Characterization of 38 polymorphic microsatellite markers for rainbow trout (Oncorhynchus mykiss)

Thirty‐eight new microsatellite markers were developed for genome mapping and population genetics studies in rainbow trout (Oncorhynchus mykiss). The amount of polymorphism, percentage of heterozygosity and ability of each marker to amplify genomic DNA from other salmonids were recorded. Five markers were observed to be duplicated in the rainbow trout genome by containing more than one allele in homozygous (clonal) fish.     

Characterization of twenty‐four microsatellite markers for rainbow trout (Oncorhynchus mykiss)

Twenty‐four new microsatellite markers were developed for genome mapping and population genetics studies in rainbow trout (Oncorhynchus mykiss). The amount of polymorphism, percentage heterozygosity and ability of each marker to amplify genomic DNA from other salmonids were recorded. Seven markers were observed to be duplicated in the rainbow trout genome by containing more than one allele in homozygous (clonal) fish.     

Mitochondrial and nuclear DNA sequence variability among populations of rainbow trout (Oncorhynchus mykiss)

Mitochondrial and nuclear DNA variability was examined to assess population genetic structure and phylogeographic relationships in rainbow trout. Single-strand conformation polymorphisms and restriction site differences within 1055 bp of the mitochondrial D-loop region and 1566 bp of nuclear DNA in six single-copy nuclear DNA regions identified 31 mitochondrial genotypes and 50 nuclear alleles. Gene trees were constructed by sequencing each variant allele or mitochondrial genotype identified. Examination of 30 populations in 10 native rainbow trout groups using an analysis of molecular variance ( AMOVA ) indicated that 65% of mitochondrial variability and 35% of nuclear variability was explained by differences among the 10 groups. Phylogenetic patterns evident in mitochondrial and nuclear DNA were not always concordant. Differences in the evolutionary patterns detected by mitochondrial and nuclear DNA may reflect the differential impact of past introgression events on variability in the two genomes.     

Relationship between stress, feeding and plasma ghrelin levels in rainbow trout, Oncorhynchus mykiss

Sexually immature rainbow trout were acclimated to small-volume (1 m³) holding tanks and then exposed to short-term stress to examine the relationship between feeding, stress, plasma ghrelin levels and other plasma stress parameters. Plasma ghrelin levels showed an increase 24 h after a single feed, plasma lactate and glucose levels decreased over the same period and plasma cortisol levels were low and constant. One hour of confinement stress resulted in elevations of plasma cortisol, glucose and lactate and depression of plasma ghrelin levels. In a separate experiment, 2 h of confinement stress also depressed feeding immediately after stress, concomitant with increases in plasma cortisol, lactate and glucose; however, in this case there was no change in plasma ghrelin concentrations. A repeat of the 2-h confinement experiment using fish that had not been acclimated to small-volume holding tanks produced a more marked elevation in plasma cortisol and a stronger suppression of feeding post-stress but in this case also, there was no change in plasma ghrelin levels. The results of this study confirm that feeding in rainbow trout is suppressed by confinement stress although the effect is transitory in this domesticated stock. Similar to that in other fishes, plasma ghrelin levels appear to be modulated by feeding status and may be influenced by stress, suggesting an orexigenic role for ghrelin in rainbow trout.     

Primary and secondary indices of stress in the progeny of rainbow trout (Oncorhynchus mykiss) selected for high and low responsiveness to stress

A series of pooled gamete matings was carried out employing eggs and milt from mature male and female rainbow trout selected for a consistently high- or low-responsiveness to stress, as indicated by post-stress plasma cortisol elevation. Development of the progeny was closely monitored and the responsiveness to stress of the progeny of high-responding parents and the progeny of low-responding parents was assessed by two methods. For a period of 14 months, at approximately monthly intervals, the plasma cortisol elevation evoked by a standardized confinement stress was determined in fish from each group, and secondly, on one occasion, the time-course of the plasma cortisol response to a 24-h period of confinement was monitored. Progeny of high-responding parents snowed a significantly greater cortisol response to stress than the progeny of low-responding parents during both testing procedures. However, when the effect of a 14-day confinement stress was examined, high-responding progeny showed a more rapid recovery of plasma cortisol levels, while levels in the low-responding progeny, although initially lower, showed a more sustained elevation. To assess the possible functional implications of these observations, circulating lymphocyte numbers, an immunologically important cortisol-sensitive component of the blood cell complement, were determined. The duration of the lymphocytopenia observed following the onset of confinement was found to be related to the initial, not the sustained, cortisol response. These data suggest that manipulation of the sensitivity to stress of fish is feasible by selective breeding, but that careful.choice of the indices employed to identify traits considered desirable is necessary.     

