Identification of the sex chromosome pair in chum salmon (Oncorhynchus keta) and pink salmon (Oncorhynchus gorbuscha)

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 the karyotypes of chum salmon (Oncorhynchus keta) and pink salmon (Oncorhynchus gorbuscha). The sex chromosome pair is a small acrocentric chromosome pair in chum salmon and the smallest metacentric chromosome pair in pink salmon. Both of these chromosome pairs are morphologically different from the sex chromosome pairs in chinook salmon (Oncorhynchus tshawytscha) and coho salmon (Oncorhynchus kisutch). The 5S rRNA genes are on multiple chromosome pairs including the sex chromosome pair in chum salmon, but at the centromeres of two autosomal metacentric pairs in pink salmon. The sex chromosome pairs and the chromosomal locations of the 5S rDNA appear to be different in all five of the North American Pacific salmon species and rainbow trout. The implications of these results for evolution of sex chromosomes in salmonids are discussed.     

Characterization of a chromosomal rearrangement responsible for producing "apparent" XY-female fall-run Chinook salmon in California

Fluorescence in situ hybridization (FISH) was used to identify the X and Y chromosomes of offspring produced by normal and "apparent" XY-female fall-run Chinook salmon (Oncorhynchus tshawytscha) from California. FISH experiments were performed using probes to 2 sex-linked loci, growth hormone pseudogene (GH-Psi), and OtY1, as well as a probe to a sex-linked microsatellite (Omy7INRA). Comparison of FISH staining patterns between the offspring produced by normal and apparent XY-females revealed that the apparent XY-female examined transmitted a "Y-like" chromosome with an attenuated OtY1 and GH-Psi signal to half of its offspring. Segregation analysis of microsatellites derived from rainbow trout (Oncorhynchus mykiss) with respect to phenotypic sex was carried out for 2 normal and 2 apparent XY-female Chinook salmon families. Inheritance patterns of Omy7INRA were consistent with this locus being closely linked to GH-Psi in males and in apparent XY-females carrying the Y-like chromosome. Another microsatellite locus (Omm1077) was closely linked to the primary sex-determining locus (SEX) in males but not to GH-Psi/OtY1 in apparent XY-females. The FISH analyses suggest that apparent XY-female fall-run Chinook salmon in California are not the product of a Y chromosome to autosome translocation. Despite the combined FISH and inheritance analyses, we were unable to differentiate between 2 alternative explanations for apparent XY-females, namely, recombination of markers between the sex chromosomes, or a Y chromosome with a dysfunctional or missing sex-determining region.     

Characterization of sex chromosomes in rainbow trout and coho salmon using fluorescence in situ hybridization (FISH)

With the aim of characterizing the sex chromosomes of rainbow trout (Oncorhynchus mykiss) and to identify the sex chromosomes of coho salmon (O. kisutch), we used molecular markers OmyP9, 5S rDNA, and a growth hormone gene fragment (GH2), as FISH probes. Metaphase chromosomes were obtained from lymphocyte cultures from farm specimens of rainbow trout and coho salmon. Rainbow trout sex marker OmyP9 hybridizes on the sex chromosomes of rainbow trout, while in coho salmon, fluorescent signals were localized in the medial region of the long arm of one subtelocentric chromosome pair. This hybridization pattern together with the hybridization of a GH2 intron probe on a chromosome pair having the same morphology, suggests that a subtelocentric pair could be the sex chromosomes in this species. We confirm that in rainbow trout, one of the two loci for 5S rDNA genes is on the X chromosome. In males of this species that lack a heteromorphic sex pair (XX males), the 5S rDNA probe hybridized to both subtelocentrics This finding is discussed in relation to the hypothesis of intraspecific polymorphism of sex chromosomes in rainbow trout.     

Identification of the Y chromosome in chinook salmon (Oncorhynchus tshawytscha)

The Y chromosome in chinook salmon, Oncorhynchus tshawytscha, was identified using fluorescence in situ hybridization (FISH) with a probe to a male-specific repetitive sequence isolated from this species. The probe highlights the distal end of the short arm of an acrocentric chromosome with a DAPI-bright interstitial band of variable size. The proximal portion of the short arm of the Y chromosome contains 5S rDNA sequences, which are also found on the short arms of six other acrocentric chromosomes in this species.     

