Rainbow trout metallothionein

Two forms of hepatic metallothionein were isolated and purified from rainbow trout injected intraperitoneally with cadmium chloride. Both forms showed similarities with mammalian metallothioneins, had a high cystein content (30 mol%), and were void of aromatic amino acids and histidine. The molecular weight was estimated to be about 6000 dalton for the apothioneins, and the thiol groups of the cysteine residues complexed with the heavy metals (Cd, Cu, Zn) in a SH/Me⁺⁺ ratio of about 2.4. The amount of copper in metallothionein from rainbow trout was very high, greater than the amount of cadmium and zinc after injections of 3 mg cadmium/kg body weight. The total metal content of cadmium, copper and zinc in metallothionein 1 and 2 were about 7 and 8 atoms per molecule respectively.     

Glutathione peroxidase and oxidative hemolysis in trout red blood cells

Red blood cells from the trout Salmo irideus contain several hemoglobin components that are prone to oxidation with production of oxygen radicals. The rate of hemolysis has been correlated to the extent of methemoglobin formation. A difference in the rate of hemolysis between red blood cells saturated with either CO or O2 was evident only when diminished glutathione peroxidase activity was observed. These results confirm the important role of this enzyme in providing protection against or repair of oxidative damage to the red cell membrane.     

Rainbow trout display a developmental shift in red muscle kinetics, swimming kinematics and myosin heavy chain isoform

Both activation and relaxation times of rainbow trout Oncorhynchus mykiss red muscle were shorter in parr than in older juveniles. Furthermore, parr red muscle had a faster maximum shortening velocity than that of older fish, as estimated with the force-clamp technique. Parr swam with higher tailbeat frequencies and lower tailbeat amplitude than did older fish across a range of length-specific steady swimming speeds. The developmental shift in contraction kinetics of red muscle and steady swimming kinematics was associated with a reduction from two or three myosin heavy chain isoforms in parr to one in older juveniles. This transition provides a mechanism to explain the variations in muscle contraction kinetics and swimming performance.     

Volume regulation by red blood cells from brown trout

Regulatory volume decrease, following physical swelling of red cells from brown trout Salmo trutta , was almost complete in oxygenated cells but much less in deoxygenated cells. There was a small, insignificant regulatory volume increase, following physical shrinkage. Amiloride had no effect on this response, indicating that hypertonic shrinkage did not activate the Na⁺/H⁺ exchanger. However, cell volume was increased markedly in shrunken cells by addition of noradrenaline, with deoxygenated cells showing complete recovery. These data show that the previously reported differences in volume regulation between the red cells of brown trout and rainbow trout Oncorhynchus mykiss are not present and that both species appear to have lost volume sensitivity of the Na⁺/H⁺ exchanger.     

Rainbow trout has two genes for growth hormone

We report the primary structures of two mRNA species (GH1 and GH2), each predicted from the cloned cDNA and genomic gene sequences, that encode growth hormone in rainbow trout (Salmo gairdneri). Both GH1 and GH2 mRNA contain open reading frames comprising 630 nucleotides and encode 210 amino acid residues, of which 11 are variant. The translated regions of mRNA are flanked by a short 5'-untranslated sequence, which is highly conserved, and a relatively long 3'-untranslated sequence, which is highly divergent. The differences at the 3'-untranslated regions suggest that the GH1 and GH2 mRNA originate from different loci. RNA blot analysis of trout pituitary RNA using an oligonucleotide probe specific for the GH2 sequence indicates that the cloned gene is expressed. The GH1 and GH2 mRNA likely are transcribed from two distinct loci, which were duplicated during tetraploidization of the salmonid genome between 50 and 100 million years ago.     

