Ectopic epithelial cell clusters in salmonid intestine are associated with inflammation.

An epizootic incidence of intestinal adenocarcinomas was reported in brood fish of Atlantic salmon (Salmo salar L.) in 2009. The condition was associated with a specific diet inducing enteritis and morphological changes. Here, two field trials of fish up to slaughter size were initiated. In Trial 1, two different feed recipes were used. Feed I was predominantly based on marine ingredients, whereas plant ingredients were limited to soy protein concentrate and wheat. Feed II was lower in fishmeal and without soya protein, which was substituted with plant proteins from other sources. In Trial 2, a commercial feed (Feed III) was included. No macroscopic tumours were observed in 300 fish (Trial 1). At the end of both trials, samples from five different segments of the gastrointestinal tract of a total of 39 fish were investigated with morphological methods. Here, we show the presence of ectopic proliferating epithelial cells only occurring in inflamed intestine and predominantly in the second segment of the mid-intestine. Presence of ectopic epithelial cells in submucosal inflammatory foci may indicate early stages in tumorigenesis, but other possibilities such as proliferative enteric disorders cannot be excluded. Together with inflammation, carcinogenesis should be a focus of investigation in future feed trials.

Vaccination of Atlantic lumpfish (Cyclopterus lumpus L.) at a low temperature leads to a low antibody response against Aeromonas salmonicida.

We present a study on the effect of water temperature on immunization of Atlantic lumpfish. In total, 360 fish were vaccinated with either 50 µl of an oil-based injection vaccine (VAX), with Aeromonas salmonicida and Vibrio salmonicida antigens, or PBS. Fish were vaccinated at three different water temperatures, 5°C, 10°C and 15°C, and sorted into six groups (N = 60). Lumpfish were weighed every 3 weeks after vaccination, sampled at 3, 6, 9 and 18 weeks post-immunization (wpi) and evaluated by modified Speilberg score, ELISA and immunoblotting. Vaccinated fish showed low antibody response against V. salmonicida. Fish vaccinated at 5°C showed significantly lower antibody response against A. salmonicida throughout the study. At higher temperatures, vaccinated fish showed significantly increased antibody responses, at 18 wpi for 10°C and at 6 and 18 wpi for 15°C. Immunoblotting demonstrated specific response against the LPS antigen of A. salmonicida in the 10°C and 15°C VAX groups. Mean body weight increased in all groups throughout the study. Vaccinated fish had low Speilberg scores with no melanization of abdominal tissue. Our results show that vaccinating lumpfish at a lower water temperature may lead to a low antibody response against A. salmonicida.

Studies on the antibody response and side effects after intramuscular and intraperitoneal injection of Atlantic lumpfish (Cyclopterus lumpus L.) with different oil-based vaccines.

Atlantic lumpfish (Cyclopterus lumpus L.) is used as a biological delousing agent for sea lice (Lepeophtheirus salmonis K.) infestations in Norwegian aquaculture. Here, we present a study on the antibody response and vaccine side effects after intramuscular and intraperitoneal injection of lumpfish with two vaccines. Both vaccines contained bacterial antigens from atypical Aeromonas salmonicida A-layer types V and VI, Vibrio anguillarum serotype O1 and Moritella viscosa sp., but one vaccine contained a vegetable oil-based adjuvant, while the other contained a mineral oil-based adjuvant. Intramuscular injection of the mineral oil-based vaccine caused a high acute mortality of fish within 48 hr after immunization. Intraperitoneal injection of the mineral oil-based vaccine resulted in a lower severity of intra-abdominal side effects than the vegetable oil-based vaccine. Intramuscular injection of the mineral oil-based vaccine resulted in a significantly higher antibody response against A. salmonicida when compared to controls and the vegetable oil-based vaccine group. The antibody response was poor against V. anguillarum and M. viscosa for all groups. Our results indicate that intramuscular injection of oil-based vaccines might be feasible for providing immunological protection for Atlantic lumpfish against bacterial diseases, especially atypical A. salmonicida, but more work is required to identity optimal adjuvants.

Coronary changes in the Atlantic salmon Salmo salar L: characterization and impact of dietary fatty acid compositions.

