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Evaluation of glutamic acid and glycine as sources of nonessential amino acids for lake trout (Salvelinus namaycush) and rainbow trout (Salmo gairdnerii).

A semi-purified test diet which contained either glutamic acid or glycine as the major source of nonessential amino acids (NEAA) was fed to lake and rainbow trout. Trout fed the diet containing glutamic acid consistently showed better growth and feed conversion efficiencies than those fed the diets containing glycine. The data indicate that these trout utilize glutamic acid more efficiently than glycine when no other major sources of NEAA are present.

Animals↗

Trout sperm motility. The transient movement of trout sperm is related to changes in the concentration of ATP following the activation of the flagellar movement.

For freshwater fish the motile period of sperm is extremely brief, even after a dilution in isotonic media. This result is in contrast to most other animals (ranging from invertebrates to mammals), in which sperm are generally motile for at least several hours. We have analyzed the reasons for the brevity of this movement by studying the relationships between the metabolism of trout sperm and the activation of their motility upon dilution. Sperm motility was not initiated when the dilution medium contained an elevated concentration of potassium (20-40 mM), but dilution in an isotonic solution of sodium chloride triggered an immediate activation of motility, and sperm swam vigorously. Motility of sperm decreased rapidly and 15 s after dilution sperm were moving slowly in small circles. Sperm became abruptly immotile at 20-30 s and flagella straightened. When millimolar concentrations of Ca2+ were also present in the dilution medium, movement did not stop abruptly, flagella kept beating and stopped only after 1-2 min. When sperm remained immotile they retained a high concentration of ATP. The activation of motility induced a rapid decrease of ATP. In the absence of calcium, and after the cessation of motility, ATP increased slowly back to its original concentration. In the presence of millimolar concentrations of calcium the concentration of ATP decreased to a very low level and remained low thereafter. The progressive decrease of the flagellar beat frequency, that had been observed during the period of trout sperm movement, might be related to the rapid exhaustion of intraflagellar ATP. Motility could be reinduced in sperm that had recovered high concentrations of ATP, demonstrating the functional integrity of the motile apparatus even after flagellar arrest. In conclusion we suggest that the maximum duration of trout sperm motility, at most 2 min (as a consequence of a depletion of ATP during the movement), is due to a low mitochondrial oxidative phosphorylation capacity.

Adenosine Triphosphate↗

The occurrence of Clostridium botulinum type E in Finnish trout farms and the prevention of toxin formation in fresh-salted vacuum-packed trout fillets.

The occurrence of C. botulinum on two Finnish rainbow trout farms were studied. C. botulinum type E toxin was detected from samples of fish intestines in 10% and 4% of the samples and in 0% and 100% of dam bottom sediments, respectively. The toxin formation of inoculated C. botulinum type E in three different brands of commercial fresh-salted vacuum-packed trout fillets was also investigated. In the brand with a salt concentration of 2.7% (a w = 0.96) and no nitrate, the toxin was formed in two weeks at 10 and 20 degrees C. If the pH of the product was lowed to 4.90 and NaCl concentration increased to 5.67% (a w = 0.94%) no toxin was formed even without the use of nitrate. By adding 0.1% of sodium nitrate to the curing solution (0.046% nitrate in the product) the toxin formation was brought to an end. According to the results of the study C. botulinum type E presents a potential health risk for man as well as for fish also in Finland. Trout products must be manufactured and stored in such a way that the possibility of toxin formation is eliminated.

Animals↗

The large high mobility group proteins of rainbow trout are localized predominantly in the nucleus and nucleoli of a cultured trout cell line.

On the basis of immunofluorescence and biochemical studies, it has been suggested that the concentration of nonhistone chromosomal protein high mobility group 1 may be higher in the cytoplasm than in the nucleus of mammalian cells (Bustin, M., and Neihart, N. K. (1979) Cell 16, 181-189). In view of the possible implications of this finding, we have examined the in situ location of trout proteins HMG-T1 and HMG-T2, which are analogous to the mammalian proteins HMG-1 and HMG-2. Antibodies prepared against purified HMG-T2 were shown to react only with HMG-T1 and HMG-T2, but not with any other chromosomal proteins from trout. This has been established using a modified immunoautoradiographic techique involving CNBr-activated paper transfers of proteins separated on regular sodium dodecyl sulfate-polyacrylamide gels. Using the indirect immunofluorescence technique to examine the subcellular location of HMG-T (T1 and T2) proteins in a cultured cell line of rainbow trout, we find that these proteins are located primarily in the nucleus of these cells. The fluorescence in the nucleolar regions is even more intense than in the nonnucleolar regions. The cytoplasmic regions show only a weak fluorescence which may be due to low levels of HMG-T proteins in the cytoplasm, since preincubation of anti-HMG-T2 with purified HMG-T2 abolishes the nuclear as well as cytoplasmic fluorescence.