Development and characterization of novel tetra‐, tri‐, and dinucleotide microsatellite markers in rainbow trout (Oncorhynchus mykiss)

We discuss the development and characterization of 40 polymorphic rainbow trout (Oncorhynchus mykiss) microsatellite loci. We used enriched libraries to isolate 14 dinucleotide, seven trinucleodide, eight compound di/tetranucleotide, and 11 tetranucleotide loci. These markers will be useful for selective breeding via marker‐assisted selection, population genetics studies, parentage analysis, and have already been used for genome mapping.     

Differences in plasma cortisol and cortisone dynamics during stress in two strains of rainbow trout (Oncorhynchus mykiss)

Plasma levels of cortisone, a steroid hormone of potential physiological significance in fish, have rarely been measured. This study examines the interrelationship between circulating levels of cortisone and the major teleost corticosteroid, cortisol, in the blood of two strains of rainbow trout subject to confinement stress, a condition know to stimulate corticosteroidogenic activity. In unstressed fish from both strains, mean plasma cortisol levels were within the range 0.4–7.5 ng ml⁻¹. Mean plasma cortisone levels were within the range 7.1–15.9 ng ml⁻¹. Plasma cortisol levels were elevated within 5 min of the onset of strees and reached peak values within 45 min, although there was a marked difference betweed the maxima observed in the two strains (strain 1:70 ng ml⁻¹; strain 2:150 ng ml⁻¹). The rate of increase of plasma cortisone levels during strees was more rapid than that of cortisol, maximum values (strain 1:100ng ml⁻¹; strain 2:160 ng ml⁻¹) being reached within 10 to 20 min of the onset of stress. This rapid stress-induced elevation of plasme cortisone has not previously been reported in fish. We suggest that rapid conversion of cortisol to cortisone during the initial response to stress accounts for the appearance of large amounts of cortisone in the blood, indicating that circulating for the appearance of large amounts of cortisone in the blood, indicating that circulating levels of cortisol alone do not fully reflect the secretory activity of the interregnal during the initial of cortisol alone do not fully the secretory activity of the interregnal during the initial phase of the stress response. The results also indicate that the rate of clearance of cortisone from the circulation may be a major factor in determining stress-stimulated levels of plasma cortisol.     

Germinal epithelium, folliculogenesis, and postovulatory follicles in ovaries of rainbow trout, Oncorhynchus mykiss (Walbaum, 1792) (Teleostei, protacanthopterygii, salmoniformes)

The rainbow trout, Oncorhynchus mykiss (Walbaum, 1792), is a salmoniform fish that spawns once per year. Ripe females that had ovulated naturally, and those induced to ovulate using salmon gonadotropin-releasing hormone, were studied to determine whether follicles were forming at the time of spawning and to describe the process of folliculogenesis. After ovulation, the ovaries of postspawned rainbow trout were examined histologically, using the periodic acid-Schiff procedure, to stain basement membranes that subtend the germinal epithelium and to interpret and define the activity of the germinal epithelium. After spawning, the ovary contained a few ripe oocytes that did not ovulate, numerous primary growth oocytes including oocytes with cortical alveoli, and postovulatory follicles. The germinal epithelium was active in postspawned rainbow trout, as determined by the presence of numerous cell nests, composed of oogonia, mitotic oogonia, early diplotene oocytes, and prefollicle cells. Cell nests were separated from the stroma by a basement membrane continuous with that subtending the germinal epithelium. Furthermore, follicles containing primary growth oocytes were connected to the germinal epithelium; the basement membrane surrounding the follicle joined that of the germinal epithelium. After ovulation, the basement membrane of the postovulatory follicle was continuous with that of the germinal epithelium. We observed consistent separation of the follicle, composed of an oocyte and surrounding follicle cells, from the ovarian stroma by a basement membrane. The follicle is derived from the germinal epithelium. As with the germinal epithelium, follicle cells derived from it never contact those of the connective tissue stroma. As with epithelia, they are always separated from connective tissue by a basement membrane.     

The bold and the shy: individual differences in rainbow trout

Boldness and shyness were investigated as 'personality' traits in hatchery-reared rainbow trout Oncorhynchus mykiss . Bold fish spent more time in an open area and were more active than shy fish and these behaviours could be used as indicators of boldness and shyness. These differences were related to learning ability in a simple conditioning task. Bold fish learned the task more quickly than shy fish.     