Chromosome painting supports lack of homology among sex chromosomes in Oncorhynchus, Salmo, and Salvelinus (Salmonidae)

The sex chromosome pair has been identified previously as the largest submetacentric pair in the genome in several species of the genus Salvelinus (eastern trouts and chars) including S. namaycush (lake trout) and as a large subtelocentric/acrocentric pair in several species of the genus Oncorhynchus (Pacific trouts and salmon). Sex chromosomes have not been identified in Salmo (Atlantic salmon and brown trout). Two paint probes, one specific for the short arm (Yp) and the other for the long arm (Yq) of the sex chromosome pair in Salvelinus namaycush were hybridized to chromosomes of Oncorhynchus mykiss (rainbow trout) and O. tshawytscha (chinook salmon) and Salmo salar (Atlantic salmon) and S. trutta (brown trout). The two probes hybridized to two different autosomal pairs in each of the Oncorhynchus species, supporting lack of homology between the sex chromosomes in the two genera. The Yp probe hybridized to interstitial regions on two different chromosome pairs in S. salar and one pair in S. trutta. The Yq probe hybridized to a different pair in both species.     

Xenoma formation during microsporidial gill disease of salmonids caused by Loma salmonae is affected by host species (Oncorhynchus tshawytscha,O. kisutch,O. mykiss) but not by salinity

Host species and salinity often affect the development of disease in aquatic species. Eighty chinook salmon Oncorhynchus tshawytscha, 80 coho salmon O. kisutch and 80 rainbow trout O. mykiss were infected with Loma salmonae. Forty of each species were reared in seawater and 40 in freshwater. The mean number of xenomas per gill filament was 8 to 33 times greater in chinook salmon than in rainbow trout (RBT). Coho salmon had a mean xenoma intensity intermediate to that of chinook salmon and RBT. In contrast to the differences between species, salinity had no significant effect on xenoma intensity in any of these host species. The onset of xenoma formation occurred at Week 5 postexposure (PE) for chinook salmon and RBT, and at Week 6 PE for coho salmon. RBT had cleared all visible branchial xenomas by Week 9 PE, whereas xenomas persisted in coho and chinook salmon at Week 9 PE. Histologically, xenomas were visible in the filament arteries of the branchial arch in chinook and coho salmon gills but were absent from RBT gills. Fewer xenomas were seen in the central venous sinusoids of RBT than in chinook and coho salmon. The lower xenoma intensity, shorter duration of infection and pathological characteristics, common to microsporidial gill disease in RBT, suggest a degree of resistance to clinical disease that is not seen in coho and chinook salmon.     

Comparative mapping reveals quantitative trait loci that affect spawning time in coho salmon (Oncorhynchus kisutch)

Spawning time in salmonids is a sex-limited quantitative trait that can be modified by selection. In rainbow trout (Oncorhynchus mykiss), various quantitative trait loci (QTL) that affect the expression of this trait have been discovered. In this study, we describe four microsatellite loci associated with two possible spawning time QTL regions in coho salmon (Oncorhynchus kisutch). The four loci were identified in females from two populations (early and late spawners) produced by divergent selection from the same base population. Three of the loci (OmyFGT34TUF, One2ASC and One19ASC) that were strongly associated with spawning time in coho salmon (p < 0.0002) were previously associated with QTL for the same trait in rainbow trout; a fourth loci (Oki10) with a suggestive association (p = 0.00035) mapped 10 cM from locus OmyFGT34TUF in rainbow trout. The changes in allelic frequency observed after three generations of selection were greater than expected because of genetic drift. This work shows that comparing information from closely-related species is a valid strategy for identifying QTLs for marker-assisted selection in species whose genomes are poorly characterized or lack a saturated genetic map.     

Susceptibility of rainbow trout Oncorhynchus mykiss,Atlantic salmon Salmo salar and coho salmon Oncorhynchus kisutch to experimental infection with sea lice Lepeophtheirus salmonis

