Individual condition and stream temperature influence early maturation of rainbow and steelhead trout, Oncorhynchus mykiss

Alternative male phenotypes in salmonine fishes arise from individuals that mature as larger and older anadromous marine-migrants or as smaller and younger freshwater residents. To better understand the processes influencing the expression of these phenotypes we examined the influences of growth in length (fork length) and whole body lipid content in rainbow trout (Oncorhynchus mykiss). Fish were sampled from the John Day River basin in northeast Oregon where both anadromous (“steelhead”) and freshwater resident rainbow trout coexist. Larger males with higher lipid levels had a greater probability of maturing as a resident at age-1+. Among males, 38% were maturing overall, and the odds ratios of the logistic model indicated that the probability of a male maturing early as a resident at age-1+ increased 49% (95% confidence interval (CI) = 23–81%) for every 5 mm increase in length and 33% (95% CI = 10–61%) for every 0.5% increase in whole body lipid content. There was an inverse association between individual condition and water temperature as growth was greater in warmer streams while whole body lipid content was higher in cooler streams. Our results support predictions from life history theory and further suggest that relationships between individual condition, maturation, and environmental variables (e.g., water temperature) are shaped by complex developmental and evolutionary influences.     

Severe, chronic proliferative kidney disease (PKD) induced in rainbow trout Oncorhynchus mykiss held at a constant 18 degrees C

Proliferative kidney disease (PKD), caused by the myxozoan parasite Tetracapsuloides bryosalmonae, is well documented as a seasonal disease of rainbow trout Oncorhynchus mykiss. Water temperatures influence the course of the infection both within the fish and the invertebrate host, the recovery of fish from the disease being accelerated with decreasing water temperatures. During this study, groups of rainbow trout were held at a constant temperature (18 degrees C) for a sustained period of time following initial exposure to T. bryosalmonae. While the majority of these fish had recovered from the clinical disease after 9 mo, 10% remained infected, showing clinical signs of disease. A histological study revealed that the majority exhibited very high parasite loads and unusually severe symptoms of PKD. This demonstrates that while most rainbow trout can recover from PKD independent of water temperature, there exists a sub-population that cannot.     

Brain distribution of myosin Va in rainbow trout Oncorhynchus mykiss

This study presents data on myosin Va localization in the central nervous system of rainbow trout. We demonstrate, via immunoblots and immunocytochemistry, the expression of myosin Va in several neuronal populations of forebrain, midbrain, hindbrain and spinal cord. The neuronal populations that express myosin Va in trout constitute a very diverse group that do not seem to have many specific similarities such as neurotransmitters used, cellular size or length of their processes. The intensity of the immunoreactivity and the number of immunoreactive cells differ from region to region. Although there is a broad distribution of myosin Va, it is not present in all neuronal populations. This result is in agreement with a previous report, which indicated that myosin Va is approximately as abundant as conventional myosin II and kinesin, and it is broadly involved in neuronal motility events such as axoplasmatic transport. Furthermore, this distribution pattern is in accordance with what was shown in rats and mice; it indicates phylogenetic maintenance of the myosin Va main functions.     

Association of Flavobacterium psychrophilum with rainbow trout (Oncorhynchus mykiss) kidney phagocytes in vitro

The capacity of virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes to associate with isolated rainbow trout (Oncorhynchus mykiss, 300-500 g) kidney phagocytes was evaluated in vitro. The results showed that F. psychrophilum was associated with the phagocytes but large differences in association were observed between the different bacterial strains examined. These differences in association with the phagocytes was not clearly related to the serotype or virulence of the bacteria, although all strains tested of the non-virulent serotype FpT showed strong association with the isolated phagocytes. A competitive association assay with treatment of the phagocytes with seven different carbohydrates, suggested a role for N-acetylneuraminic acid (sialic acid) in the binding of F. psychrophilum to phagocytes. A significant dose dependent inhibition of the association was observed with sialic acid. Treatment of F. psychrophilum with sodium-metaperiodate showed that carbohydrate components play a role in the adhesion of the bacteria to the phagocytes. The results indicate that the binding of F. psychrophilum to rainbow trout kidney phagocytes can be mediated by opsonin independent cell-receptor adhesion. All tested strains seemed to be non-cytotoxic for rainbow trout kidney phagocytes in vitro suggesting that a phagocyte toxin is not necessary for the virulence of F. psychrophilum     

