Copper exposure and the degeneration of olfactory receptors in rainbow trout (Oncorhynchus mykiss)

Abstract

Sublethal concentrations of copper in water cause the degeneration of olfactory receptors in rainbow trout (Oncorhynchus mykiss). Receptor cell loss has been correlated to the loss of olfaction in fish and may cause difficulties in olfactory mediated behaviors such as migration. This study investigated the effects of three levels of copper (100, 75 and 50 mg L^?1) on the olfactory epithelium of rainbow trout. Twenty fish randomly allocated between three exposure groups and one control were exposed for 24 hours under static renewal conditions. Light and scanning electron microscopic observations of olfactory tissue were taken to determine the extent of degeneration of receptors. In addition, levels of copper and zinc in the brain tissues were analyzed to determine if the olfactory route was a significant route of copper exposure and transfer to fish brain tissue. Results indicate that degeneration of receptors is related to the concentration of copper. Levels of copper in brain were found to be below detection of the instrument. Levels of zinc were extremely variable ranging from 52 to 132 ng zinc g^?1 brain tissue.     

Two novel muramidases from skin mucosa of rainbow trout (Oncorhynchus mykiss)

Two novel antibacterial muramidases were purified to homogeneity from skin exudates of rainbow trout (Oncorhynchus mykiss). Unusually, one has an acidic isoelectric point and it is the first anionic muramidase to be reported for fish. Its molecular mass is 14,268 Da, as determined by mass spectrometry. The other muramidase is cationic with a mass of 14,252 Da. Partial N-terminal amino acid sequencing and peptide mapping strongly point to it being a c-type lysozyme, the first to be purified and characterised from skin of a salmonid. Its optimum pH ranges from 4.5 to 5.5 and its optimum temperature, at pH 5.0, is 33-49 degrees C, although it still exhibits activity at 5 degrees C. It is strongly bactericidal to the Gram-(+) bacterium Planococcus citreus, with a minimum bactericidal concentration of 100 U ml(-1), but is neither chitinolytic nor haemolytic. These two muramidases probably contribute to epithelial defence of the fish against microbes, either alone or in synergism with antibacterial peptides.     

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%).     

Plasma amino acid levels in rainbow trout (Oncorhynchus mykiss)

The time course of plasma amino acid concentrations was studied in adult rainbow trout (300 g mean body weight). After a starvation period of 2 days fish were force-fed either with fish protein concentrate or a mixture of acidic casein and Na-caseinate at a rate of 0.32% CP (N' 6.25) of body weight. Peak levels occurred for feeding fish protein concentrate 6–12 h and for the casein mix 18 h post-feeding. The increase of the essential amino acids was closely correlated to the amino acid profile of the test proteins, whereas the concentration differences of the non-essential amino acids were at no time correlated to the amino acid pattern of fish protein concentrate or even negatively correlated in case of casein. The limiting amino acids in the test proteins were determined by ranking the average concentration increases (decreases) of the individual essential amino acids. Accordingly, arginine and histidine were most deficient in casein; in fish protein concentrate tryptophan seems to be the first limiting amino acid, followed by isoleucine.     

Immunohistochemical localization of rainbow trout ladderlectin and intelectin in healthy and infected rainbow trout (Oncorhynchus mykiss)

In the present study, the pattern of immuno-reactive ladderlectin and intelectin in healthy rainbow trout is compared to rainbow trout infected with a variety of infectious agents. In healthy rainbow trout, both proteins were localized to individual epithelial cells of the gill and intestine and both proteins were clearly demonstrated within cytoplasmic granules of polymorphonuclear leucocytes and macrophages/monocytes found in blood vessels, hepatic sinusoids, renal interstitium, mucosal epithelium and submucosa of normal intestine. In tissue from infected rainbow trout, there was an overall relative increase in both lectins compared to healthy fish and both proteins were detected in extra-cellular spaces surrounding bacteria, fungi and protozoa. Increased distribution and density of both RTLL and RTInt was demonstrated along mucosal surfaces and within inflammatory leucocytes in infected tissues and immune related organs. These findings represent one of the few examples of in vivo association of defence lectins and infectious agents.     

Immune-complex trapping in the splenic ellipsoids of rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss), immunised with horseradish peroxidase, were given horseradish peroxidase intravenously, and the trapping of antigen in the spleen was followed 1, 24, and 48 h after injection. After 1 h, the localisation of horseradish peroxidase indicated that the antigen had been extensively trapped in the walls of the splenic ellipsoids. The colocalisation of horseradish peroxidase with rainbow trout immunoglobulin M and complement factor 3 was shown with a double immunofluorescence technique and suggested that horseradish peroxidase was trapped in the form of immune complexes. After 24 and 48 h, very little horseradish peroxidase was detected in the ellipsoids, and horseradish peroxidase was mainly found in association with large cells with prominent cytoplasmic extensions. In nonimmunised fish given horseradish peroxidase intravenously, antigen was not detected in ellipsoids. Thus, the observed difference between immunised and nonimmunised trout suggests a specific role for the splenic ellipsoids in rapid immune-complex trapping and invites speculation on its significance in a secondary immune response.     