Physiological, immunological and biochemical parameters of blood and mucus, as well as skin histology, were compared in 3 salmonid species (rainbow trout Oncorhynchus mykiss, Atlantic salmon Salmo salar and coho salmon O. kisutch) following experimental infection with sea lice Lepeophtheirus salmonis. The 3 salmonid species were cohabited in order to standardize initial infection conditions. Lice density was significantly reduced on coho salmon within 7 to 14 d, while lice persisted in higher numbers on rainbow trout and Atlantic salmon. Lice matured more slowly on coho salmon than on the other 2 species, and maturation was slightly slower on rainbow trout than on Atlantic salmon. Head kidney macrophages from infected Atlantic salmon had diminished respiratory burst and phagocytic capacity at 14 and 21 d post-infection (dpi), while infected rainbow trout macrophages had reduced respiratory burst and phagocytic capacities at 21 dpi, compared to controls. The slower development of lice, coupled with delayed suppression of immune parameters, suggests that rainbow trout are slightly more resistant to lice than Atlantic salmon. Infected rainbow trout and Atlantic salmon showed increases in mucus lysozyme activities at 1 dpi, which decreased over the rest of the study. Mucus lysozyme activities of infected rainbow trout, however, remained higher than controls over the entire period. Coho salmon lysozyme activities did not increase in infected fish until 21 dpi. Mucus alkaline phosphatase levels were also higher in infected Atlantic salmon compared to controls at 3 and 21 dpi. Low molecular weight (LMW) proteases increased in infected rainbow trout and Atlantic salmon between 14 and 21 dpi. Histological analysis of the outer epithelium revealed mucus cell hypertrophy in rainbow trout and Atlantic salmon following infection. Plasma cortisol, glucose, electrolyte and protein concentrations and hematocrit all remained within physiological limits for each species, with no differences occurring between infected and control fish. Our results demonstrate that significant differences in mucus biochemistry and numbers of L. salmonis occur between these species.     

Identification of the sex chromosomes of brown trout (Salmo trutta) and their comparison with the corresponding chromosomes in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)

Males are the heterogametic sex in salmonid fishes. In brown trout (Salmo trutta) the sex-determining locus, SEX, has been mapped to the end of linkage group BT-28, which corresponds to linkage group AS-8 and chromosome SSA15 in Atlantic salmon (Salmo salar). We set out to identify the sex chromosomes in brown trout. We isolated Atlantic salmon BAC clones containing microsatellite markers that are on BT-28 and also on AS-8, and used these BACs as probes for fluorescent in situ hybridization (FISH) analysis. SEX is located on the short arm of a small subtelocentric/acrocentric chromosome in brown trout, which is consistent with linkage analysis. The acrocentric chromosome SSA15 in Atlantic salmon appears to have arisen by a centric fusion of 2 small acrocentric chromosomes in the common ancestor of Salmo sp. We speculate that the fusion process that produced Atlantic salmon chromosome SSA15 disrupted the ancestral sex-determining locus in the Atlantic salmon lineage, providing the impetus either for the relocation of SEX or selection pressure for a novel sex-determining gene to arise in this species. Thus, the sex-determining genes may differ in Atlantic salmon and brown trout.     

DNA homologies between the rainbow trout, chum salmon and coho salmon

The thermal stabilities of hybrid duplexes between the DNAs from three salmonid fish species were monitored as measures of DNA homology. The chum salmon, Oncorhynchus keta, and coho salmon, Oncorhynchus kisutch, had more DNA homology with each other than either had with the rainbow trout, Salmo gairdnerii. Morphological, ecological and protein similarities between the coho salmon and the rainbow trout may be due to parallel or convergent evolution.     

Isolation and characterization of Edwardsiella tarda from fall chinook salmon (Oncorhynchus tshawytscha).

A new bacterial pathogen of chinook salmon (oncorhynchus tshawytscha) was isolated from fish in Oregon's Rogue River. The bacteria are biochemically and serologically related to strains of Edwardsiella tarda. Initially isolated from chinook salmon, the bacteria were also pathogenic for steelhead and rainbow trout (Salmo gairdneri), and channel catfish (Ictalurus punctatus). The 50% lethal doses for chinook salmon, steelhead trout, and channel catfish injected intraperitoneally and maintained in 18 degrees C water were 4.1 x 10(6), 5.6 x 10(6), and 4.0 x 10(5) respectively. When chinook salmon and rainbow trout were injected intraperitoneally and held in 12 degrees C water, the mean lethal doses were 6.4 x 10(7) and 1.7 x 10(6), respectively. The invasiveness of the organism was low in steelhead trout exposed to the bacteria by the waterborne route. The optimum growth temperature of the bacteria in brain heart infusion broth was approximately 35 degrees C. The guanine plus cytosine content of DNA obtained from E. tarda isolated from salmon was 59 mol%.     

