Antibacterial proteins in rainbow trout, Oncorhynchus mykiss

Antibacterial proteins are an important part of the innate immune system for all animals. They have been extensively studied in mammals, amphibians and invertebrates, but have received only scant attention in fish. Their expression and processing, however, provide a way of monitoring defence vigour during development or with seasonal changes in physiology. The aim of the present work was to identify and characterise antibacterial proteins in rainbow trout. In vitro analyses of extracts of the peripheral blood leucocytes, head kidney leucocytes and mucus from adult unstimulated (non-immune) fish showed marked antibacterial activity against Gram positive bacteria. Fractionation by ion exchange chromatography and RP-HPLC of head kidney extracts showed the presence of two forms of lysozyme but no constitutively expressed antimicrobial proteins of < 10 kDa. By contrast, chromatographic analyses of mucus revealed at least four antibacterial proteins. Two are conventional lysozymes, a third is an unusual lysozyme-like protein with a low isoelectric point, and the fourth is a highly hydrophobic, cationic peptide of c. 3 kDa.     

Exposure to thyroid hormone in ovo affects otolith crystallization in rainbow trout Oncorhynchus mykiss

Calcareous otoliths in the inner ears of fishes are necessary for proper hearing and vestibular function. Sagittal otoliths are usually composed of the calcium carbonate polymorph aragonite but may contain the polymorph vaterite, a phenomenon called otolith crystallization. The causes of otolith crystallization are poorly understood. Thyroid hormone (TH) can influence the chemical microenvironment and structure of the inner ear, suggesting that TH may influence otolith crystallization. The present study examined the effect of exogenous TH treatment on sagittal otolith crystallization and growth in larval and juvenile rainbow trout, Oncorhynchus mykiss. In the first experiment, 110?C179?day-old fish raised from TH-treated oocytes had significantly fewer sagittal otoliths containing the crystalline calcium carbonate polymorph vaterite as compared to untreated fish. Vaterite-containing otoliths were significantly longer than those containing the typical polymorph aragonite, although there was no effect of TH treatment on otolith length. In the second experiment, juveniles immersed in an exogenous solution of TH for 6?weeks had slightly longer otoliths (relative to fish length) than age-matched controls, but this effect was not significant. This juvenile population had a very high percentage (88.3?%) of vaterite sagittae overall and this percentage did not change significantly with treatment, suggesting the switch from aragonite to vaterite occurred prior to inclusion of the fish in the study. These results suggest that early manipulation of TH levels may affect calcium carbonate deposition on the otolith but that later TH exposure is unable to restore typical otolith composition.     

Investigating fear in rainbow trout (Oncorhynchus mykiss) using the conditioned-suppression paradigm

Trout learned the operant task of pendulum-pressing for a food-reward in a mean of 4.3 sessions lasting 1 hr. In a separate phase, fish also learned—through classical conditioning—to associate a neutral light cue with an aversive stimulus. When again allowed to pendulum-press for food, after aversive classical conditioning, there was a drop in the rate of responding. The mean rate dropped from 3.6-2.9 responses per min. Most important, when the light-stimulus was superimposed on a steady bout of pendulum-pressing, trout ceased to press the pendulum and did not resume activity until termination of the light-stimulus (mean number of responses during a 3-min interval immediately prior to light-stimulus = 14.3 vs. during 3-min light-stimulus = 0.1). Psychologists have used this decrease in operant responding, or “conditioned emotional response,” as a tool to examine the psychological nature of this type of aversive conditioning. In this study, the fish demonstrated various results under this paradigm similar to those shown by “higher” nonhuman animals, therefore challenging the view of fish as unconscious, nonsentient animals.     

