The effects of early chronic exposure to sublethal copper on the olfactory discrimination of rainbow trout,Oncorhynchus mykiss

Synopsis The impact of a long-term sublethal copper exposure on the olfactory discrimination performance was examined in rainbow trout,Oncorhynchus mykiss. Two fish groups, one from the 14th day post-fertilization and the other from hatching, were exposed continuously to 22 g Cu²⁺·1^–1 for 41 and 37 weeks, respectively. Preference/avoidance responses to different odor conditions (own rearing water/well water; own rearing water/heterospecific water) were evaluated during the 8th month after hatching. The 2 odor conditions appear reliable criteria to evaluate the fish discrimination ability. When controls were given a choice between own rearing water against either well water or heterospecifc water, they significantly preferred their own rearing water, whereas both copper-exposed groups showed no preference. The behavioral response of exposed fish indicates impairment of their olfactory discrimination ability; however, it cannot be determined if this reflects a loss of olfactory sensitivity or an olfactory hyposensitivity. Behavioral tests performed 2 and 10 weeks after removal of copper, showed some functional recovery of the olfactory discrimination ability which could be related to the renewal property of olfactory receptor cells. Results demonstrate that a long-term sublethal exposure to copper, as it commonly occurs in the natural condition, may result in olfactory dysfunction with potential impacts on fish survival and reproduction.     

Effects of exposure to sublethal propiconazole on intestine-related biochemical responses in rainbow trout,Oncorhynchus mykiss

The effect of long-term (30 days) exposure to PCZ (0.2, 50, and 500 μg l^?1) on intestine-related biochemical markers in rainbow trout was investigated. Multiple biomarkers were measured, including digestive enzymes (proteolytic enzymes and amylase), antioxidant responses (TBARS, CP, SOD, CAT, GR and GPx) and energy metabolic parameters (RNA/DNA ratio, Na⁺-K⁺-ATPase). Exposure to 500 μg l^?1 PCZ led to significantly inhibited (p < 0.01) proteolytic enzyme and amylase activity. Activities of the antioxidant enzymes SOD, CAT, and GPx gradually increased at lower PCZ concentrations (0.2 and 50 μg l^?1). At the highest concentration (500 μg l^?1), oxidative stress was apparent as significant higher (p < 0.05) lipid peroxidation and protein carbonyls, associated with an inhibition of antioxidant enzymes activity. Moreover, energy metabolic parameters (RNA/DNA ratio, Na⁺-K⁺-ATPase) were significantly inhibited (p < 0.01) in the intestines of fish exposed to 500 μg l^?1 PCZ, compared with controls. We suggest that long-term exposure to PCZ could result in several responses in intestine-related biochemical markers, which potentially could be used as indicators for monitoring residual PCZ present in the aquatic environment.     

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.     

Rhesus glycoprotein and urea transporter genes are expressed in early stages of development of rainbow trout (Oncorhynchus mykiss)

The objective of this study was to determine if the genes for the putative ammonia transporters, Rhesus glycoproteins (Rh) and the facilitated urea transporter (UT) were expressed during early development of rainbow trout, Oncorhynchus mykiss Walbaum. We predicted that the Rh isoforms Rhbg, Rhcg1 and Rhcg2 would be expressed shortly after fertilization but UT expression would be delayed based on the ontogenic pattern of nitrogen excretion. Embryos were collected 3, 14 and 21 days postfertilization (dpf), whereas yolk sac larvae were sampled at 31 dpf and juveniles at 60 dpf (complete yolk absorption). mRNA levels were quantified using quantitative polymerase chain reaction and expressed relative to the control gene, elongation factor 1alpha. All four genes (Rhbg, Rhcg1, Rhcg2, UT) were detected before hatching (25-30 dpf). As predicted, the mRNA levels of the Rh genes, especially Rhcg2, were relatively high early in embryonic development (14 and 21 dpf), but UT mRNA levels remained low until after hatching (31 and 60 dpf). These findings are consistent with the pattern of nitrogen excretion in early stages of trout development. We propose that early expression of Rh genes is critical for the elimination of potentially toxic ammonia from the encapsulated embryo, whereas retention of the comparatively benign urea molecule until after hatch is less problematic for developing tissues and organ systems.     