The sockeye salmon neo-Y chromosome is a fusion between linkage groups orthologous to the coho Y chromosome and the long arm of rainbow trout chromosome 2

Unlike other Pacific salmon, sockeye salmon (Oncorhynchus nerka) have an X(1)X(2)Y sex chromosome system, with females having a diploid chromosome number of 2n = 58 and males 2n = 57 in all populations examined. To determine the origin of the sockeye Y chromosome, we mapped microsatellite loci from the rainbow trout (O. mykiss; OMY) genetic map, including those found on the Y chromosomes of related species, in kokanee (i.e. non-anadromous sockeye) crosses. Results showed that 3 microsatellite loci from the long arm of rainbow trout chromosome 8 (OMY8q), linked to SEX (the sex-determining locus) in coho salmon (O. kisutch), are also closely linked to SEX in the kokanee crosses. We also found that 3 microsatellite loci from OMY2q are linked to those markers from OMY8q and SEX in kokanee, with both linkage groups fused to form the neo-Y. These results were confirmed by physical mapping of BAC clones containing microsatellite loci from OMY8q and OMY2q to kokanee chromosomes using fluorescence in situ hybridization. The fusion of OMY2q to the ancestral Y may have resolved sexual conflict and, in turn, may have played a large role in the divergence of sockeye from a shared ancestor with coho.     

The cutthroat trout Y chromosome is conserved with that of rainbow trout

Five genetic markers previously shown to be located on the sex chromosomes of rainbow trout (Oncorhynchus mykiss) were tested for linkage with the sex locus of Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri) in a genetic cross created from a rainbow x cutthroat male hybrid. We show that the sex locus of both rainbow and cutthroat trout is on the same homologous linkage group. Fluorescence in situ hybridization (FISH) using a probe for the microsatellite marker Omm1665, which maps close to the sex locus of Yellowstone cutthroat trout, was used to identify the Y chromosome of cutthroat trout in the hybrid. The Y chromosome of cutthroat trout is sub-telocentric and lacks a DAPI band found on the short arm of the Y chromosome of some rainbow trout males.     

Y chromosome phylogeny for cutthroat trout (Oncorhynchus clarkii) subspecies is generally concordant with those of other markers

Sequence divergence was evaluated in the non-recombining, male-specific OmyY1 region of the Y chromosome among the subspecies of cutthroat trout (Oncorhynchus clarkii) in the western United States. This evaluation identified subspecies-discriminating OmyY1-haplotypes within a ～1200 bp region of the OmyY1 locus and localized the region to the end of the Y chromosome by FISH analysis. OmyY1 sequences were aligned and used to reconstruct a phylogeny of the cutthroat trout subspecies and related species via maximum-parsimony and Bayesian analyses. In the Y-haplotype phylogeny, clade distributions generally corresponded to the geographic distributions of the recognized subspecies. This phylogeny generally corresponded to a mitochondrial tree obtained for these subspecies in a previous study. Both support a clade of trout vs. Pacific salmon, of rainbow trout, and of a Yellowstone cutthroat group within the cutthroat trout. In our OmyY1 tree, however, the cutthroat “clade”, although present topologically, was not statistically significant. Some key differences were found between trees obtained from the paternally-inherited OmyY1 vs. maternally-inherited mitochondrial haplotypes in cutthroat trout compared to rainbow trout. Other findings are: The trout OmyY1 region evolves between 3 and 13 times slower than the trout mitochondrial regions that have been studied. The Lahontan cutthroat trout had a fixed OmyY1 sequence throughout ten separate populations, suggesting this subspecies underwent a severe population bottleneck prior to its current dispersal throughout the Great Basin during the pluvial phase of the last ice age. The Yellowstone group is the most derived among the cutthroat trout and consists of the Yellowstone, Bonneville, Colorado, Rio Grande and greenback subspecies. Identification of subspecies and sex with this Y-chromosome marker may prove useful in conservation efforts.     

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.     

Tissue astaxanthin and canthaxanthin distribution in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

A comparative investigation of tissue carotenoid distribution between rainbow trout, Oncorhynchus mykiss, and Atlantic salmon, Salmo salar, was undertaken to identify the relative efficiency of utilization of astaxanthin and canthaxanthin. Higher apparent digestibility coefficients (ADCs) (96% in trout vs. 28-31% in salmon; P<0.05), and pigment retention efficiencies (11.5-12.5% in trout vs. 5.5% in salmon; P<0.05), for both astaxanthin and canthaxanthin, were observed for rainbow trout. Astaxanthin deposition was higher than canthaxanthin in rainbow trout, while the reverse was true for Atlantic salmon, suggesting species-specificity in carotenoid utilization. The white muscle (95% in trout vs. 93% in salmon) and kidneys (0.5% in trout vs. 0.2% in salmon) represented higher proportions of the total body carotenoid pool in rainbow trout than in Atlantic salmon (P<0.05), whereas the liver was a more important storage organ in Atlantic salmon (2-6% in salmon vs. 0.2% in trout; P<0.05). The liver and kidney appeared to be important sites of carotenoid catabolism based on the relative proportion of the peak chromatogram of the fed carotenoid in both species, with the pyloric caecae and hind gut being more important in Atlantic salmon than in the rainbow trout. Liver catabolism is suspected to be a critical determinant in carotenoid clearance, with higher catabolism expected in Atlantic salmon than in rainbow trout.     