Rainbow trout neutrophils are responsible for non-specific cytotoxicity

This is the first report that rainbow trout (Oncorhynchus mykiss) neutrophils are responsible for non-specific cytotoxicity. A monoclonal antibody (mab) for rainbow trout leucocytes was produced. Using this mab (TTL-5E9), neutrophils (5E9-positive cells) were isolated from the pronephros by a panning technique. The isolated neutrophils showed high viability (approximately 95%) and purity (92-95%), and were functional in cytotoxic activity assays. The neutrophils demonstrated significantly higher cytotoxic activities against YAC-1 target cells than the other cells (5E9-negative cells, predominantly lymphocytes). The number of neutrophils contaminating the 5E9-negative fraction and their non-specific cytotoxicities were positively correlated. These findings demonstrate that rainbow trout neutrophils possess non-specific cytotoxic activities.     

Rainbow trout leukocyte culture: A simplified method

Summary The addition of 10% fetal bovine serum to Leibovitz’s L-15 culture medium resulted in marked growth of peripheral blood leukocytes from rainbow trout,Salmo gairdneri. Culture medium without serum or with 20% homologous serum did not induce substantial growth. In contrast to what has been reported by others, oxygenation of the culture medium was found not to be required for excellent cell growth.     

Rainbow trout (Oncorhynchus mykiss) shift the age composition of circulating red blood cells towards a younger cohort when exposed to thermal stress

Freshwater fish, such as the rainbow trout, are commonly exposed to temperature fluctuations in their aquatic environment. Exposure to increased temperatures places fish under respiratory stress and increases the likelihood of protein misfolding and degradation that could eventually lead to cell death. Previously, we showed that genes associated with the cellular stress response, apoptosis and hematopoiesis are upregulated in the red blood cells (RBCs) of rainbow trout post-thermal stress, leading to the hypothesis that a tightly regulated interaction between cell repair and cell death is occurring after heat stress. To test this hypothesis, we tracked changes in age class composition and markers of apoptosis in circulating RBCs within individual trout during exposure to and recovery from acute thermal stress. RBCs did not show any indication of apoptosis or necrosis following acute heat stress; however, we observed significant increases in numbers of early, juvenile and dividing RBCs. We also observed a shift in the composition of the circulating RBCs towards a younger cohort following heat shock through release of stored cells from the spleen and an increase in the maturation rate of early RBCs. These results suggest that the genes activated by increased temperature provided sufficient protection against thermal stress in the RBC, subsequently preventing the triggering of the cell death cascade.     

Rainbow trout leukocyte culture: a simplified method.

The addition of 10% fetal bovine serum to Leibovitz's L-15 culture medium resulted in marked growth of peripheral blood leukocytes from rainbow trout, Salmo gairdneri. Culture medium without serum or with 20% homologous serum did not induce substantial growth. In contrast to what has been reported by others, oxygenation of the culture medium was found not to be required for excellent cell growth.     

Rainbow trout sleeping disease virus is an atypical alphavirus

Sleeping disease (SD) is currently a matter of concern for salmonid fish farmers in most parts of the world. A viral etiology of SD has recently been suspected, since virus-like particles have been observed in infected rainbow trout cells. In salmonid-derived cell lines, the maximal rate of virus production was observed at 10 degrees C, while little virus was produced at 14 degrees C. Through biochemical, physicochemical, and morphological studies, SD virus (SDV) was shown to be an enveloped virus of roughly 60 nm in diameter. The genome consists of 12 kb of RNA, with the appearance of a 26S subgenomic RNA during the time course of SDV replication. The screening of a random-primed cDNA library constructed from the genomic RNA of semipurified virions facilitated the identification of a specific SDV cDNA clone having an open reading frame related to the alphavirus E2 glycoproteins. To extend the comparison between SDV structural proteins and the alphavirus protein counterparts, the nucleotide sequence of the total 4.1-kb subgenomic RNA has been determined. The 26S RNA encodes a 1,324-amino-acid polyprotein exhibiting typical alphavirus structural protein organization. SDV structural proteins showed several remarkable features compared to other alphaviruses: (i) unusually large individual proteins, (ii) very low homology (ranging from 30 to 34%) (iii) an unglycosylated E3 protein, and (iv) and E1 fusion domain sharing mutations implicated in the pH threshold. Although phylogenetically related to the Semliki Forest virus group of alphaviruses, SDV should be considered an atypical member, able to naturally replicate in lower vertebrates.     