Effect of induced triploidy on fin regeneration of juvenile rainbow trout, Oncorhynchus mykiss

Triploidy was induced in the rainbow trout in order to evaluate whether the altered numbers and sizes of triploid cells could modify fin regeneration. Amputation of one lobe of the tail fin of diploid and triploid juveniles resulted in regeneration for experimentals and controls. Nevertheless, both rate and frequency of regeneration in triploids were significantly increased as compared with diploids, a fact that can be attributed to the increased nuclear and cellular volume in a wide range of tissues, whereas the cell numbers were reduced. These data suggest that a great deal of interesting and important research could be done using triploid animals as experimental models for studying the regeneration of appendages.     

Domestication shapes morphology in rainbow trout Oncorhynchus mykiss

In this study, clonal lines from North American resident and migratory populations of rainbow trout Oncorhynchus mykiss adapted to different geographical conditions and with different domestication histories were characterized morphologically. Lines reared in a common‐garden experiment were characterized for external shape and meristic values, searching for a general pattern of morphological variation due to exposure to captive conditions. A sharp distinction was identified between wild and captive lines. The body profile was deeper in captive lines, with longer dorsal and anal fins and shorter and deeper caudal peduncles. Highly significant differences were also identified in meristic values among the lines but no consistent relation between meristic values and domestication status was detected. This morphological characterization will facilitate the selection of lines with divergent phenotypes for subsequent quantitative trait loci analysis, aimed at identifying genome regions linked with morphological adaptive response to captive conditions.     

虹鳟对时间和地点的学习记忆(英文)

为确定虹鳟(Oncorhynchus mykiss)是否能将日投放两次食物的不同时间和地点联系起来,在30天内,于09:00-10:00将食饵投放在水族箱一侧,于15:00-16:00将食饵投放在水族箱另一侧.在第21天和28天,不放食饵以确定虹鳟对时间-地点的学习记忆.结果表明,虹鳟将不同时间和地点联系起来以获取食物,并且在水族箱两个投放食饵之处均表现出预取食活动,提示该物种具有控制时间-地点学习任务的内在机制.     

Cardiorespiratory responses to haemolytic anaemia in rainbow trout Oncorhynchus mykiss

To quantify cardiorespiratory response to experimental anaemia in rainbow trout Oncorhynchus mykiss, a 24 h phenylhydrazine treatment was used to reduce haematocrit to almost one third of its initial value over 4–5 days. In response, relative blood velocity in the ventral aorta (an index of cardiac output) progressively increased to more than double to its normocythaemic value and there was no significant change in routine oxygen uptake. Thus, the primary compensatory response to anaemia was an increase in cardiac output.     

Differences in metabolic response to Loma salmonae infection in juvenile rainbow trout Oncorhynchus mykiss and brook trout Salvelinus fontinalis

Routine and post-exercise metabolic rates were measured for juvenile rainbow trout Oncorhynchus mykiss and brook trout Salvelinus fontinalis infected with the microsporidium gill parasite Loma salmonae under laboratory conditions. Rainbow trout increased routine and post-exercise metabolic rate in response to infection compared with controls. Brook trout, on the other hand, lowered routine metabolic rate without effecting post-exercise metabolic rate compared to controls. The result of these 2 different strategies may either reflect defense of metabolic scope or a difference in the rate of recovery of the excess post-exercise oxygen consumption between the 2 species in response to the same infection.     

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.     

A blue variant in the rainbow trout, Oncorhynchus mykiss Walbaum

A blue variant of the rainbow trout, which appeared in a French fish farm, displayed an iridescent body color that was cobalt blue on the back, lighter on the undersides, and silvery on the belly and which held up to adult stage. This color was supposed to result from a Tyndall effect involving a structural arrangement of melanin pigments because it disappeared when it was associated with a depigmenting gene. This blue variant appeared to be governed by an autosomal recessive gene. Blue fry survival and body weight were about 25% less than those of wild-type sibs, but no major problem was observed in further breeding performances, including reproduction. These features do not correspond with those of the blue variants previously described in the rainbow trout.     