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.     

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.     

Acid-base balance during social interactions in rainbow trout (Oncorhynchus mykiss)

Socially subordinate rainbow trout (Oncorhynchus mykiss) experience chronic stress that impacts upon a variety of physiological functions, including Na(+) regulation. Owing to the tight coupling between Na(+) and Cl(-) uptake and, respectively, H(+) and HCO(3)(-) loss at the gill, ionoregulatory changes associated with social status may affect acid-base regulation. The present study assessed the responses of dominant, subordinate and control trout to hypercapnia (1% CO(2)) to test this hypothesis. Social status appeared to impact net acid excretion (J(net)H(+)) as subordinate individuals failed to increase net acid flux in response to hypercapnia. However, blood acid-base status was found to be unaffected by social status before or during hypercapnic exposure, indicating that subordinate fish were as effective as dominant or control trout in achieving compensation for the acid-base disturbance induced by hypercapnic exposure. Compensation in all groups involved decreasing Cl(-) uptake in response to hypercapnia. The branchial activities of both Na(+),K(+)-ATPase (NKA) and V-type H(+)-ATPase were affected by social interactions and/or exposure to hypercapnia. Branchial NKA activity was higher but V-ATPase activity was lower in control fish than in dominant or subordinate trout. In addition, control and subordinate but not dominant trout exposed to 24h of hypercapnia exhibited significantly higher branchial V-ATPase activity than fish maintained in normocapnia. Collectively, the data suggest that subordinate trout are able to regulate blood pH during a respiratory acidosis.     

Hydrogen sulfide stimulates catecholamine secretion in rainbow trout (Oncorhynchus mykiss)

We tested the hypothesis that endogenously produced hydrogen sulfide (H(2)S) can potentially contribute to the adrenergic stress response in rainbow trout by initiating catecholamine secretion from chromaffin cells. During acute hypoxia (water Po(2) = 35 mmHg), plasma H(2)S levels were significantly elevated concurrently with a rise in circulating catecholamine concentrations. Tissues enriched with chromaffin cells (posterior cardinal vein and anterior kidney) produced H(2)S in vitro when incubated with l-cysteine. In both tissues, the production of H(2)S was eliminated by adding the cystathionine beta-synthase inhibitor, aminooxyacetate. Cystathionine beta-synthase and cystathionine gamma-lyase were cloned and sequenced and the results of real-time PCR demonstrated that with the exception of white muscle, mRNA for both enzymes was broadly distributed within the tissues that were examined. Electrical field stimulation of an in situ saline-perfused posterior cardinal vein preparation caused the appearance of H(2)S and catecholamines in the outflowing perfusate. Perfusion with the cholinergic receptor agonist carbachol (1 x 10(-6) M) or depolarizing levels of KCl (1 x 10(-2) M) caused secretion of catecholamines without altering H(2)S output, suggesting that neuronal excitation is required for H(2)S release. Addition of H(2)S (at concentrations exceeding 5 x 10(-7) M) to the perfusion fluid resulted in a marked stimulation of catecholamine secretion that was not observed when Ca(2+)-free perfusate was used. These data, together with the finding that H(2)S-induced catecholamine secretion was unaltered by the nicotinic receptor blocker hexamethonium, suggest that H(2)S is able to directly elicit catecholamine secretion via membrane depolarization followed by Ca(2+)-mediated exocytosis.     

Morphometric characterization of the first blastomeres of rainbow trout (Oncorhynchus mykiss)

Summary In the following investigation the morphometric characteristics of the first two blastomeres of rainbow trout (Oncorhynchus mykiss) were determined. Embryos were incubated at 9°C and then fixed in a Stockard solution every 30 min starting from 8.5 to 12.5 h of incubation post fertilization. Embryonic discs were extracted and microphotographs were taken with Q Capture Pro 5.0 software using a stereomicroscope Olympus SZX7. The average size of the blastodiscs was 941.22 ± 160.42 μm. The first cleavage finished after approximately 12 h of incubation. The first two blastomeres were regularly symmetrical in their morphology. Blastomere 1 had an average length (L) of 942.68 ± 105.56 μm and width (W) of 467.34 ± 64.33 μm. Blastomere 2 had an average length of 887.60 ± 101.65 and width of 454.49 ± 47.25 μm (n = 91). Significant differences were found between the length and width of blastomeres 1 and 2. The proportion between the length of blastomeres 1 and 2 was 0.94 ± 0.07 (n = 91); between the width of blastomeres 1 and 2 it was 0.88 ± 0.11 (n = 91); and the width/length ratio was 0.51 ± 0.09 (n = 182). It was concluded that rainbow trout blastomeres tend to be asymmetrical in length with a higher dispersion of widths.     

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.