Consumption of fatty acids from fishes is widely regarded as beneficial for preventing cardiovascular disorders. Nevertheless, salmonids themselves are victims of vascular diseases. As the pathogenesis and nature of these changes are elusive, they are here addressed using novel morphological and transcriptional approaches. Coronary arteries of wild Atlantic salmon Salmo salar L., (n = 12) were investigated using histological and immunohistochemical techniques, and RT-qPCR was employed to investigate expression of stretch-induced genes. In an experimental trial, fish were fed diets with different fatty acids composition, and histological features of the coronary arteries (n = 36) were investigated. In addition, the heart fatty acid profile (n = 60) was analysed. There were no differences in morphological or immunological features between wild fish and groups of experimental fish. Arteriosclerotic lesions consisted of smooth muscle cells in dissimilar differential stages embedded in considerable amounts of extracellular matrix in a similar fashion to what is seen in early stages of human atherosclerosis. No fat accumulations were observed, and very few inflammatory cells were present. In affected arteries, there was an induction of stretch-related genes, pointing to a stress-related response. We suggest that salmon may have a natural resistance to developing atherosclerosis, which corresponds well with their high investment in lipid metabolism.

Characterization of myocardial lesions associated with cardiomyopathy syndrome in Atlantic salmon, Salmo salar L., using laser capture microdissection.

Cardiomyopathy syndrome (CMS) in Atlantic salmon, Salmo salar L., is characterized by focal infiltration in the spongy myocardium and endocardium of the heart. The origin of the mononuclear infiltrate is unknown. Using experimentally infected fish, we investigated localization of the causative agent, piscine myocarditis virus (PMCV), within the heart and characterized the cell population associated with myocardial lesions. Cellular and transcriptional characteristics in the lesions were compared with adjacent non-infiltrated tissues using laser capture microdissection, RT-qPCR and immunohistochemistry. Our results reveal that PMCV is almost exclusively present in myocardial lesions. The inflammatory infiltrate comprises a variety of leucocyte populations, including T cells, B cells, MHC class II(+) and CD83(+) cells, most likely of the macrophage line. Correlation analyses demonstrated co-ordinated leucocyte activity at the site of the virus infection. Cellular proliferation and/or DNA repair was demonstrated within the myocardial lesions. Different cell populations, mainly myocytes, stained positive for proliferating cell nuclear antigen (PCNA). Densities of endothelial cells and fibroblasts were not significantly increased. The simultaneous presence of PMCV and various inflammatory cells in all myocardial lesions analysed may indicate that both viral lytic and immunopathological effects may contribute to the pathogenesis of CMS.

CD4 homologues in Atlantic salmon.

In mammals CD4 is a membrane glycoprotein on Th cells with four extracellular immunoglobulin-like (Ig-like) domains (D1-D4). It functions as a co-receptor during immune recognition between the TCR and the MHC II/peptide complex. The cytoplasmic domain binds p56lck, a protein kinase responsible for phosphorylating CD3 which is the first interaction in a cascade leading to T cell activation. We have previously reported a CD4-2 gene in rainbow trout (Oncorhynchus mykiss) which was found adjacent to the CD4-1 gene by synteny analysis. There are two subtypes (a and b) of CD4-2 in rainbow trout, with two Ig-like extracellular domains. Here we present the homologues of mammalian CD4 in Atlantic salmon (Salmo salar): CD4-1 with four extracellular domains and CD4-2a and CD4-2b with two extracellular domains. A Southern blot analysis shows two copies of the CD4-1 gene in the genomic DNA of the closely related rainbow trout. The genes for CD4-1 and CD4-2 have been sequenced and show typical traits for CD4 genes, such as the code for the first domain (D1) being divided between two exons and the other domains being largely coded for by single exons. The corresponding translated cDNAs show little (13-17%) identity to higher vertebrates and are approximately 37% similar to other translated, teleost sequences but are 89% identical to the closely related rainbow trout. However they exhibit conserved features such as the Lck binding motif in their cytoplasmic domains and the order of variable and constant type Ig-like domains. qRT-PCR data are presented describing the differential tissue expression of these genes together with other T cell markers (TCR and CD3) in several individuals.

MHC class II+ cells in the gills of Atlantic salmon (Salmo salar L.) affected by amoebic gill disease.