Haemoglobin isomorph abundances in splenectomized rainbow trout, Oncorhynchus mykiss (Walbaum)

Rainbow trout, Oncorhynchus mykiss (Walbaum) were rendered acutely anaemic by immersion in phenylhydrazine HC1 at 5° C. Following splenectomy or sham operation erythropoiesis was induced by gradual warming. No consistent or significant differences in red cell numbers, haemoglobin, haematocrit, mean erythrocytic volume or haemoglobin or haemoglobin isomorph system organization were evident during or following recovery. It is unlikely, therefore, that reported acclimatory adjustments in haemoglobin isomorph abundances can be attributed to site-related differences in erythroid cell lineages.     

Genetic characterization of hybridization and introgression between anadromous rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki)

Interspecific hybridization represents a dynamic evolutionary phenomenon and major conservation problem in salmonid fishes. In this study we used amplified fragment length polymorphisms (AFLP) and mitochondrial DNA (mtDNA) markers to describe the extent and characterize the pattern of hybridization and introgression between coastal rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki). Hybrid individuals were initially identified using principle coordinate analysis of 133 polymorphic AFLP markers. Subsequent analysis using 23 diagnostic AFLP markers revealed the presence of F1, rainbow trout backcross, cutthroat trout backcross and later-generation hybrids. mtDNA analysis demonstrated equal numbers of F1 hybrids with rainbow and cutthroat trout mtDNA indicating reciprocal mating of the parental types. In contrast, rainbow and cutthroat trout backcross hybrids always exhibited the mtDNA from the recurrent parent, indicating a male hybrid mating with a pure female. This study illustrates the usefulness of the AFLP technique for generating large numbers of species diagnostic markers. The pattern of hybridization raises many questions concerning the existence and action of reproductive isolating mechanisms between these two species. Our findings are consistent with the hypothesis that introgression between anadromous populations of coastal rainbow and coastal cutthroat trout is limited by an environment-dependent reduction in hybrid fitness.     

Arteriosclerosis in steelhead trout, Oncorhynchus mykiss (Walbaum): a developmental analysis

Arteriosclerosis in Oncorhynchur mykiss has been examined histologically in parr, smolts, spawning adults and post-spawning adults captured during downstream migration. All stages of fish from the Clearwater River in Idaho are characterized by the presence of lesions in the main vessel on the bulbus arteriosus. The incidence is lowest in parr, increases significantly in smolts, and is present in 100% of spawning fish. Regression of lesions was not observed in either hatchery or wild fish after spawning.     

A molecular analysis of hybridization between native westslope cutthroat trout and introduced rainbow trout in southeastern British Columbia, Canada

Restriction site variation in the Ikaros gene intron was used to assess the incidence of westslope cutthroat trout ( Oncorhynchus clarki lewisi ), rainbow trout ( O. mykiss ) and interspecific hybrids at 11 localities among eight streams tributary to the upper Kootenay River system in south-eastern British Columbia, Canada. Out of 356 fish assayed by this technique, hybrids ( n =16) were found at seven of the 11 sites across five different streams. Rainbow trout ( n =6) were found at two of the 11 sites. Analysis of hybrids with a second genetic marker (heat shock 71 intron) indicated that most represented either backcrosses to both westslope cutthroat and rainbow trout, or post F₁ hybrids. Mitochondrial DNA analysis indicated that hybrid matings occur between male rainbow trout and female westslope cutthroat trout and vice versa. Comparison of present hybridization in five tributaries relative to an allozyme-based analysis in the mid-1980s, that documented hybrids in only a single tributary of seven that were common to the two studies, suggests that hybridization and introgression has increased in upper Kootenay River tributaries. The present analysis is a conservative estimate of genetic interaction between the species because introgression was not tested in the majority of samples. Identification of genetically pure westslope cutthroat trout populations, and why they might be resistant to introgression from rainbow trout, are crucial conservation priorities for this unique subspecies of cutthroat trout.     

Diagnostic single nucleotide polymorphisms for identifying westslope cutthroat trout (Oncorhynchus clarki lewisi), Yellowstone cutthroat trout (Oncorhynchus clarkii bouvieri) and rainbow trout (Oncorhynchus mykiss)

We describe 12 diagnostic single nucleotide polymorphism (SNP) assays for use in species identification among rainbow and cutthroat trout: five of these loci have alleles unique to rainbow trout (Oncorhynchus mykiss), three unique to westslope cutthroat trout (O. clarkii lewisi) and four unique to Yellowstone cutthroat trout (O. clarkii bouvieri). These diagnostic assays were identified using a total of 489 individuals from 26 populations and five fish hatchery strains.     

