.e. 68%) between the two measures. rSMR status was correlated with life history strategy: salmon fry with a high SMR in June were more likely to become smolts during the autumn than those with a low SMR.