Amoebic gill disease (AGD) is characterised by the association of Neoparamoeba sp. with hyperplastic gill tissue of affected fishes, however, the identity and role of host cells associated with AGD lesions are not known. Here, we investigated cells with an immunological role that were associated with AGD lesions by locating cellular MHC class II beta chain. A tank housing Atlantic salmon (Salmo salar) was inoculated with Neoparamoeba sp., and MHC class II beta chain expression in the gills was qualitatively assessed by immunohistochemistry. In AGD-naïve control fish, MHC class II+ cells were detected basolateral to the interlamellar epithelium as well as upon the interlamellar and secondary epithelium. In the gills of AGD affected fish MHC class II+ cells were observed in both affected and unaffected tissue. Within AGD lesions, numerous MHC class II+ cells were present and these cells exhibited variable levels of expression suggesting that like mammals, MHC class II expression is highly regulated. The presence of MHC class II+ cells within gill lesions is indicative of immune cell trafficking and these cells could contribute in an antigen presentation capacity to the development of an antibody response in fish chronically affected by AGD.

Characterisation of salmon and trout CD8alpha and CD8beta.

The genes and corresponding cDNAs of both alpha and beta chains of the Atlantic salmon (Salmo salar) CD8 molecule have been sequenced and characterized. In addition, the cDNAs for alpha and beta chains of brown trout (Salmo trutta) and for the beta chain in rainbow trout (Oncorhynchus mykiss) have been sequenced. The cDNAs code for signal sequences which are preceded by short 5' UTRs. These are followed by typical immunoglobulin superfamily variable sequences all of which contain two conserved cysteines for the intra-chain disulphide bond. The hinge regions display conserved cysteines for dimerisation and several O-glycosylation motifs for each predicted protein. The domain sharing the highest sequence identity with mammals is the single pass transmembrane domain for all sequences. In salmon, each domain is predominantly coded for by a single exon except the cytoplasmic/3' UTR domains, which are coded for by 3 and 2 exons for the alpha and beta genes, respectively. In the alpha gene, the second cytoplasmic exon may be spliced out to form an alternative shorter transcript which if expressed would exhibit a truncated cytoplasmic tail. A splice variant found for the salmon beta gene introduces a stop codon after only 40 amino acids. Overall amino acid identities between salmonid sequences were higher than 90%, whereas they shared only 15-20% identity with species such as, chicken and human. Analysis of the expression patterns of the two salmon genes using quantitative RT-PCR shows a very high expression in the thymus. This is mirrored by the expression of the TCRalpha gene, which is known to be co-expressed with CD8 on mammalian T cells. This is the first report of a sequence for CD8beta in a teleost and together with the CD8alpha sequence, it encodes the ortholog of the CD8 co-receptor molecule on mammalian T cells.

Vaccine-associated granulomatous inflammation and melanin accumulation in Atlantic salmon, Salmo salar L., white muscle.

The purpose of this study was to investigate the nature of variably sized pigmented foci encountered in fillets of farmed Atlantic salmon, Salmo salar L. The material was sampled on the fillet production line and on salmon farms from fish with an average size of 3 kg from various producers. The fish had been routinely vaccinated by injection. Gross pathology, histology, immunohistochemistry using antisera against major histocompatibility complex (MHC) class II beta chain and transmission electron microscopy (TEM) were used to characterize the changes. Macroscopically, melanized foci were seen penetrating from the peritoneum deep into the abdominal wall, sometimes right through to the skin, and also embedded in the caudal musculature. Histological investigation revealed muscle degeneration and necrosis, fibrosis and granulomatous inflammation containing varying numbers of melano-macrophages. Vacuoles, either empty or containing heterogeneous material, were frequently seen. The presence of abundant MHC class II+ cells indicated an active inflammatory condition. TEM showed large extracellular vacuoles and leucocytes containing homogeneous material of lipid-like appearance. The results showed that the melanized foci in Atlantic salmon fillet resulted from an inflammatory condition probably induced by vaccination. The described condition is not known in wild salmon and in farmed salmon where injection vaccination is not applied.

Granulomatous uveitis associated with vaccination in the atlantic salmon.