Cellular responses in the skin of rainbow trout (Oncorhynchus mykiss) exposed to Rhine water

Trout, Oncorhynchus mykiss , were exposed to water from the Rhine for 24 days and their skin examined by light and electron microscopy. Relative to control fish mitotic figures were common and seen throughout the epidermis. Pavement cells in fish exposed to Rhine water contained significantly more secretory vesicles than control fish. Necrotic pavement cells were apparent after 24 h, and apoptotic cells from day 4 on. Mucous secretion was intense and the differentiation of mucous cells was stimulated. Some of these cells synthesized mucus of high electron density, probably of a serous composition. Leucocytes invaded the dermis and epidermis, and towards the end of the experiment many apoptotic and necrotic lymphocytes were found. In the dermis fibroblasts were abundant and actively producing collagen. Pigment containing cytoplasmic extensions of melanocytes penetrated into the epidermis. After 14 and 24 days of exposure many pigment cells, melanocytes, iridocytes and xanthocytes became apoptotic. Most of these changes are known from fish exposed to heavy metals, acid water or other stressful treatments, indicating that exposure to Rhine water is a stressful experience for trout.     

A comparison of the effect of long-term starvation on responses to low-temperature stress by juvenile rainbow (Oncorhynchus mykiss) and brown (Salmo trutta) trout reveal different responses in the two species

Biometric parameters and oxidative stress indicators were measured in liver and muscle samples from rainbow trout and brown trout juveniles exposed to a 45-day starvation period at low water temperature. As a general tendency, hepatic antioxidant enzyme activities in both species increased with fasting to eliminate the harmful effects of reactive oxygen species (ROS). However, the metabolic response to food deprivation in the muscle of each species was different. Lipid peroxidation levels in both species increased with starvation. We concluded that (1) low water temperature enhances ROS production in salmonids because of increased polyunsaturated fatty acid content in the cell membrane; (2) starvation significantly impaired the growth parameters of brown trout, yet the reverse was found for rainbow trout; and (3) despite this negative interaction, brown trout juveniles can physiologically tolerate oxidative stress caused by starvation and can therefore be cultivated under stressful conditions even in their early life stages.     

Novel molecular markers differentiate Oncorhynchus mykiss (rainbow trout and steelhead) and the O. clarki (cutthroat trout) subspecies

A suite of 26 PCR‐based markers was developed that differentiates rainbow (Oncorhynchus mykiss) and coastal cutthroat trout (O. clarki clarki). The markers also differentiated rainbow from other cutthroat trout subspecies (O. clarki), and several of the markers differentiated between cutthroat trout subspecies. This system has numerous positive attributes, including: nonlethal sampling, high species‐specificity and products that are easily identified and scored using agarose gel electrophoresis. The methodology described for developing the markers can be applied to virtually any system in which numerous markers are desired for identifying or differentiating species or subspecies.     

Uptake of vitellogenin into oocytes during early vitellogenic development in the rainbow trout, Oncorhynchus mykiss (Walbaum)

Uptake of ³H-vitellogenin (³H-VTG) into oocytes of various sizes was investigated during early vitellogenic development in the rainbow trout, Oncorhynchus mykiss (Walbaum). Females were injected with ³H-VTG and uptake into oocytes of different sizes (<0.4,0.4–0.59, 0.6–0.79, 0.8–0.99 and 1.0 1.2 mm in diameter) measured. Oocytes measuring less than 0.6 mm in diameter appeared unable to sequester VTG and were therefore considered pre-vitellogenic. Oocytes measuring 0.6 mm or more all sequestered VTG. The larger the oocyte, the more ³H-VTG it sequestered, even when uptake was expressed per unit surface area. The latter observation could be due to an increase in the number of VTG receptors per unit surface area, an increase in the rate of turnover of the VTG receptor, greater access of VTG to the receptors as oocytes grow, or a combination of any of these factors. The data suggest that the ability to sequester VTG is developmentally regulated.     

Poststress levels of lysozyme and cortisol in adult rainbow trout: heritabilities and genetic correlations

Lysozyme response in stressed rainbow trout was compared with measurements of poststress cortisol activity. Estimates of heritability of lysozyme and cortisol were both moderate-to-high and both traits displayed positive genetic correlations in pair-wise comparisons of stress exposures. Genetic correlations between lysozyme and cortisol in stressed rainbow trout tended to be negative, although insignificant. Neither lysozyme, nor cortisol exhibited significant correlations with serum haemolytic activity. It is concluded that the data do not confirm earlier suggestions that lysozyme should be superior to cortisol in consistency of stress response in rainbow trout.