Determination of relative protein degradation activity at different life stages in rainbow trout (Oncorhynchus mykiss)

Rainbow trout were reared from 5 g to ~ 400 g on a diet formulated to supply the required protein from either fishmeal or plant proteins. The fish were sampled at every weight doubling and liver and muscle samples were obtained. From these tissue samples RNA and protein were isolated and analyzed for the expression of a number of muscle regulatory and protein degradation genes and enzymatic activity for proteins involved in the caspase, calpain, and ubiquitin-proteasome pathways for protein proteolysis. Only MyoD2 showed significant differences in expression between the two diets, while no significant changes over the course of the experiment were determined for MyoD2 or the other muscle factors. For the degradation genes significant changes in expression were determined for calpain1 and calpastatin. Calpastatin also showed a significant increase in expression over the course of the experiment in the muscle of fish fed a fishmeal diet and significant decrease in expression in the liver of fish fed the fishmeal based diet. Differences in proteasome enzyme activity were found between diets in the liver and muscle of fish and for caspase-3 activity in muscle. Significant changes in activity over the course of the experiment were noted for proteasome and calpain activity in the liver and muscle. These findings suggest that diets replacing fishmeal with plant material can have some effects on protein turnover in muscle and that some degradation pathways are differentially regulated during the growth of rainbow trout.     

Water chloride provides partial protection during chronic exposure to waterborne silver in rainbow trout (Oncorhynchus mykiss) embryos and larvae

Rainbow trout embryos and larvae were continuously exposed (at 12.5 degrees C) to waterborne silver in a flow-through setup, from fertilization to swim-up, at nominal silver concentrations of 0, 0.1, or 1.0 microg/L total silver (as AgNO(3)) at three different water Cl(-) levels (30, 300, and 3,000 microM, added as KCl). Exposures were conducted in synthetic soft water (hardness 20 mg CaCO(3)/L generated from reconstituted reverse osmosis freshwater). Continuous exposure to 1.0 microg/L total silver for 58 d at 30 microM water Cl(-) resulted in a pronounced ionoregulatory disturbance (as indicated by a reduction in whole body Na(+),K(+)-ATPase activity, unidirectional Na(+) uptake [Jin Na(+)], and whole body Na(+) and Cl(-) levels) and a reduction in extractable protein and wet weight. Thus, the mechanism of chronic silver toxicity appears to be similar to that observed during acute silver exposure in juvenile and adult fish, specifically an ionoregulatory disturbance. Higher water Cl(-) levels (300 and 3,000 microM Cl(-)) offered some degree of protection from the ionoregulatory disturbance, with only minor protective effects in terms of mortality. The protective effects of water Cl(-) on the toxicity of silver (as AgNO(3)) appear to be far less during chronic than during acute exposure. Mortality and larval Na(+) concentration, Jin Na(+), and Na(+),K(+)-ATPase activity all appear to be correlated with silver body burden and calculated water Ag(+) during chronic silver exposure. Thus, there appears to be potential to model chronic toxicity but not simply by recalibration of an acute model. A chronic model must be based on real chronic data because the protective effects of various ligands appear to be quantitatively very different from those in the acute situation.     

The effect of humic substances on the toxicity of aluminium to adult rainbow trout, Oncorhynchus mykiss (Walbaum)

Some physiological parameters were measured in adult rainbow trout during a 10-day exposure to 180 μg Al_total l⁻¹ in acid water (pH 4.7) with or without humic substances (10 mg l⁻). The fish were acclimatized to pH 5.0 for 7 days prior to the experimental treatments.
Chemical analyses revealed that, in the presence of human substances, 74–80% of the A1 was organic bound, while in the absence of humic substances most of the Al(987percnt;) occurred in the inorganic form.
Al bound to humic substances (13–150 μg l⁻¹) did not alter the plasma NaCl-concentration, nor the haematocrit value, of rainbow trout during an exposure period of 10 days. This contrasts with the high death rate obtained within 2–3 days when most of the A1 (175 μg l⁻¹) was in the inorganic form. The lethality was accompanied by a 25% decrease in the plasmaconcentration of NaCl and a doubling of the haematocrit value. Bulk analysis revealed that when the metal was present in inorganic forms the total Al content of the gills (75 μg A1 g⁻¹ wet weight) was 15 times higher than when it was present as bound to the humic substances. These experiments showed that the accumulation of A1 at the gills was accompanied by physiological disturbances, both being a function of the chemical speciation of Al.     

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.     