This study addressed histologic and immunopathologic changes in ocular tissues and investigated the distribution of major histocompatibility class II (MHC class II)-positive cells in Atlantic salmon (Salmo salar) suffering from severe postvaccination disease. Twenty-nine fish with generalized inflammation, probably a result of vaccination, were investigated. One individual that had escaped vaccination was included in the study. Material was investigated by cultivation methods for fungi and bacteria. Histology using conventional staining procedures and immunohistochemistry with antisera against MHC class II beta chain were performed. No growth was observed from the cultivation investigations. Histology revealed occlusion of the lumen in the larger choroid vessels and in the choriocapillaris, inflammatory infiltrations and loss of structure in the choroid rete, and, in some cases, aggregations of multinucleated giant cells (MGC) and Splendore-Hoeppli material. Immunohistochemistry demonstrated massive MHC class II+ cellular infiltrations in the uveal tract. Such infiltrations were also seen in the ventral ciliary cleft, a condition that is associated with glaucoma. Immunoreactive cells included dendritelike cells, epithelioid cells, and MGCs. The endothelia of smaller vessels were frequently MHC class II+, and immunoreactive infiltrations were seen in the optic nerve in several individuals. No pathologic changes were detected in the unvaccinated individual. In conclusion, generalized inflammatory reactions in fish may lead to severe ocular inflammation, occlusion of uveal vessels, and perivascular changes with MHC class II+ upregulation in cells in the uveal tract and optic nerve.

Vaccination induces major histocompatibility complex class II expression in the Atlantic salmon eye.

The aim of the study was to investigate the presence, distribution and density of major histocompatibility complex (MHC) class II+ cells in the ocular tissues of the Atlantic salmon, Salmo salar, prior to and following vaccination. Eyes were collected 14 days prior to and at 4, 11, 25 and 39 days and 4 months subsequent to vaccination with a commercial fish vaccine. A quantitative analysis was performed in sections on the number of immunopositive cells in the retinal layers. In all groups, MHC class II+ cells were detected in the area of the limbus but not in the central parts of the cornea. In the uvea, immunopositive cells were present in unvaccinated and vaccinated fish. Abundant immunopositive cells were identified in the choroid rete (or choroid gland) in all groups as well as in the ventral ciliary cleft, where macrophage-like MHC class II+ cells were seen. Quantitative histology of the retina revealed a significant increase in MHC class II+ cells in the outer plexiform layer (OPL) and the inner nuclear layer (INL) 4 days following vaccination. Positive cells were detected in all layers of the retina with the exception of the photoreceptor layer.

Production of rabbit antisera against recombinant MHC class II beta chain and identification of immunoreactive cells in Atlantic salmon (Salmo salar).

In the present work, rabbit antisera recognising the Atlantic salmon (Salmo salar) MHC class II beta chain polypeptide were produced and used in immunoblotting, immunohistochemistry and immunogold electron microscopy. A construct encoding the beta1 and beta2 domains fused to the E. coli protein thioredoxin was used to express the recombinant MHC class II beta chain. Immunoblotting revealed a band of approximately 30kDa in total protein samples from head kidney, spleen, gills, thymus and blood leukocytes, while being absent in muscle. The distribution of MHC class II positive cells was immunohistochemically demonstrated in Atlantic salmon epithelial and haemopoietic tissues. Ultrastructural demonstration of immunoreactive organelles in mid-kidney cells was performed by immunogold electron microscopy. The results indicate expression in lymphocytes, macrophages, epithelial cells and endritic-like cells. This is the first study to address morphological MHC class II expression in a fish species.

Presence of IgM in cutaneous mucus, but not in gut mucus of Atlantic salmon, Salmo salar. Serum IgM is rapidly degraded when added to gut mucus.

In the first part of this study, cutaneous mucus of Atlantic salmon (Salmo salar) was shown to contain IgM, i.e. molecules composed of approximately 72 and 27 kDa subunits and reactive with polyclonal antisera and monoclonal antibodies made against serum IgM. Attempts to detect IgM-like molecules in gut mucus were negative, indicating the IgM is present, at best, in very small amounts. The degradation of serum IgM in mucosal secretions was examined in the second part of this study. Purified IgM from serum was rapidly digested in gut mucus at 4 degrees C. Intermediate 58, 52, 38, 35, 33 and 18 kDa breakdown fragments appeared when analysed in immunoblots. The transient fragments were further degraded to small fragments. HPLC analysis showed that only half of the added serum IgM was intact after 30 min of digestion, and after 4 h intact IgM could not be detected. Serum IgM was not degraded in cutaneous mucus, even after 17 h of incubation.

Diversity of the immunoglobulin heavy chain in the Atlantic salmon (Salmo salar L.) is contributed by genes from two parallel IgH isoloci.