Animals↗

Differentiation of soluble proteins in cataracts caused by deficiencies of methionine, riboflavin or zinc in diets fed to Atlantic salmon, Salmo salar, rainbow trout, Salmo gairdneri, and lake trout, Salvelinus namaycush.

Water soluble extracts from cataractous and normal salmonid lenses were subjected to thin-layer isoelectric focusing. The cataracts resulted from deficiencies of specific nutrients in diets fed to three species of salmonids as follows: (1) rainbow trout (Salmo gairdneri) fed a diet deficient in riboflavin; (2) Atlantic salmon (Salmo salar) fed a diet deficient in methionine; and (3) rainbow trout and lake trout (Salvelinus namaycush) fed a diet deficient in zinc. In all tests, the protein pattern of cataractous lenses differed from that of normal lenses. Each cataract caused by the different dietary deficiencies had a specific pattern of soluble lens proteins. Even though the general patterns of the soluble lens proteins for all three salmonids though the general patterns of the soluble lens proteins for all three salmonids studied were similar, a specific pattern was unique for each of the species.

Animals↗

Characterization of rainbow trout terminal deoxynucleotidyl transferase structure and expression. TdT and RAG1 co-expression define the trout primary lymphoid tissues.

One component of antigen receptor diversity shared by all gnathostomes characterized to date is mediated by a unique DNA polymerase, terminal deoxynucleotidyl transferase (TdT), which generates significant functional diversity during immunoglobulin and T-cell receptor rearrangement. To gain further insight into the evolutionary origin(s) of this unique enzyme and the immune system, a thymic cDNA clone encoding TdT was isolated from rainbow trout. The 2.3 kilobase (kb) full-length clone contained an open reading frame of 1 506 base pairs with a deduced protein product of Mr 57 000. Sequence comparisons demonstrate that TdT has been highly conserved in both sequence (>70% aa similarity, >50 aa identity) and overall structure during the course of vertebrate evolution. An amino acid alignment of all known TdT sequences (chicken, Xenopus, mouse, human, cattle, and trout) reveals that some, but not all, structural motifs believed to be critical for TdT activity have been conserved. The TdT alignment, in conjuction with the recently solved crystal structure for rat beta-polymerase, supports the hypothesis that both may have evolved from a common ancestral repair gene. In addition, four PKC phosphorylation sites are conserved, and hence may be involved in TdT regulation. Because TdT contributes to the generation of junctional diversity in antigen receptors of immature lymphocytes, its expression serves as a developmental marker for the sites of teleost lymphopoiesis. Northern blot (2.3 kb message) analysis shows that TdT mRNA is highly expressed within the thymus and to a lesser extent in the pronephros. In addition, reverse transcriptase-polymerase chain reaction analysis detected transcipts of both RAG1 and TdT in the thymus, pronephros, mesonephros, spleen, and intestine, but not within muscle, liver, or brain. Finally, TdT cDNA was amplified from embryos at 20 days post-fertilization (pf), which correlates with the appearence of the thymus and pronephros anlage during trout ontogeny.

Amino Acid Sequence↗

A stromal cell line from rainbow trout spleen, RTS34ST, that supports the growth of rainbow trout macrophages and produces conditioned medium with mitogenic effects on leukocytes.

A rainbow trout spleen cell line, RTS34, was developed from a long-term hemopoietic culture. This cell line consisted of a mixed stromal cell layer with an associated cell population of macrophage-like cells that formed proliferative foci and released nonadherent progeny cells into the culture medium. A stromal cell line, RTS34st, was isolated from the RTS34 cell line. RTS34st cultures contained cells with fibroblast-like and epithelial-like morphologies and showed enhanced [3H]thymidine incorporation in response to either FBS or rainbow trout serum. The combination of FBS and trout serum was synergistic. Conditioned medium from RTS34st stimulated thymidine incorporation by peripheral blood and head kidney leukocytes, but not by leukocytes from the spleen. In addition, RTS34st provided a hemopoietic inductive microenvironment for immature precursor cells, selectively supporting the growth of macrophage-like cells. Therefore, RTS34st appears useful for studying the different roles of the stroma in regulating hemopoiesis in fish.

Animals↗

Respiratory effects of hexachlorocyclopentadiene on intact rainbow trout (Salmo gairdneri) and on oxidative phosphorylation of isolated trout heart mitochondria.