Bacterial kidney disease as a model for studies of cell mediated immunity in rainbow trout (Oncorhynchus mykiss)

A cell mediated immune (CMI) response was measured in vitro to heat-killed and to paraformaldehyde fixed Renibacterium salmoninarum (Rs) in rainbow trout (Oncorhynchus mykiss) experimentally challenged with live Rs. The mitogenic response to the T lymphocyte mitogen Concanavalin A (Con A) was reduced during samplings 4 to 6 weeks after immersion, but no effect of the response to the B lymphocyte mitogen lipopolysaccharide (LPS) was detected. The subpopulation of lymphocytes, detected by the monoclonal antibody 1C2, was decreased from the 4th week to the 5th week of infection, and remained at the decreased level up to 10 weeks post immersion. The proportion of Immunoglobulin (Ig) bearing lymphocytes was not affected during the Rs infection period. The humoral antibody level to heat-stable Rs-antigens was increased up to 10 weeks after immersion but after 27 weeks was reduced to a level similar to that of the non-challenged fish. An anamnestic response was demonstrated in challenged fish, as intraperitoneal injection of heat-treated Rs bacteria into Rs challenged fish elicited a stronger humoral antibody response compared with injection into non-challenged fish.     

Immunohistochemical localization of rainbow trout ladderlectin and intelectin in healthy and infected rainbow trout (Oncorhynchus mykiss)

In the present study, the pattern of immuno-reactive ladderlectin and intelectin in healthy rainbow trout is compared to rainbow trout infected with a variety of infectious agents. In healthy rainbow trout, both proteins were localized to individual epithelial cells of the gill and intestine and both proteins were clearly demonstrated within cytoplasmic granules of polymorphonuclear leucocytes and macrophages/monocytes found in blood vessels, hepatic sinusoids, renal interstitium, mucosal epithelium and submucosa of normal intestine. In tissue from infected rainbow trout, there was an overall relative increase in both lectins compared to healthy fish and both proteins were detected in extra-cellular spaces surrounding bacteria, fungi and protozoa. Increased distribution and density of both RTLL and RTInt was demonstrated along mucosal surfaces and within inflammatory leucocytes in infected tissues and immune related organs. These findings represent one of the few examples of in vivo association of defence lectins and infectious agents.     

Tissue-specific distribution patterns of retinoids and didehydroretinoids in rainbow trout Oncorhynchus mykiss

Retinoids (vitamin A) are known to be involved in many key biological functions in mammals, such as embryonic development, reproduction or vision. Besides standard vitamin A forms, freshwater fish tissues contain high levels of didehydroretinoids or vitamin A₂ forms. However, the tissue distribution, metabolism and function of both standard and particularly the didehydroretinoids are still poorly known in fish. In this study, we have quantified the levels of retinoids, including retinol, retinaldehyde, retinyl palmitate and their corresponding didehydro forms, as well as the levels of the active polar retinoids all-trans-, 9-cis- and 13-cis-retinoic acid in distinct tissues of juvenile rainbow trout. Our results indicate that the liver is clearly the main retinoid storage tissue in juvenile rainbow trout. Didehydroretinoids were dominant over retinoids in all analyzed tissues with the exception of plasma. Additionally, significant differences among tissues were observed between retinoids and didehydroretinoids, such as differences in the ester profiles and the proportions between free and esterified forms, suggesting that mechanisms that favor the utilization or storage of one of the other groups of compounds might exist in fish. Our data also show the presence of polar retinoids in different tissues of fish at the fmol/g scale. Overall, this study clearly demonstrates the presence of tissue-specific patterns of accumulation of both polar and nonpolar retinoids in fish tissues. The biological relevance of these findings should be the focus of future studies.     

Patterns of transgene inheritance in rainbow trout (Oncorhynchus mykiss).

There have been very few studies of the inheritance of introduced genes (transgenes) in fish. We have followed the inheritance of the mammalian fusion gene MTrGH from founder generation transgenics (originating from eggs microinjected with the MTrGH DNA) to offspring in crosses with control fish. Initial screening of the founder generation transgenics was by analysing DNA from blood samples. Only three out of six fish which carried the novel gene in blood DNA transmitted it to their offspring, despite the presence of the gene in DNA extracted from the sperm of all four male fish in this group. The frequency of transgenics in the progeny groups from the three fish which transmitted the gene varied widely: in one of these groups more than one type of MTrGH restriction pattern was found. These results suggest widespread mosaicism in founder generation transgenics.     

