Ubiquitin genes in rainbow trout (Oncorhynchus mykiss)

Ubiquitin is a small protein involved in intracellular proteolysis. It is highly conserved throughout eukaryotic phyla and has been detected in such diverse species as yeast, barley, Drosophila and man. A previous study showed that chromatin of rainbow trout testis contains free ubiquitin with a sequence similar to that of other phyla. In the present study, which focused on rainbow trout but included eleven other species, it is shown that fish ubiquitin genetic organisation and expression are similar to those of other phylogenetic groups through the following set of observations: (a) Multiple loci were detected, (b) These loci encode repeats of ubiquitin, (c) Although the DNA sequences are not conserved, the encoded amino acid sequences are fully conserved, (d) The expression of ubiquitin was influenced by cell culture conditions and viral infection.     

Identification of new participants in the rainbow trout (Oncorhynchus mykiss) oocyte maturation and ovulation processes using cDNA microarrays

Background  

The hormonal control of oocyte maturation and ovulation as well as the molecular mechanisms of nuclear maturation have been thoroughly studied in fish. In contrast, the other molecular events occurring in the ovary during post-vitellogenesis have received far less attention.     

Genome organization of glutamine synthetase genes in rainbow trout (Oncorhynchus mykiss)

Unlike mammals, bony fish appear to possess multiple genes encoding glutamine synthetase (GS), the nitrogen metabolism enzyme responsible for the conversion of glutamate and ammonia into glutamine at the expense of ATP. This study reports on the development of genetic markers for each of the four isoforms identified thus far in rainbow trout (Oncorhynchus mykiss) and their genome localization by linkage mapping. We found that genes coding for GS01, GS02, GS03, and GS04 map to four different linkage groups in the trout genome, namely RT-24, RT-23, RT-08, and RT-13, respectively. Linkage groups RT-23 and RT-13 appear to represent distinct chromosomes sharing duplicated marker regions, which lends further support to the previous suggestion that GS02 and GS04 may be duplicate gene copies that evolved from a whole-genome duplication in the trout ancestor. In contrast, there is at present no further evidence that RT-24 and RT-08 share ancestrally homologous segments and additional genomic studies will be needed to clarify the evolutionary origin of genes coding for GS01 and GS03.     

Monoclonal antibodies to lymphocytes of rainbow trout (Oncorhynchus mykiss)

Monoclonal antibodies (Mabs) to lymphocytes of rainbow trout have been developed by immunisation with synthetic peptides, prepared from selected parts of the alpha- and beta-gene sequences of the T-cell receptor (TCR). Mab 1C2 (TCR beta immunisation) identified lymphocytes in blood (11%), spleen (18%) and in thymus (9%) in flow cytometry analysis (FCM). Immune complexes of lymphocytes coupled to Mab 1C2 was used for further immunisations resulting in numerous supernatants reactive with lymphocytes in FCM, of which Mabs 7A5 and 8H4 were selected for further characterisation. Mab 7A5 identified 31% of lymphocytes in blood and 9% in the spleen. Mab 8H4 labelled 61% and 85% of lymphocytes in the same organs. Mab 8H4 reacted with the majority of the lymphocytes in the thymus (98%). Mabs 1C2, 7A5 and 8H4 recognised surface markers on both Ig(-) and Ig(+) lymphocytes in peripheral blood and in spleen in double staining experiments. An increased proportion of Ig(-) lymphocytes were identified when Ig(+) lymphocytes were eliminated by immunomagnetic separation. No cross-reactivity of Mabs 1C2, 7A5 or 8H4 to anti-thrombocyte Mabs was detected. Mab 1C2 captured molecules of about 40 and also of 55-60kDa, in an immunoprecipitation assay. Mab 7A5 recognised an antigen of approximately 75-80kDa and Mab 8H4 identified proteins of about 70, 100 and 150kDa. Immunohistochemical staining by Mab 8H4 of fixed thymus, revealed a strong labelling of lymphoid cells in the outer zones of thymus. The 8H4 positive lymphoid cells surrounds circular structures, which were not labelled by Mab 8H4. These distinctly appearing structures have a similar shape as nurse cells described in mammals.     