Coronary vascular volume remodelling in rainbow trout Oncorhynchus mykiss

The 34% increase in relative ventricular mass (M_rv) resulting from chronic anaemia (induced by an intraperitoneal injection of phenylhydrazine hydrochloride) was accompanied by a 117% increase in coronary vascular volume of diploid rainbow trout Oncorhynchus mykiss. Coronary vascular volume of normocythemic triploid fish was similar to that of normocythemic diploid fish despite a larger M_rv. These observations, in combination with previous studies, suggest that the vascularity of compact myocardium in O. mykiss can vary independently of M_rv.     

Culturing and characterization of astrocytes isolated from juvenile rainbow trout (Oncorhynchus mykiss)

An access to brain cell cultures from fish would enable screening of possible neurotoxic chemicals contaminating the aquatic environment. In the present study, a protocol for a successful routine isolation and culturing of brain cells from juvenile rainbow trout was worked out. The coating material was shown to be of importance for cell proliferation. Cells grow better on a surface coated with laminin than on those coated with poly-L-lysine (PLL), poly-D-lysin (PDL) or poly-L-ornithine (PLO). The best cell growth was obtained on double-coated surfaces (PLL, PDL or PLO plus laminin). On such a culture substrate and with a seeding density of 1 x 10(7) cells/cm(2) confluence was obtained within 3-4 weeks at an incubation temperature of 18 degrees C. Approximately 95% of the cells were identified as astrocytes on the basis of a positive staining with antibodies against the astrocyte specific glial protein (GFAP). No oligodendrocytes or fibroblasts were identified in the cultures, and despite several efforts, neurons did not grow under the culture conditions used. When challenged with ligands known to awake a calcium transient in mammalian astrocytes, 44% of the cells responded to ATP with an increase in [Ca 2+](i), 38% to norepinephrine, 27% to 5-hydroxytryptamine, 7% to histamine and 6% to glutamate. Kainate, quisqualate and gamma-aminobutyric acid did not awake a calcium transient in the cells. Using a proper protocol, it is thus quite easy to get an almost pure culture of astrocyte, whereas neurones proved to very difficult to culture.     

Specific binding of [3H]ketanserin to hypothalamus membranes of juvenile rainbow trout, Oncorhynchus mykiss

This study examines the existence and pharmacological specificity of [3H]ketanserin binding in hypothalamus of juvenile rainbow trout. Hypothalamic membranes were incubated with [3H]ketanserin (selective 5HT2 antagonist) under several experimental conditions; reactions were terminated by filtration and bound radioactivity was counted by liquid scintillation spectroscopy. Tissue dilution experiments revealed that specific [3H]ketanserin binding (B(sp)) was tissue dependent; 1 hypothalamus equivalent per tube (1100 +/- 115 cpm/mg protein) was subsequently used throughout the rest of this study. In association experiments, B(sp) increased progressively with time, achieved equilibrium binding levels (1192 +/- 120 cpm/mg protein) within 80 min, and remained stable for at least 60 min thereafter; k(obs), and k(+1) were 0.032 and 0.048 min(-1) x nM(-1), respectively. In dissociation experiments, B(sp) completely dissociated within 20 min following addition of excess ketanserin; k(-1) and t1/2 were 0.0803 min(-1) and 8.7 min, respectively. B(sp) was saturable (2500 +/- 256 cpm/mg protein); Scatchard-calculated values for the equilibrium dissociation constant (K(D)) and capacity (Bmax) were 0.48 nM, and 125 fmol/mg protein, respectively. B(sp) was differentially displaced by structurally related competitors, with a rank order of potency of ketanserin = mianserin > ritanserin > serotonin (5HT) = spiperone > methiothepin mesylate > metergoline = DOI ((+/-)-2-5-dimethoxy-4-iodoamphetamine hyrobromide) > 2-methyl-5HT > alpha-methyl-5HT > 5HIAA (5-hydroxyindole acetic acid) = reserpine. These findings provide pharmacological evidence for the presence of a 5HT2-like receptor subtype in the trout hypothalamus.     