Quantitative evaluation of macrophage aggregates in brook trout Salvelinus fontinalis and rainbow trout Oncorhynchus mykiss

Macrophage aggregates (MAs) occur in various organs of fishes, especially the kidney, liver and spleen, and contain melanin, ceroid/lipofuscin and hemosiderin pigments. They have been used as indicators of a number of natural and anthropogenic stressors. Macrophage aggregates occur in salmonids but are poorly organized, irregularly shaped, and are generally smaller than those in derived teleosts. These features complicate quantification, and thus these fishes have seldom been used in studies correlating MAs with environmental stressors. To alleviate these complications, we developed color filtering algorithms for use with the software package ImagePro Plus (Media Cybernetics) that select and quantify pigmented area (i.e. colors ranging from gold to brown to black) in tissue sections. Image analysis results compared well with subjective scoring when tested on brook trout Salvelinus fontinalis and rainbow trout Oncorhynchus mykiss captured from high-elevation lakes or hatcheries. Macrophage aggregate pigments correlated positively with age and negatively with condition factor. Within individual fish, pigmentation correlated positively among organs, suggesting that the kidney, liver or spleen are suitable indicator organs. In age-matched fishes, MA pigments were not different between hatcheries and lakes in the organs examined. Between lakes, differences in pigments were observed in the kidney and spleen, but were not explained by age, condition factor, sex or maturation state. Our results indicate that quantification of the area occupied by MA pigments is an efficient and accurate means of evaluating MAs in salmonid organs and that organ pigmentation correlates with age and condition factor, as seen in studies with more derived fishes.     

The influence of chronic anaemia on catecholamine secretion in the rainbow trout (Oncorhynchus mykiss)

The effect of long-term (7 day) anaemia on catecholamine release was examined in rainbow trout (Oncorhynchus mykiss) in vivo during acute exposure to hypoxia and in situ using a perfused post-cardinal vein preparation. The first goal was to distinguish among reductions in blood O₂ partial pressure, O₂ concentration and haemoglobin percentage saturation as potential stimuli for, or correlates of, catecholamine secretion during hypoxia. The second goal was to elucidate the role of these factors in promoting enhanced chromaffin cell responsiveness in trout subjected to chronic hypoxia (Montpetit and Perry 1998). Anaemic fish (haematocrit lowered from 28.4±2.4% to 11.9±1.6%) displayed a marked reduction in haemoglobin-O₂ binding affinity [P ₅₀ (P _aO₂ at 50% Hb-O₂ saturation) was increased from 14.7 mm Hg to 24.3 mm Hg]. Upon exposure to hypoxia, the anaemic fish released catecholamines into their circulation at higher values of arterial O₂ partial pressure (∼52 mm Hg versus ∼18 mm Hg) and haemoglobin O₂ saturation (<70% versus <55%) than did control fish. In addition, anaemic fish achieved significantly greater circulating levels of total catecholamines (noradrenaline plus adrenaline) during acute hypoxia (294.8±67.3 versus 107.0±35.6 nmol l⁻¹). These results do not support the view that catecholamine release is triggered by a reduction in haemoglobin O₂ saturation or arterial PO₂, per se. Nor are they consistent with the idea that catecholamine release occurs at a threshold value of arterial PO₂ corresponding to a critical reduction in blood O₂ concentration. The effects of the non-selective cholinergic receptor carbachol on catecholamine secretion from chromaffin tissue were assessed using perfused posterior cardinal vein preparations derived from control or anaemic fish. For adrenaline secretion, there was no statistically significant change in the ED₅₀ (dose eliciting 50% response). For noradrenaline secretion however, preparations originating from anaemic fish displayed an enhanced responsiveness to carbachol as indicated by a significant 4.5-fold reduction in the carbachol ED₅₀ value from 2.53 × 10⁻⁶ mol kg⁻¹ to 5.67 × 10⁻⁷ mol kg⁻¹. These results demonstrate that anaemia-induced hypoxaemia, in the absence of any lowering of PO₂, is able to modulate the responsiveness of chromaffin cells to cholinergic stimulation. Accepted: 21 April 1999     

Routes of entry of Piscirickettsia salmonis in rainbow trout Oncorhynchus mykiss.