Acclimated normal rainbow trout were exposed to 130 ppb hexachlorocyclopentadiene (HEX) in a flow-through well water circuit which was designed to permit measurements of oxygen consumption by the fish. Compared to preHEX values, HEX increased oxygen consumption rates by 186 +/- 24% (means +/- SEM), with maximum oxygen consumption rates being reached in approximately 84 min after HEX exposure. Oxygen consumption subsequently decreased, and all HEX-exposed fish died within 6.5 hr of exposure. Fish exposed to HEX-free vehicle (acetone) showed no changes of oxygen consumption. When added to normal isolated trout heart mitochondria, HEX appeared to uncouple oxidative phosphorylation, with calculated respiratory control ratios being decreased 50% from control values at a HEX concentration of 0.41 microM. We postulate that one important mechanism of HEX intoxication in the intact animal may be due to increased oxygen consumption and impaired oxidative ATP synthesis due to the mitochondrial uncoupling action of the toxicant.

Animals↗

Opsonic effect of rainbow trout (Salmo gairdneri) antibody on phagocytosis of Yersinia ruckeri by trout leukocytes.

Partly purified peripheral blood leukocytes from normal rainbow trout (Salmo gairdneri) were used to study the influence of specific antibody on phagocytic uptake and intracellular killing of Yersinia ruckeri, a bacterial pathogen of trout. Specific antibody exerted a significant opsonic effect on the rate of phagocytic ingestion of the bacteria but did not affect the rate of intracellular killing. The results are discussed with reference to the current understanding of fish antibody function and phagocytosis by fish leukocytes.

Animals↗

Trout ovulatory proteins are partially responsible for the anti-proteolytic activity found in trout coelomic fluid.

After ovulation in salmonids, the eggs are held in the peritoneal cavity and bathed in coelomic fluid. Using a chromogenic peptide substrate, the anti-protease activity of brook trout coelomic fluid was measured. Trypsin, chymotrypsin, and pancreatic elastase activities were significantly inhibited by coelomic fluid containing 5.0, 10.0, and 25.0 microgram of total protein, respectively. Using subtractive cDNA cloning, we have previously characterized a set of ovarian proteins called TOPs (trout ovulatory proteins) that are secreted into the coelomic fluid after ovulation. TOPs are most homologous to mammalian antileukoprotease, a heat- and acid-stable serine protease inhibitor. On the basis of this homology, we hypothesized that the anti-trypsin activity observed in the coelomic fluid was related to the presence of TOPs. In the present study, this hypothesis was supported by the acid- and heat-stability of the anti-trypsin activity present in coelomic fluid. Coelomic fluid could be heated to 50 degrees C or treated at a pH less than 5.2 without a significant decrease in the inhibitory activity. Further, coelomic fluid from which TOPs were immunoprecipitated had significantly less anti-trypsin activity than nonimmunoprecipitated controls. We propose that TOP proteins are uniquely produced by the ovary and secreted into the coelomic fluid to act as protease inhibitors following ovulation.

Animals↗

Quantitative evaluation of macrophage aggregates in brook trout Salvelinus fontinalis and rainbow trout Oncorhynchus mykiss.

Macrophage aggregates (MAs) occur in various organs of fishes, especially the kidney, liver and spleen, and contain melanin, ceroid/lipofuscin and hemosiderin pigments. They have been used as indicators of a number of natural and anthropogenic stressors. Macrophage aggregates occur in salmonids but are poorly organized, irregularly shaped, and are generally smaller than those in derived teleosts. These features complicate quantification, and thus these fishes have seldom been used in studies correlating MAs with environmental stressors. To alleviate these complications, we developed color filtering algorithms for use with the software package ImagePro Plus (Media Cybernetics) that select and quantify pigmented area (i.e. colors ranging from gold to brown to black) in tissue sections. Image analysis results compared well with subjective scoring when tested on brook trout Salvelinus fontinalis and rainbow trout Oncorhynchus mykiss captured from high-elevation lakes or hatcheries. Macrophage aggregate pigments correlated positively with age and negatively with condition factor. Within individual fish, pigmentation correlated positively among organs, suggesting that the kidney, liver or spleen are suitable indicator organs. In age-matched fishes, MA pigments were not different between hatcheries and lakes in the organs examined. Between lakes, differences in pigments were observed in the kidney and spleen, but were not explained by age, condition factor, sex or maturation state. Our results indicate that quantification of the area occupied by MA pigments is an efficient and accurate means of evaluating MAs in salmonid organs and that organ pigmentation correlates with age and condition factor, as seen in studies with more derived fishes.

Age Factors↗

The ecology of endoparasitic helminth infections of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) in Scotland.