Rainbow trout (Onchorhynchus mykiss) hepatic glucose transporter

We report identification of a rainbow trout hepatic glucose transporter sharing 58% and 52% amino acid identity with avian and mammalian GLUT2 sequences, respectively. The functionality of OnmyGLUT2 was assessed by expression in rainbow trout embryos. We also measured the transport of hexose in isolated rainbow trout hepatocytes. Inhibition of 3-O-methylglucose uptake by cytochalasin B, phloretin and 2-deoxy-D-glucose suggested the existence of a functional facilitative transporter in these cells. Expression of OnmyGLUT2 was found in the liver, kidney and intestine.     

Rainbow trout (Oncorhynchus mykiss) neuropeptide Y.

Neuropeptide Y (NPY) has been isolated from brain extracts of the rainbow trout (Oncorhynchus mykiss) and subjected to structural analyses. Plasma desorption mass spectroscopy estimated the molecular mass of the purified peptide as 4303.9 Da. Automated Edman degradation unequivocally established the sequence of a 36 amino acid residue peptide as: Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Pro-Glu-Glu-Leu-Ala-Lys-Tyr-Tyr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr. The molecular mass calculated from this sequence (4304 Da) is consistent with that obtained by mass spectroscopy. The presence of a C-terminal amide was established by radioimmunoassay. Rainbow trout NPY is identical in primary structure to coho salmon (Oncorhynchus kisutch) pancreatic polypeptide (PP). These data may indicate that, in this group of salmonid fishes, a single member of the NPY/PP peptide family is expressed in both neurons and peripheral endocrine cells.     

Glycine uptake by trout (Salmo trutta) red blood cells

The present study demonstrates the presence of different amino acid carriers in the membrane of trout red cells. Most glycine is taken up through the Na⁺-dependent system ASC, although the nearly specific Gly system is also active. Besides these carriers, glycine is taken up by means of Na⁺-independent transporters, system l being the most important. A system asc of high affinity and low capacity has been found, and band 3 is unable to transport glycine under physiological conditions. These results suggest that although all these carriers are already present in primitive vertebrates, several differences exist in their properties with respect to those found in mammalian cells.We would like to express our sincere thanks to Mr. Antonino Clemente (Piscifactoria de Bagà, Medi Natural, Generalitat de Catalunya) for his help and logistical assistance and to Mr. Robin Rycroft for his editorial help.This work was supported by a grant of Comisió Interdepartamental de Recerca i Technologia (AR90-3.3394). M.A.G. is recipient of a fellowship from the Generalitat de Catalunya.     

Physiological salines and the mechanical properties of trout red blood cells

Suspensions of rainbow trout erythrocytes in different physiological salines were compared with respect to their haematological and filtration properties.
A method is described for the suspension of erythrocytes in Cortland saline which has proved suitable for studies of their mechanical properties over periods of several hours.
Significant differences were found between whole blood samples taken during cannulation and after several days recovery, particularly mean cell volume, frequency distribution of red cell volumes and the pore passage time through nucleopore filters. These differences were also found using red cell suspensions of the same bloods. The pore passage time of whole blood sampled during cannulation or its suspensions is less than that of recovery blood although its mean cell volume is greater.     