Immunoglobulin heavy chain (IgH) variable (V) region cDNAs from the Atlantic salmon, Salmo salar L., have been isolated and analysed with respect to diversity and transcription of the two parallel IgH isoloci in this species. A total of nine V(H) families were defined according to the 80% identity criterion, of which seven were highly related (>80% identity) to the V(H) families defined in rainbow trout and arctic charr. The variability of the CDR1 and 2 was low, although mutational hot-spot consensus sequences were accumulated in these regions. The CDR3 showed largest variability, expressing at least eight different groups of D motifs diversified by fusion of the D motifs, possible N and P nucleotide insertions and exonuclease activity. Presumably functional transcripts expressing D motifs in all three reading frames were identified for two of the motifs. The cDNAs were mapped to either of the two parallel loci, and sequence analysis revealed that the repertoire of V(H) segments was contributed by transcription of genes from both of the IgH isoloci. Transcription of genes from both isoloci generated no obvious effects on variability in the CDR3 of the Atlantic salmon IgH chains, although one additional J(H)-segment with altered N-terminal was generated by the process of duplication and divergence. Thus, the issue of biological significance of the two IgH isoloci remains unclear.

Expression and analysis of recombinant salmon parvalbumin, the major allergen in Atlantic salmon (Salmo salar).

The parvalbumin from white muscle of Atlantic salmon was previously found to be a major allergen, and designated Sal s1. Two distinct cDNAs, 14.1 and 24.1, which comprise the entire parvalbumin-encoding regions, were cloned, revealing transcripts from two different parvalbumin genes. In the present study, the protein-coding regions of these cDNAs were subcloned into an Escherichia coli expression vector (pET-19b). Both proteins were expressed and the generated target proteins were localized in both soluble and insoluble fractions of the expression host. The recombinant products in the soluble fraction were purified using the His tag-purification system and analysed on Western blots with anti-salmon parvalbumin polyclonal rabbit sera and sera from patients allergic to fish. Both recombinant products (His10-14.1 and His10-24.1) reacted positively with salmon parvalbumin-specific immunoglobulin G (IgG) from rabbits, and with specific immunoglobulin E (IgE) from the sera of six fish-allergic patients. The allergenicity of His10-14.1 was confirmed using enzyme-linked immunosorbent assay (ELISA). The 14.1 cDNA of salmon parvalbumin was shown to be the dominant type represented in a muscle cDNA library.

Molecular cloning and phylogenetic analysis of the Atlantic salmon immunoglobulin D gene.

A gene homologous to the IgD heavy chain (delta) gene in channel catfish (Ictalurus punctatus) was found 0.9 kb downstream of the IgM heavy chain (mu) gene in Atlantic salmon (Salmo salar). As in catfish, the first constant mu exon is spliced into the delta transcripts. In agreement with the tetraploid ancestry of the salmonid fish family there are two highly similar delta genes in Atlantic salmon. Characterization of these genes showed that they encode seven 'unique' Ig domains, three of which are tandem duplicated, i.e. like delta1-(delta2-delta3-delta4)*-(delta2- delta3-delta4)-delta5-delta6-d elta7. Sequence analysis indicates that delta1-delta7 arose from two duplication events. Accordingly, salmon delta can be reduced to a unit of three Ig domains corresponding to the three C-terminal domains of a prototypic Ig molecule. The ancestral three-domain unit is apparently best conserved in delta1-delta5-delta6. Phylograms indicate a relationship between teleost and mammalian IgD mainly because of the similarity between the teleost delta5 and human delta2. The corresponding domain in mouse IgD has been deleted during evolution. The teleost delta1 and delta6 sequences are most similar to domains of other non-IgM isotypes, including those in cartilaginous fishes.

The putative polymerase sequence of infectious salmon anemia virus suggests a new genus within the Orthomyxoviridae.