Two hundred and forty brown trout (Salmo trutta) and 49 rainbow trout (Oncorhynchus mykiss), obtained from 21 locations in Central Scotland between October 1990 and August 1993, were examined for endoparasitic helminth infections. Crepidostomum farionis (Digenea) was the most widely distributed helminth species, followed by Eubothrium crassum (Cestoda), Diphyllobothrium dendriticum and D. ditremum (Cestoda), Neoechinorhynchus rutili (Acanthocephala), Echinorhynchus truttae (Acanthocephala), Eustrongylides sp. (Nematoda), Capillaria salvelini (Nematoda), Cyathocephalus truncatus (Cestoda), Raphidascaris acus (Nematoda) and Cystidicola farionis (Nematoda), in that order. The prevalences and intensities of each helminth infection were recorded. No evidence was found to indicate that even fish with the highest worm burdens (e.g. 339 plerocercoids of Diphyllobothrium spp.) were experiencing any obvious morbidity. An analysis of pairs of associations between species of helminths revealed a significantly positive association between N. rutili and C. farionis (P < 0.01). The results are discussed in terms of patterns in helminth communities in freshwater fish.

Acanthocephala↗

Seasonal changes in osmoregulation, cortisol, and cortisol receptor activity in the gills of parr/smolt of steelhead trout and steelhead-rainbow trout hybrids, Oncorhynchus mykiss.

Steelhead and hybrid trout (rainbow trout male, steelhead female) presmolts and smolts, Oncorhynchus mykiss, exposed to 30/1000 seawater (SW) for 24 hr manifested progressively lower plasma sodium values from December to May. The increase in plasma sodium after 24 hr in SW diminished significantly after March, with the hybrids (which were heavier in weight than the steelheads) reaching their peak osmoregulatory ability in the middle of April, and the steelheads in late May. In fresh water there was little difference in plasma osmotic concentration but steelhead plasma chloride fell significantly from March 30 levels to May 23 levels, whereas hybrid plasma chloride levels did not change during this time. In steelheads but not hybrids, plasma cortisol correlated negatively with plasma sodium of fish exposed 24 hr in SW. However, in both steelheads and hybrids, plasma sodium values 24 hr after SW exposure had an inverse correlation with cytosol cortisol from the gills. Again, in both steelheads and hybrids, plasma values of fish exposed 24 hr in SW correlated positively with cytosol receptor activity. In hybrids, the nuclear extract receptor activity, Nmax, increased significantly until late March and then dropped significantly thereafter. In steelheads, there were no significant differences but Nmax followed the same trends as occurred in hybrids. Thus seasonal changes in cortisol and cortisol receptors in steelhead occur during smoltification and preparation for marine life and suggest an important role for cortisol. In hybrids there appears to be a reduction in seasonality.

Animals↗

Fin erosion on rainbow trout on commercial trout farms in the United Kingdom.

Fish weighing less than 30 g and more than 100 g were sampled from 38 rearing units on 10 commercial farms growing rainbow trout for the table market. A fin index was calculated for each of the eight rayed fins on 949 trout by dividing their length by the standard length of the fish. There was a large range in the indices of all eight fins. The fin indices of the small and large fish were compared, controlling for farm effect. With the exception of the dorsal fin, all the indices were larger for the small fish than for the large fish, but the magnitude of the difference was greater for some fins than others. In comparison with the fins of wild fish, the pectoral and dorsal fins appeared to be most eroded and the damage to these fins was evident even in the small fish. The erosion of the caudal, anal and ventral (or pelvic) fins was more prominent in the larger fish. Variations in the fin indices of the caudal, anal and ventral fins suggested that there was little variation between rearing units on the same farm, but that there was significant variation between individual fish in the same rearing units, and between fish on different farms.

Animals↗

Two complementary bioassays for screening the estrogenic potency of xenobiotics: recombinant yeast for trout estrogen receptor and trout hepatocyte cultures.