Glucose metabolism by trout (Salmo trutta) red blood cells

Summary Glucose metabolism has been studied in Salmo trutta red blood cells. From non-metabolizable analogue (3-O-methyl glucose and l-glucose) uptake experiments it is concluded that there is no counterpart to the membrane transport system for glucose found in mammalian red blood cells. Once within the cells, glucose is directed to CO₂ and lactate formation through both the Embden-Meyerhoff and hexose monophosphate shunts; lactate appears as the most important endproduct of glucose metabolism in these cells. From experiments under anaerobic conditions, and in the presence of an inhibitor of pyruvate transfer to mitochondria, most of the CO₂ formed appears to derive from the hexose monophosphate pathway. Appreciable O₂ consumption has been detected, but there is no clear relationship between this and substrate metabolism. Key enzymes of glucose metabolism hexokinase, fructose-6-phosphate kinase and, probably, pyruvate kinase are out of equilibrium, confirming their regulatory activity in Salmo trutta red blood cells. The presence of isoproterenol, a catecholamine analogue, induces important changes in glucose metabolism under both aerobic and anaerobic conditions, and increases the production of both CO₂ and lactate. From the data presented, glucose appears to be the major fuel for Salmo trutta red blood cells, showing a slightly different pattern of glucose metabolism from rainbow trout red blood cells.Abbreviations EM Embden-Meyerhoff pathway - G6D glucose-6-phosphate dehydrogenase - GOT glutamate oxalacetate transaminase - GPI glucose phosphate isomerase - HK hexokinase - HMS hexose monophosphate shunt - IP isoproterenol - LDH lactate dehydrogenase - MCB modified Cortland buffer - OMG 3-O-methyl glucose - PFK fructose-6-phosphate kinase - PK pyruvate kinase - RBC red blood cells - TAC tricarboxylic acid cycle     

Regulation of Cl-dependent K transport by oxy-deoxyhemoglobin transitions in trout red cells.

The oxygenation of trout red cells opens a Cl-dependent K pathway inhibited by furosemide, and by inhibitors of the erythrocyte anion exchanger such as DIDS and niflumic acid. The trigger is the deoxy-oxy conformational change of hemoglobin. The binding of carbon monoxide to heme, which induces a similar conformational change, mimics the effect of oxygen. The possible mechanisms enabling molecular oxygen to control the transport protein are discussed. This oxygenation-activated K transport appears to play a regulatory role in the control of the extracellular K concentration.     

Inhibition of adrenergic proton extrusion in rainbow trout red cells by nitrite-induced methaemoglobinaemia

Summary The effects of nitrite-induced methaemoglobinaemia on adrenergic proton extrusion from rainbow trout red blood cells were studied using the pH-stat method. In control conditions adrenergic proton extrusion was completely inhibited by amiloride and was greater in deoxygenated than in oxygenated erythrocytes. Nitrite-induced methaemoglobinaemia was associated with a pronounced reduction in the catecholamine-stimulated proton efflux from both deoxygenated and oxygenated erythrocytes. In deoxygenated erythrocytes the initial proton efflux upon catecholamine stimulation decreased by 60–70%, while the percentage of methaemoglobin in the red cells increased from the control level of 1–3% to 20%. In oxygenated erythrocytes the decrease was 30% at the same methaemoglobin percentage range. It is suggested that the pronounced influence of nitriteinduced methaemoglobinaemia on adrenergic proton efflux results from an inhibition of the red cell sodium/proton exchanger by the R-like haemoglobin conformations.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - RBC red blood cell     

Rainbow trout offspring with different resistance to viral haemorrhagic septicaemia

To study immunological and immunogenetical parameters related to resistance against viral haemorrhagic septicaemia (VHS), attempts to make gynogenetic strains of rainbow trout selected for high and low resistance to VHS were initiated in 1988. The first gynogenetic generation of inbreeding resulted in the more resistant offspring E8 and the low resistance offspring K3; the K3 offspring having the same high mortality as the susceptible reference strain of outbred trout in infection trials. A second gynogenetic generation derived from the E8 strain resulted in some low resistance offspring, and two gynogenetic families in which all, or nearly all, fish survived challenge with VHS virus. In this study, an attempt to associate the distribution of different MHC class II genotypes with low and high resistance gynogenetic offspring was performed. Two different MHC haplotypes could be distinguished, and in both low and high resistance families all three genotypes were found, which could be explained by the fact that the mother fish carried the heterozygous genotype. Although no significant differences in MHC II genotypes were found between the high and low resistance offspring, a significantly different distribution of haplotypes in the low resistance offspring was observed, that could not be explained by a one- or two-locus model.