Fecundity and egg size variation in North American Pacific salmon (Oncorhynchus)

We examined regional and latitudinal variation in fecundity and egg weight for five species of Pacific salmon ( Oncorhynchus ) along the Pacific coast of North America. Data were examined for 24 chum salmon, 15 pink salmon, 34 sockeye salmon, 44 chinook salmon, and 40 coho salmon populations from published sources, unpublished Canadian hatchery records, our own laboratory investigations, and other unpublished sources. Substantial regional variation in fecundity and egg weight was observed, with salmon on the Queen Charlotte Islands and Vancouver Island in British Columbia generally having lower fecundity and larger egg size than nearby mainland populations. The relative distance of freshwater migration to the spawning grounds generally had a marked effect on both fecundity and egg size, with populations spawning in the upper portions in the drainages of large rivers like the Fraser River in British Columbia having reduced fecundity and egg size compared with coastal spawning populations. Fecundity was generally higher and egg size generally lower in more northern populations of sockeye, chinook, and coho salmon compared with southern ones. We suggest that egg size tends to be lower in northern populations of some species as a result of increased fecundity due to their older ages at maturity and a limited amount of energy that can be expended on egg production.     

An electrophoretic analysis of plasma proteins from juvenile Oncorhynchus tshawytscha (Walbaum)

The plasma proteins of juvenile spring chinook salmon, Oncorhynchus tshawytscha , were examined as a function of development. Two-way gel electroprioresis of plasma proteins was conducted on samples taken from fish before initiation of downstream migration, from fish classified as actively migrating and from fish that were prevented from migrating. Analysis of the samples indicated that plasma proteins of fish changed depending on the migratory status of the fish. The results were consistent with the notion that some of the factors normally associated with smoltification in species such as steelhead trout, Salmo gairdneri , and coho salmon, O. kisutch , may well be temporally separated in Idaho O. ishawytscha.     

A comparative analysis of the rainbow trout genome with 2 other species of fish (Arctic charr and Atlantic salmon) within the tetraploid derivative Salmonidae family (subfamily: Salmoninae).

We updated the genetic map of rainbow trout (Oncorhynchus mykiss) for 2 outcrossed mapping panels, and used this map to assess the putative chromosome structure and recombination rate differences among linkage groups. We then used the rainbow trout sex-specific maps to make comparisons with 2 other ancestrally polyploid species of salmonid fishes, Arctic charr (Salvelinus alpinus) and Atlantic salmon (Salmo salar) to identify homeologous chromosome affinities within each species and ascertain homologous chromosome relationships among the species. Salmonid fishes exhibit a wide range of sex-specific differences in recombination rate, with some species having the largest differences for any vertebrate species studied to date. Our current estimate of female:male recombination rates in rainbow trout is 4.31:1. Chromosome structure and (or) size is associated with recombination rate differences between the sexes in rainbow trout. Linkage groups derived from presumptive acrocentric type chromosomes were observed to have much lower sex-specific differences in recombination rate than metacentric type linkage groups. Arctic charr is karyotypically the least derived species (i.e., possessing a high number of acrocentric chromosomes) and Atlantic salmon is the most derived (i.e., possessing a number of whole-arm fusions). Atlantic salmon have the largest female:male recombination ratio difference (i.e., 16.81:1) compared with rainbow trout, and Arctic charr (1.69:1). Comparisons of recombination rates between homologous segments of linkage groups among species indicated that when significant experiment-wise differences were detected (7/24 tests), recombination rates were generally higher in the species with a less-derived chromosome structure (6/7 significant comparisons). Greater similarity in linkage group syntenies were observed between Atlantic salmon and rainbow trout, suggesting their closer phylogenetic affinities, and most interspecific linkage group comparisons support a model that suggests whole chromosome arm translocations have occurred in the evolution of this group. However, some possible exceptions were detected and these findings are discussed in relation to their influence on segregation distortion patterns. We also report unusual meiotic segregation patterns in a female parent involving the duplicated (homeologous) linkage group pair 12/16 and discuss several models that may account for these patterns.