Since 1989, Piscirickettsia salmonis, the causal agent of piscirickettsiosis, has killed millions of farmed salmonids each year in southern Chile. The portal of entry for the pathogen was investigated by use of selected experimental infections in juvenile rainbow trout (12 g). The methods used were intraperitoneal injection, subcutaneous injection, patch contact on skin, patch contact on gills, intestinal intubation and gastric intubation. Cumulative mortalities at Day 33 post-inoculation were 98, 100, 52, 24, 24, and 2%, respectively. It was shown that intact skin and gills could be penetrated by P. salmonis. The high mortality obtained in subcutaneously injected fish indicated that skin injuries could facilitate the invasion of this pathogen. Results suggested that the main entry sites are through the skin and gills and that the oral route may not be the normal method by which P. salmonis initiates infection of salmonids.     

Chromosome rearrangements and survival of androgenetic rainbow trout (Oncorhynchus mykiss)

The purpose of this work was to quantify the impact of spontaneous and X-radiation-induced chromosome rearrangements on survival rate of androgenetic rainbow trout (Oncorhynchus mykiss). Various doses of X irradiation (50, 150, 250, 350 Gy) were used for inactivation of nuclear DNA in oocytes. After the irradiation, eggs were inseminated with normal sperm from 4 males derived from a strain characterized by Robertsonian rearrangements and length polymorphism of the Y chromosome. The haploid zygotes were exposed to a high hydrostatic pressure (7000 psi) to duplicate the paternal DNA. Neither Robertsonian chromosome polymorphism nor the Y chromosome morphology impaired the viability of the androgenetic embryos and alevins. Moreover, survival of eyed embryos of the androgenetic rainbow trout increased significantly with increasing doses of oocyte X irradiation. After 6 months of rearing, only specimens from the 250 and 350 Gy variants survived. The number of fingerlings with remnants of the maternal genome in the forms of chromosome fragments was higher in the 250 Gy group. Intraindividual variation of chromosome fragment number was observed, and some individuals exhibited haploid/diploid mosaicism and body malformations. Individuals irradiated with less than 250 Gy died, presumably because of the conflict between intact paternally derived chromosomes and the residues of maternal genome in the form of chromosome fragments.     

Major histocompatibility class II genes in rainbow trout (Oncorhynchus mykiss) exhibit temperature dependent downregulation

Major histocompatibility (MH) class II receptors are expressed on the surface of specialized antigen-presenting cells in vertebrate immune systems. Their function is to present peptides derived from exogenous pathogens to CD4+ T cells. Variation in the level of expression of these genes has been linked to pathogenesis in various diseases. Very little has been published on the function of MH class II receptors in teleost fish to date. In this study, we have produced polyclonal antibodies recognizing MH class II alpha and beta proteins of rainbow trout and employed them to characterize the expression pattern of these genes. Deglycosylation using N-glycosidase F and endoglycosidase H showed that MH class II alpha is glycosylated in rainbow trout. MH class II beta was also found to be glycosylated as reported previously. Results from Northern blotting revealed that the expression of these genes was not affected by exposure of rainbow trout to temperature of 5°C. However, at 2°C, downregulation of MH class II alpha and beta genes was evident at both the mRNA and protein levels as assessed by Northern and Western blotting, respectively. Because MH class II antigens play an important role in generating an immune response to bacterial and fungal pathogens, downregulation of these genes at low temperature could account for the susceptibility of fish to low temperature-related diseases such as bacterial cold-water disease and winter saprolegniosis.     

Marker-assisted estimation of quantitative genetic parameters in rainbow trout,Oncorhynchus mykiss

Estimation of quantitative genetic parameters conventionally requires known pedigree structure. However, several methods have recently been developed to circumvent this requirement by inferring relationship structure from molecular marker data. Here, two such marker-assisted methodologies were used and compared in an aquaculture population of rainbow trout (Oncorhynchus mykiss). Firstly a regression-based model employing estimates of pairwise relatedness was applied, and secondly a Markov Chain Monte Carlo (MCMC) procedure was employed to reconstruct full-sibships and hence an explicit pedigree. While both methods were effective in detecting significant components of genetic variance and covariance for size and spawning time traits, the regression model resulted in estimates that were quantitatively unreliable, having both significant bias and low precision. This result can be largely attributed to poor performance of the pairwise relatedness estimator. In contrast, genetic parameters estimated from the reconstructed pedigree showed close agreement with ideal values obtained from the true pedigree. Although not significantly biased, parameters based on the reconstructed pedigree were underestimated relative to ideal values. This was due to the complex structure of the true pedigree in which high numbers of half-sibling relationships resulted in inaccurate partitioning of full-sibships, and additional unrecognized relatedness between families.     