Real-time quantitative polymerase chain reaction (QPCR) to identify Myxobolus cerebralis in rainbow trout Oncorhynchus mykiss

This study describes the development of a TaqMan real-time quantitative polymerase chain reaction (QPCR) technique using the heat-shock protein 70 (Hsp 70) and 18S ribosomal DNA (18S rDNA) sequences to identify Myxobolus cerebralis and attempt to quantify infection severity within rainbow trout fry Oncorhynchus mykiss. Rainbow trout for this study were exposed to M. cerebralis under natural river conditions and examined for infection by histology, polymerase chain reaction (PCR) and QPCR analysis at 900 Celsius temperature units (CTUs) following exposure. Detection sensitivity by QPCR was shown to be equal to traditional PCR but greater than histopathology. Primer/probe combinations developed for this study were capable of specifically detecting M. cerebralis DNA in infected fish tissue and single triactinomyxon (TAM) spores with a sensitivity of 12.5 and 6.3 pg microl(-1) of DNA for the Hsp 70 and 18S rDNA sequences, respectively. A strong relationship between QPCR and infection severity was found for the Hsp 70 probe when parasite copy number and histology scores of 0-4 were compared (R2 = 0.96, p = 0.003). However, a reduction in copy number was observed at higher histology scores for the 18S probe (scores of 4 and 5) and the Hsp 70 probe (score of 5). The results of this study demonstrate that QPCR analysis is an effective tool for detecting M. cerebralis in fish tissue and may provide a relative indication of infection severity.     

Effects of dietary astaxanthin supplementation on reproductive characteristics of rainbow trout (Oncorhynchus mykiss)

The effect of dietary astaxanthin supplementation on reproductive characteristics was investigated in five groups of female rainbow trout broodstock fed diets containing either 0.07, 12.46, 33.33, 65.06 or 92.91 mg astaxanthin kg^?1, respectively, and two groups of male rainbow trout broodstock fed diets supplemented with 0.07 and 33.33 mg astaxanthin kg^?1, respectively, for 6 months in an artificial photoperiod system until sexual maturation. The eggs from each group of female broodstock were divided into two equal batches. One batch was fertilized with homogenized sperm of four males fed diets with 0.07 mg astaxanthin kg^?1 and the other portion with sperm of four males fed diets with 33.3 mg astaxanthin kg^?1. The females produced eggs with astaxanthin concentrations ranging from 2.03 to 29.79 mg kg^?1. Dietary astaxanthin supplementation had positive effects on investigated reproductive traits. Significant differences in rate of fertilization, percentage of eyed and hatched eggs, and mortality of eyed eggs were found between treatments (P < 0.05), but no significant difference was found on percentage of mortality before hatching (P > 0.05). A significant difference (P < 0.05) in fertilization rate was found for male groups fed 0.07 and 33.3 mg astaxanthin kg^?1. The astaxanthin content in the eggs and fertilization rate, eyed‐egg percentage and percentage hatch were significantly correlated (P < 0.05). It is concluded that dietary supplements of astaxanthin are required for optimum reproduction in rainbow trout.     

Peripheral serotonin dynamics in the rainbow trout (Oncorhynchus mykiss)

Serotonin (5-hydroxytryptamine, 5-HT) occurs in a wide range of tissues throughout the body of the rainbow trout. Results reported here indicate that the main peripheral sources of serotonin are the intestinal tract and the gill epithelium (levels above 1500 ng/g). The high intestinal serotonin concentration is mostly due to serotoninergic nerve fibres, which are present at high density in the intestinal wall. Only about 2% of serotonin is associated with mucosal enterochromaffin cells. In the remaining tissues studied serotonin concentration was below 160 ng/g: the highest concentrations were seen in the anterior and posterior kidneys, followed by the liver, heart, and spleen. 5-Hydroxyindolacetic acid (5-HIAA) levels, except in plasma, were generally lower than serotonin levels, and were below our detection limits in heart, spleen and posterior kidney. Acute d-fenfluramine treatment (5 or 15 mg/kg i.p.) significantly increased 5-HIAA/5-HT ratio in the anterior intestine, pyloric caeca and plasma. Serotonin released from intestinal serotoninergic fibres in response to d-fenfluramine treatment is metabolized locally, and only a small part reaches the blood, from where it can be taken up and metabolized by other peripheral tissues, such as the liver and gill epithelium. The non-metabolized serotonin pool in the blood appears to be located extracellularly, not intracellularly as in mammals. In view of these findings, we present an overview of peripheral serotonin dynamics in rainbow trout.     