Effect of long-term silver exposure on survival and ionoregulatory development in rainbow trout (Oncorhynchus mykiss) embryos and larvae,in the presence and absence of added dissolved organic matter

Developing rainbow trout were chronically exposed to silver (as AgNO(3)) from fertilization to swim-up, in moderately hard water (120 mg CaCO(3)(x)l(-1)) in the presence and absence of an additional 12 mg C/L of dissolved organic carbon (DOC, as humic acid, Aldrich). Nominal silver concentrations were 0, 0.1 and 10 microg l(-1) total silver in a flow-through set-up maintained at 12 degrees C. The objectives of the study were to investigate the possible protective effects of DOC on growth, mortality, time to hatch and swim-up, and sublethal ionoregulatory disturbances during chronic exposure to ionic silver. Throughout development, there was a large increase in % daily mortality at 10 microg(x)l(-1) total silver (in the absence of DOC), that was associated with an ionoregulatory disturbance, in particular a 35% reduction in whole body Na(+) just prior to hatch. At nominal 10 microg(x)l(-1) total silver, the presence of additional DOC (reducing dissolved silver to 4.7+/-0.3 microg l(-1)) resulted in a significant reduction in % daily mortality up to hatch, demonstrating a protective effect of DOC. Interestingly, DOC did not appear to mitigate the ionoregulatory disturbance, with the exception of whole body [Cl(-)] on day 44 of exposure. Exposure to 0.1 microg(x)l(-1) total silver (in the absence of DOC) resulted in a statistically significant reduction in growth, and DOC did not prevent an ionoregulatory disturbance [based upon (J(in) Na(+)), whole body Na(+),K(+) ATPase activity and whole body (Na(+))] at this silver concentration relative to controls+DOC. DOC exerted a direct effect on growth and ionoregulatory development that complicates interpretation of the data, however, these data indicate that protective effects of DOC (in the form of Aldrich humic acid) during chronic silver exposure appear to be less than that observed during acute exposure. The ultimate goal of this and future studies is to develop a model that can predict chronic toxicity on a site-specific basis, taking into account protective effects of various ligands present in different waters, as is presently being employed for some metals during acute exposure.     

Uptake of vitellogenin into oocytes during early vitellogenic development in the rainbow trout, Oncorhynchus mykiss (Walbaum)

Uptake of ³H-vitellogenin (³H-VTG) into oocytes of various sizes was investigated during early vitellogenic development in the rainbow trout, Oncorhynchus mykiss (Walbaum). Females were injected with ³H-VTG and uptake into oocytes of different sizes (<0.4,0.4–0.59, 0.6–0.79, 0.8–0.99 and 1.0 1.2 mm in diameter) measured. Oocytes measuring less than 0.6 mm in diameter appeared unable to sequester VTG and were therefore considered pre-vitellogenic. Oocytes measuring 0.6 mm or more all sequestered VTG. The larger the oocyte, the more ³H-VTG it sequestered, even when uptake was expressed per unit surface area. The latter observation could be due to an increase in the number of VTG receptors per unit surface area, an increase in the rate of turnover of the VTG receptor, greater access of VTG to the receptors as oocytes grow, or a combination of any of these factors. The data suggest that the ability to sequester VTG is developmentally regulated.     

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%).     

The influence of chronic anaemia on catecholamine secretion in the rainbow trout (Oncorhynchus mykiss)

The effect of long-term (7 day) anaemia on catecholamine release was examined in rainbow trout (Oncorhynchus mykiss) in vivo during acute exposure to hypoxia and in situ using a perfused post-cardinal vein preparation. The first goal was to distinguish among reductions in blood O₂ partial pressure, O₂ concentration and haemoglobin percentage saturation as potential stimuli for, or correlates of, catecholamine secretion during hypoxia. The second goal was to elucidate the role of these factors in promoting enhanced chromaffin cell responsiveness in trout subjected to chronic hypoxia (Montpetit and Perry 1998). Anaemic fish (haematocrit lowered from 28.4±2.4% to 11.9±1.6%) displayed a marked reduction in haemoglobin-O₂ binding affinity [P ₅₀ (P _aO₂ at 50% Hb-O₂ saturation) was increased from 14.7 mm Hg to 24.3 mm Hg]. Upon exposure to hypoxia, the anaemic fish released catecholamines into their circulation at higher values of arterial O₂ partial pressure (∼52 mm Hg versus ∼18 mm Hg) and haemoglobin O₂ saturation (<70% versus <55%) than did control fish. In addition, anaemic fish achieved significantly greater circulating levels of total catecholamines (noradrenaline plus adrenaline) during acute hypoxia (294.8±67.3 versus 107.0±35.6 nmol l⁻¹). These results do not support the view that catecholamine release is triggered by a reduction in haemoglobin O₂ saturation or arterial PO₂, per se. Nor are they consistent with the idea that catecholamine release occurs at a threshold value of arterial PO₂ corresponding to a critical reduction in blood O₂ concentration. The effects of the non-selective cholinergic receptor carbachol on catecholamine secretion from chromaffin tissue were assessed using perfused posterior cardinal vein preparations derived from control or anaemic fish. For adrenaline secretion, there was no statistically significant change in the ED₅₀ (dose eliciting 50% response). For noradrenaline secretion however, preparations originating from anaemic fish displayed an enhanced responsiveness to carbachol as indicated by a significant 4.5-fold reduction in the carbachol ED₅₀ value from 2.53 × 10⁻⁶ mol kg⁻¹ to 5.67 × 10⁻⁷ mol kg⁻¹. These results demonstrate that anaemia-induced hypoxaemia, in the absence of any lowering of PO₂, is able to modulate the responsiveness of chromaffin cells to cholinergic stimulation. Accepted: 21 April 1999     