Lectin histochemistry of rainbow trout (Oncorhynchus mykiss) gill and skin

In order to characterize the glycoconjugate residues in skin and gills of the adult rainbow trout, the binding pattern of five biotinylated lectins with different carbohydrate specificities was examined. In the skin, mucous cells revealed binding sites for PNA and SBA; filament-containing cells were additionally labelled with Con A. However, the basal cell layer showed no reaction. In the gill, subpopulations of mucous cells reacted with Con A, PNA, SBA and UEA-I. This broader spectrum of glycoconjugates in gill mucous cells compared with the epidermal mucous cells could point to the additional function of gill mucus in ion and osmoregulation. Lectin binding sites were less common in the respiratory epithelial cells of the secondary lamellae than in those of the primary lamellae. Chloride cells revealed mannose, galactose and fucose residues. Immature chloride cells, as indicated by a comparison with Na⁺/K⁺ ATPase immunolabelling, reacted with Con A; subpopulations of them reacted with PNA, SBA and UEA-I. The results form the basis for further investigations in which these cell populations can be analysed under different environmental conditions This revised version was published online in November 2006 with corrections to the Cover Date.     

Purification and characterization of calpain and calpastatin from rainbow trout, Oncorhynchus mykiss

Although the calpain system has been studied extensively in mammalian animals, much less is known about the properties of μ-calpain, m-calpain, and calpastatin in lower vertebrates such as fish. These three proteins were isolated and partly characterized from rainbow trout, Oncorhynchus mykiss, muscle. Trout m-calpain contains an 80-kDa large subunit, but the  26-kDa small subunit from trout m-calpain is smaller than the 28-kDa small subunit from mammalian calpains. Trout μ-calpain and calpastatin were only partly purified; identity of trout μ-calpain was confirmed by labeling with antibodies to bovine skeletal muscle μ-calpain, and identity of trout calpastatin was confirmed by specific inhibition of bovine skeletal muscle μ- and m-calpain. Trout μ-calpain requires 4.4 ± 2.8 μM and trout m-calpain requires 585 ± 51 μM Ca²⁺ for half-maximal activity, similar to the Ca²⁺ requirements of μ- and m-calpain from mammalian tissues. Sequencing tryptic peptides indicated that the amino acid sequence of trout calpastatin shares little homology with the amino acid sequences of mammalian calpastatins. Screening a rainbow trout cDNA library identified three cDNAs encoding for the large subunit of a putative m-calpain. The amino acid sequence predicted by trout m-calpain cDNA was 65% identical to the human 80-kDa m-calpain sequence. Gene duplication and polyploidy occur in fish, and the amino acid sequence of the trout m-calpain 80-kDa subunit identified in this study was 83% identical to the sequence of a trout m-calpain 80-kDa subunit described earlier. This is the first report of two isoforms of m-calpain in a single species.     

Whirling disease: host specificity and interaction between the actinosporean stage of Myxobolus cerebralis and rainbow trout Oncorhynchus mykiss

Scanning electron microscopic studies were conducted on rainbow trout Oncorhynchus mykiss in the first 60 min after their exposure to the triactinomyxon spores of Myxobolus cerebralis. The results demonstrated that as early as 1 min post exposure the whole process, from the attachment of the triactinomyxon spores to the complete penetration of their sporoplasm germs, had occurred. The triactinomyxon spores sought out the secretory openings of mucous cells of the epidermis, the respiratory epithelium and the buccal cavity of trout and used them as portals of entry. Exposure experiments of the triactinomyxon spores of M. cerebralis to non-salmonid fish, such as goldfish Carassius auratus, carp Cyprinus carpio, nose Chondrostoma nasus, medaka Oryzias latipes, guppy Poecilia reticulata and also the amphibian tadpole Rana pipiens as well as to rainbow trout fry indicated a specificity for salmonids. Attempts to activate the triactinomyxon spores by exposure to mucus prepared from cyprinid and salmonid fish showed no significant differences from those conducted in tap water. The results suggest that the simultaneous presence of both mechano- and chemotactic stimuli was required for finding the salmonid fish host.