Muscular and brain cholinesterase sensitivities to azinphos methyl and carbaryl in the juvenile rainbow trout Oncorhynchus mykiss

The organophosphate azinphos methyl (AzMe) and the carbamate carbaryl are the insecticides mostly used in the irrigated valley of Río Negro and Neuquén, Patagonia, Argentina. Juvenile rainbow trout were exposed to AzMe and carbaryl and the sensitivity of skeletal muscular cholinesterase (ChE) and the time course of inhibition and recovery were evaluated. EC50 values demonstrated that AzMe was a stronger in vivo inhibitor of muscular ChE (1.05+/-0.23 microg/L) than carbaryl (270+/-62.23 microg/L). Muscular ChE was significantly less sensitive to both insecticides than brain ChE. EC50 values obtained for muscular ChE were closer than those for brain ChE to the respective pesticide lethal concentrations, pointing out the relevance of the muscular enzyme in determining acute toxicity. The recovery process of ChE activity after carbaryl exposure (500 microg/L) was fast, whereas no significant recovery was observed with AzMe (1 microg/L) after 21 days in uncontaminated media. Brain and muscular ChE were inhibited and showed a significant but not complete recovery after three consecutive 48-h exposures to AzMe (1 microg/L) followed by a recovery period of 7 days. This scheme mimics the periodical application of the insecticides in the region and suggests a certain probability of a sustained ChE inhibition under field conditions, affecting fish development and survival.     

Interaction between dietary calcium supplementation and chronic waterborne zinc exposure in juvenile rainbow trout, Oncorhynchus mykiss

This study investigated the effects of dietary Ca2+ on branchial Ca2+ and Zn2+ uptake, new and total zinc accumulation in target tissues (gill, liver and kidney), calcium and zinc homeostasis, and acute tolerance to waterborne zinc in fish chronically exposed to waterborne zinc. Juvenile rainbow trout (Oncorhynchus mykiss) were maintained on a calcium-enriched diet [41.2 mg vs. 21.2 mg (control) calcium/g dry wt. of food] and chronic waterborne zinc exposure (2.3 micromol/L), both separately and in combination, for 28 days. Calcium-supplemented diet in the absence of waterborne zinc significantly reduced branchial Ca2+ and Zn2+ influx rates, and new and total zinc accumulations in target tissues relative to control. However it did not protect against the acute zinc challenge. In contrast, waterborne zinc exposure significantly increased branchial Ca2+ and Zn2+ influx rates, new and total zinc concentrations in target tissues, and acute zinc tolerance relative to control. Interestingly, no such changes in any of these parameters were recorded in fish treated simultaneously with elevated dietary Ca2+ and waterborne zinc, except acute zinc tolerance which was highest among all the treatments. Thus, we conclude that the interactions between elevated dietary Ca2+ and waterborne zinc can protect freshwater fish against waterborne zinc toxicity.     

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.     

Streptococcus iniae type II infections in rainbow trout Oncorhynchus mykiss

Clinical and pathological findings (anorexia, hemorrhage, lethargy, loss of orientation and exophthalmia) indicated that Streptococcus iniae type II is responsible for a fatal disease in rainbow trout. Histopathological findings revealed that S. iniae type II produces a systemic disease, including a diffuse necrotizing myositis. The distribution of viable bacteria in infected tissues substantiated the pathological findings, confirming that S. iniae type II is responsible for a generalized septic disease of rainbow trout.     

Formation of chromosome aberrations in androgenetic rainbow trout Oncorhynchus mykiss

Residues of maternal nuclear DNA in the form of chromosome fragments were observed in the healthy and morphologically normal androgenetic rainbow trout Oncorhynchus mykiss. A hypothetical model for formation of chromosome re‐arrangements caused by the incomplete maternal nuclear DNA inactivation in the androgenetic rainbow trout was proposed in the present paper.