The infectious salmon anemia virus (ISAV) is an orthomyxovirus-like virus infecting teleosts. The disease caused by this virus has had major economic consequences for the Atlantic salmon farming industry in Norway, Canada, and Scotland. In this work, we report the cloning and sequencing of an ISAV-specific cDNA comprising 2,245 bp with an open reading frame coding for a predicted protein with a calculated molecular weight of 80.5 kDa. The putative protein sequence shows the core polymerase motifs characteristic of all viral RNA-dependent RNA polymerases. Comparison of the conserved motifs with the corresponding regions of other segmented negative-stranded RNA viruses shows a closer relationship with members of the Orthomyxoviridae than with viruses in other families. The putative ISAV polymerase protein (PB1) has a length of 708 amino acids, a charge of +22 at neutral pH, and a pI of 9.9, which are consistent with the properties of the PB1 proteins of other members of the family. Calculations of the distances between the different PB1 proteins indicate that the ISAV is distantly related to the other members of the family but more closely related to the influenza viruses than to the Thogoto viruses. Based on these and previously published results, we propose that the ISAV comprises a new, fifth genus in the Orthomyxoviridae.

Molecular phylogeny of microsporidians with particular reference to species that infect the muscles of fish.

Ribosomal DNA from eight species of microsporidians infecting fish have been sequenced. Seven of these species infect the skeletal muscle of fish (Pleistophora spp.) and one species infects migratory mesenchyma cells (Glugea anomala). These sequences, in addition to other available microsporidian rDNA sequences from a broad range of host taxa, have been used in phylogenetic analysis. This analysis revealed that muscle-infecting microsporidians from fish are a polyphyletic group, indicating that characters supposed to be important in the classification of the genus Pleistophora have to be re-evaluated. One character that probably has a polyphyletic origin is the amorphous coat, which has been extensively used in the definition of this genus. Furthermore, our results showed that the insect parasitizing Pleistophora spp. are not related to the true pleistophorans parasitic in skeletal muscle of fish. Phylogenetic analysis of small subunit rDNA sequences revealed disagreements between the molecular phylogeny and classifications based upon ultrastructure. Many of the morphological characters claimed to be important in microsporidian classifications appeared to have arisen several times during evolution: for example, the diplokaryon and sporophorous vesicles.

The impact of ancestral tetraploidy on antibody heterogeneity in salmonid fishes.

The immunoglobulin heavy chain locus in teleost fish is structurally similar to that in mammals, comprising a series of variable gene segments upstream of two constant region genes coding for IgM and IgD. Atlantic salmon have been shown to possess two distinct heavy chain loci, related to the tetraploid ancestry of this fish family. The nature (and results) of the evolutionary processes following the tetraploidization event are the focus of this review. Salmonid fish did not return quickly to a diploid state, but are still in the process of re-establishing disomic inheritance. Thus, a specific locus in one species may still be endowed with four alleles, while it may have been converted to a pair of isoloci in another species. Analyses of immunoglobulin heavy chain genes in Atlantic salmon (Salmo salar) have strongly indicated that the ancestral heavy chain locus was subjected to tetrasomy throughout the radiation of the genera Oncorhynchus and Salmo, and that disomic inheritance was established in the Salmo lineage in the comparatively recent past. The introduction of disomic inheritance at these loci has resulted in two subsets of IgM and IgD heavy chains in Atlantic salmon.

Structure and organization of the immunoglobulin M heavy chain genes in Atlantic salmon, Salmo salar.

To determine the structure and organization of the germline immunoglobulin M heavy chain (IgH) genes in Atlantic salmon, Salmo salar, relevant clones from a genomic library (of one individual fish) have been characterized. Two closely related IgH constant region genes, CHA and CHB, have been sequenced completely. In addition, an allotypic variant of CHA was identified and partially sequenced. Five joining (JH) elements were found in a distance of 0.5-1.6 kb upstream of the first constant exon (CH1), in both CHA and CHB, substantiating the hypothesis that the entire gene complex is duplicated; possibly a remnant of a tetraploid event in the salmonid ancestor. An octamer motif (ATGTATTT, and its reverse complementary sequence) was found to be dispersed in the JH-CH1 region, but not elsewhere, signifying a role in these loci. Four closely related variable (VH) genes which were subcloned from three distinct lambda clones showed the classical structure of a two exon unit split by a 100 bp intron. The split-intron and a few hundred base pairs of the flanking sequences of the genes were highly similar. Three of the four genes were interrupted by stop codons and/or frame shifts, indicating a high proportion of VH-pseudogenes in this species. Based on the present results, and comparison with sequences of rainbow trout, Oncorhynchus mykiss, it is likely that the IgH loci have remained tetrasomicly inherited throughout the radiation of the genus Salmo and Oncorhynchus, and that the duplicated loci have gone into a disomic inheritance pattern in the comparatively recent past.