Testis transplantation in male rainbow trout (Oncorhynchus mykiss)

The objective of the present study was to establish a procedure for the transplantation of an intact testis from one male rainbow trout (Oncorhynchus mykiss) to another individual and evaluate the reproductive function of the transplanted testis at sexual maturity. Isogenic (cloned) male rainbow trout were produced by crossing a completely homozygous male (YY) with a homozygous female (XX) to eliminate any problem of tissue rejection. Transplantation was performed on four pairs of sexually immature animals (n = 8); each served both as a donor and recipient. The left testis was removed by making a ventral midline incision to expose the body cavity and gonads. The left testis was disconnected at the anterior and posterior points of attachment and transferred to the recipient fish where it was placed in position adjacent to the pyloric cecae. The right testis was left intact. After 4 wk, the fish were injected (i.p.) twice weekly for 8 or 9 wk with salmon pituitary extract (1.5 mg/kg) to induce precocious sexual maturation. A similar number of untreated fish were maintained as controls. Following this treatment, all the fish were killed, and the right (intact) and left (transplanted) testes were removed, weighed, and sampled for sperm. Although the mean weights of the left, transplanted testes were significantly (P: < 0.05) smaller than the intact testes (transplants = 1.2 g; intact = 3.9 g), transplanted testes were present in all animals, had increased in mass, and were sexually mature containing sperm. The mean fertility, as measured by the proportion of eggs completing first cleavage, of sperm derived from transplanted testes (92%) was no different from the sperm obtained from intact testes (84%). Similarly, there was no difference in the number of embryos attaining the eyed stage of development, after 18 days of incubation, that were derived from transplanted (84%) or intact testes (85%).     

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     

Polymorphisms in MHC class Ia genes and resistance to IHNV in rainbow trout (Oncorhynchus mykiss)

Infectious haematopoietic necrosis virus (IHNV) is detrimental to the farming of rainbow trout (Oncorhynchus mykiss) and other salmonids in the Northern hemisphere. The major histocompatibility complex (MHC) plays a key role in immune response in invertebrates, as evidenced by the close correlation of MHC polymorphisms with disease resistance/susceptibility. To analyse the correlation between rainbow trout resistance and susceptibility to IHNV and genetic variation in exon 2 of MHC class Ia gene, UBA, we employed two approaches, namely, polymerase chain reaction-single strand conformation polymorphism analysis and cloning/sequencing. From 102 resistant and 82 susceptible individuals, a total of 12 alleles in UBA exon 2 (GenBank: JX136662–JX136673) were identified, including 11 novel alleles. The maximum number of these alleles in a single individual was four, suggesting that UBA exon 2 most likely resides on at least two loci in the genome. Most of the variations in UBA exon 2 were located in the peptide-binding region and were determined to have been subject to positive selection during evolution. Correlation analysis revealed that Onmy-UBA*0111 and Onmy-UBA*0107 are highly associated with IHNV susceptibility (P = 0.001), whereas Onmy-UBA*0101, Onmy-UBA*0102, and Onmy-UBA*0103 are highly related to IHNV resistance (P = 0.000). In addition, the three resistant alleles were predominant in the IHNV disease-resistant population; thus, these molecular markers can be used for anti-IHNV breeding of rainbow trout.     

Tissue-specific accumulation and speciation of selenium in rainbow trout (Oncorhynchus mykiss) exposed to elevated dietary selenomethionine

The toxicity of selenium in fish is influenced by its chemical speciation and the exposure route. In the natural environment, selenium exposure to fish occurs primarily in the form of selenomethionine in diet. Thus, the main objective of this study was to examine the tissue-specific selenium burden and speciation in fish exposed to elevated dietary selenomethionine. Rainbow trout (Oncorhynchus mykiss) were treated with dietary selenomethionine (40 μg g(-1) dry mass) for 2 weeks, and at the end of the exposure different tissue samples were collected to assess the tissue-specific distribution and speciation of selenium. We used synchrotron-based X-ray absorption near edge spectroscopy (XANES) to determine the selenium speciation profile. Selenomethionine, selenocysteine and selenocystine were found to be the predominant form of selenium in all of the tissues; however their relative proportion varied across different tissues. In general, the organs primarily involved in selenium handling in fish (e.g., liver, kidney) accumulated a higher percentage of selenocystine. We also found that dietary selenomethionine exposure resulted into a marked increase in selenium burden of all major tissues in fish including the brain. Collectively, our findings provide new insights into the tissue-specific distribution and speciation of selenium in fish exposed to selenomethionine via diet.     

Dietary phosphorus-responsive genes in the intestine, pyloric ceca, and kidney of rainbow trout