A relation between the chemical structure of a xenobiotic and its steroidal action has not yet been clearly established. Thus, it is not possible to define the estrogenic potency of different xenobiotics. An assessment may be accomplished by the use of different bioassays. We have previously developed a yeast system highly and stably expressing rainbow trout estrogen receptor (rtER) in order to analyze the biological activity of the receptor. The recombinant yeast system appears to be a reliable, rapid and sensitive bioassay for the screening and determination of the direct interaction between ER and estrogenic compounds. This system was used in parallel with a more elaborate biological system, trout hepatocyte aggregate cultures, to examine the estrogenic potency of a wide spectrum of chemicals commonly found in the environment. In hepatocyte cultures, the vitellogenin gene whose expression is principally dependent upon estradiol was used as a biomarker. Moreover, competitive binding assays were performed to determine direct interaction between rtER and xenobiotics. In our study, 50% of the 49 chemical compounds tested exhibited estrogenic activity in the two bioassays: the herbicide diclofop-methyl; the fungicides biphenyl, dodemorph, and triadimefon; the insecticides lindane, methyl parathion, chlordecone, dieldrin, and endosulfan; polychlorinated biphenyl mixtures; the plasticizers or detergents alkylphenols and phthalates; and phytoestrogens. To investigate further biphenyl estrogenic activity, its principal metabolites were also tested in both bioassays. Among these estrogenic compounds, 70% were able to activate rtER in yeast and hepatocytes with variable induction levels according to the system. Nevertheless, 30% of these estrogenic compounds exhibited estrogenic activity in only one of the bioassays, suggesting the implication of metabolites or different pathways in the activation of gene transcription. This paper shows that it is important to combine in vivo bioassays with in vitro approaches to elucidate the mechanism of xenoestrogen actions.

Animals↗

Polycyclic aromatic hydrocarbons as inducers of cytochrome P4501A enzyme activity in the rainbow trout liver cell line, RTL-W1, and in primary cultures of rainbow trout hepatocytes.

In order to investigate cell-specific differences in the response of in vitro models to environmental toxicants, we compared the capacity of nine polycyclic aromatic hydrocarbons (PAHs) to induce cytochrome P4501A (CYPIA) in primary rainbow trout (Oncorhynchus mykiss) hepatocytes and a rainbow trout liver cell line, RTL-W1. Induction of CYPIA was estimated from the catalytic activity of 7-ethoxyresorufin-O-deethylase (EROD) and compared by median effective concentration (EC50) values, induction spans, and benzo[a]pyrene induction equivalency factors for inducing PAHs. The influence of culture conditions was investigated with respect to the presence or absence of serum and varying exposure times. Both in vitro systems lead to an identical classification of the PAHs in noninducing (anthracene, fluoranthene, phenanthrene, and pyrene) and inducing compounds with a similar ranking of inducing PAHs. Mean EC50 values in RTL-W1 cells were, respectively, 343 and 266 nM for benzo[a]anthracene, 57 and 92 nM for BaP, 134 and 283 nM for benzo[b]fluoranthene, 455 and 270 nM for chrysene, and 98 and 116 nM for 3-methylcholanthrene. Compared to primary hepatocytes, the RTL-W1 cell line was more sensitive in its EROD response to the presence or absence of serum and to the increase in exposure time, which led to higher EC50 values.

Animals↗

Injection of the mouse MT-1 gene into rainbow trout eggs and assay of trout fry for resistance to cadmium and zinc toxicity.

The mouse MT-1 gene, cloned into the plasmid pBR 322, has been injected into fertilised eggs of the rainbow trout, Salmo gairdneri. About 10(6) copies of the construct, either circular or linearized, have been injected into 1 cell stage eggs within 4 hours of fertilisation, and fry hatched from such eggs are being assayed for evidence of integration of the injected sequence. In addition, with a view to the development of an assay which can be used to monitor for expression of the injected gene, uninjected fry have been exposed to various concentrations of cadmium chloride or zinc sulphate, with or without prior exposure to inducing doses of these compounds. Whereas a distinct protection is evident following induction with zinc, no such protection has been detected with cadmium induction.

Animals↗

Details of retropositional genome dynamics that provide a rationale for a generic division: the distinct branching of all the pacific salmon and trout (Oncorhynchus) from the Atlantic salmon and trout (Salmo).

Salmonid species contain numerous short interspersed repetitive elements (SINEs), known collectively as the HpaI family, in their genomes. Amplification and successive integration of individual SINEs into the genomes have occurred during the evolution of salmonids. We reported previously a strategy for determining the phylogenetic relationships among the Pacific salmonids in which these SINEs were used as temporal landmarks of evolution. Here, we provide evidence for extensive genomic rearrangements that involved retropositions and deletions in a common ancestor of all the Pacific salmon and trout. Our results provide genetic support for the recent phylogenetic reassignment of steelhead and related species from the genus Salmo to the genus Oncorhynchus. Several other informative loci identified by insertions of HpaI SINEs have been isolated, and previously proposed branching orders of the Oncorhynchus species have been confirmed. The authenticity of our phylogenetic tree is supported both by the isolation of more than two informative loci per branching point and by the congruence of all our data, which suggest that the period between successive speciations was sufficiently long for each SINE that had been amplified in the original species to become fixed in all individuals of that species.

Animals↗