Identification of phosphorus (P)-responsive genes is important in diagnosing the adequacy of dietary P intake well before clinical symptoms arise. The mRNA abundance of selected genes was determined in the intestine, pyloric ceca, and kidney of rainbow trout fed low-P (LP) or sufficient-P (SP) diet for 2, 5, and 20 days. The LP-to-SP ratio (LP/SP) of mRNA abundance was used to evaluate the difference in gene expression between LP and SP fish, and to compare the response with bone and serum P, which are conventional indicators of P status. The LP/SP of intestinal, cecal, and renal type II sodium-phosphate cotransporter (NaPi-II) mRNA abundance changed from approximately 1-2 (day 2) to approximately 1.4-4 (day 5) and to approximately 2-10 (day 20). The LP/SP of renal NaPi-II, vitamin D 24-hydroxylase, and vitamin D receptor mRNA abundance correlated inversely with serum P on day 5 but not on day 2 and day 20. In another study, differentially expressed genes between LP and SP fish were examined by subtractive hybridization, confirmed by Northern blot, and evaluated by t-test and correlation with serum and bone P concentrations. About 30 genes were identified as dietary P responsive at day 20, including intestinal meprin and cysteinesulfinic acid decarboxylase, renal S100 calcium-binding protein and mitochondrial P(i) carrier, and cecal apolipoprotein E, somatomedin B-related protein, and NaPi-II. The LP/SP of mRNA abundance of renal mitochondrial P(i) carrier and intestinal cysteinesulfinic acid decarboxylase changed significantly by day 2, and intestinal meprin by day 5. Hence, these genes and NaPi-II are among the earliest steady-response genes capable of predicting P deficiency well before the onset of clinical deficiency.     

Differences in MHC class I genes between strains of rainbow trout (Oncorhynchus mykiss)

In rainbow trout there is only one dominant classical MHC class I locus, Onmy-UBA, for which four very different allelic lineages have been described. The purpose of the present study was to determine if Onmy-UBA polymorphism could be used for strain characterisation. This was performed by lineage-specific PCR investigation of 30 fish, each of the Nikko and Donaldson strains, and by sequence analysis of 25 of the amplified DNA fragments. Two new MHC class I lineages were detected in addition to the four previously described lineages, thus six distinct lineages were observed within the fish examined (Sal-MHCIa*A-F). The distribution of lineages appeared to be strain-specific. For example, the lineage Sal-MHCIa*A was very common in the Nikko strain but could not be detected in the Donaldson strain. Analysis of MHC class I variation may help to elucidate relationships between strains and the roles of MHC alleles in disease resistance.     

Effects of dietary Ergosan on cutaneous mucosal immune response in rainbow trout (Oncorhynchus mykiss)

The effects of dietary Ergosan on the growth performance and mucosal immunity in rainbow trout skin were investigated. 60 rainbow trout (100-110 g) were randomly assigned to 2 groups in triplicates and fed one of the experimental diet formulated with 5 g kg⁻¹ Ergosan or control diet for 50 days. Results showed that on the 45th day of feeding trial, Ergosan supplementation significantly enhanced the growth performance compared to control group. Various enzyme activities, namely lysozyme, protease, alkaline phosphatase and esterase in treatment group were also enhanced on the 45th and 50th day. Skin mucus in Ergosan-fed fish showed the agglutination of erythrocytes while in control group, no visible agglutination was shown. In addition, skin mucus in treatment group showed strong antibacterial activity against Yersinia ruckeri. In conclusion, the major immune components of rainbow trout mucus that are involved in the non-specific immunity were enhanced by administration of Ergosan in 5 g kg⁻¹.     

Cloning and characterization of the rainbow trout (Oncorhynchus mykiss) type II interleukin-1 receptor cDNA.

A homologue of mammalian type II interleukin-1 receptor (IL-1RII) was isolated from a rainbow trout cDNA library by differential hybridization using a suppression subtractive hybridization generated probe enriched for sequences upregulated after immune stimulation. The trout cDNA has an ORF encoding 441 amino acids, and represents the first piscine IL-1 receptor described. The predicted amino-acid sequence has 29 and 26% identity with human and mouse IL-1RII, respectively. The trout IL-1 receptor has a domain organization similar to that of mammalian type II receptor, with a short cytoplasmic tail of 24 amino acids. These results suggest that type II receptor is also present in lower vertebrates, and therefore the duplication of an ancestral gene that generated type I and type II IL-1 receptors occurred prior to the time mammals emerged.     

Molecular cloning of an IL-8-like CXC chemokine and tissue factor in rainbow trout (Oncorhynchus mykiss) by use of suppression subtractive hybridization

Suppression subtractive hybridization (SSH) was performed to construct a Rainbow trout cDNA library enriched in sequences up-regulated in head kidney leukocytes after lipopolysaccharide (LPS) and tumour necrosis factor alpha (TNFalpha) stimulation. Random sequencing of fifty clones allowed the identification of a Rainbow trout interleukin 8 (IL-8)-related CXC chemokine, as well as the Rainbow trout tissue factor (TF) precursor. Expression of both the IL-8-like chemokine and TF is induced after LPS and TNFalpha stimulation, indicating that they are associated with inflammatory responses in fish, as has been suggested in mammals. These results confirm the potential of SSH to identify cytokines and immuno-regulatory genes in fish.     

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     

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.