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.     

The dynamics of oocyte growth during vitellogenesis in the rainbow trout (Oncorhynchus mykiss)

This report describes the dynamics of oocyte growth during vitellogenesis in a population of virgin female rainbow trout. Indices of ovarian development increased dramatically during the period of study: the gonadosomatic index (GSI) increased over 50-fold, reaching a peak of 20 just before ovulation; the mean oocyte diameter increased from less than 1 mm to 5.4 mm; and plasma levels of vitellogenin increased from less than 1.5 mg/ml to 25 mg/ml. There were no changes in the numbers of developing oocytes (measuring 0.5 mm or greater in diameter) from the time when the majority of oocytes undergoing secondary development had entered vitellogenesis in August to ovulation in February (averaging 4000 oocytes per fish). The increase in ovary weight during vitellogenesis was, therefore, due to an increase in the size of oocytes rather than to recruitment of more maturing oocytes. The numbers of vitellogenic oocytes in the ovary during the entire study also suggested that atresia of vitellogenic oocytes does not play a prominent role in determining fecundity. During early vitellogenesis, the volume of maturing oocytes within an ovary varied by as much as 250-fold. From September onwards, when all oocytes to be ovulated that season had entered vitellogenesis, a gradual uniformity in size began to develop, such that at ovulation, in February, all the eggs were very similar in size (there was less than a 2-fold variation in volume). The pattern of growth of oocytes in an ovary during vitellogenesis suggests that growth between oocytes is closely coordinated.     

The effect of egg size on yolk utilization and growth of rainbow trout alevins (Oncorhynchus mykiss Walbaum)

Changes in body weight and yolk weight were studied in rainbow trout alevins derived from large eggs (diameter >4 mm) and small eggs (egg diameter <4 mm) from the same female. Maximal body weight was reached later, and was higher, for alevins derived from large than from small eggs. The yolk was absorbed more quickly by the alevins of small eggs. Percentage of body water increased in alevins during fasting up to a limit of 91%. Although the limit was the same for both groups of alevins, it was reached more quickly by alevins from small eggs. Relative water content of the yolk did not seem to be influenced by egg size. These results suggest that original egg size had both a quantitative and qualitative effect on the early development of alevins.     

Expression of four glutamine synthetase genes in the early stages of development of rainbow trout (Oncorhynchus mykiss) in relationship to nitrogen excretion

The incorporation of ammonia into glutamine, catalyzed by glutamine synthetase, is thought to be important in the detoxification of ammonia in animals. During early fish development, ammonia is continuously formed as yolk proteins and amino acids are catabolized. We followed the changes in ammonia and urea-nitrogen content, ammonia and urea-nitrogen excretion, glutamine synthetase activity, and mRNA expression of four genes coding for glutamine synthetase (Onmy-GS01-GS04) over 3-80 days post fertilization and in adult liver and skeletal muscle of the rainbow trout (Oncorhynchus mykiss). Both ammonia and urea-nitrogen accumulate before hatching, although the rate of ammonia excretion is considerably higher relative to urea-nitrogen excretion. All four genes were expressed during early development, but only Onmy-GS01 and -GS02 were expressed at appreciable levels in adult liver, and expression was very low in muscle tissue. The high level of expression of Onmy-GS01 and -GS03 prior to hatching corresponded to a linear increase in glutamine synthetase activity. We propose that the induction of glutamine synthetase genes early in development and the subsequent formation of the active protein are preparatory for the increased capacity of the embryo to convert the toxic nitrogen end product, ammonia, into glutamine, which may then be utilized in the ornithine-urea cycle or other pathways.     

Immunostimulant effects of Cotinus coggyria on rainbow trout (Oncorhynchus mykiss)

In this study, non-specific immune effects of tetra (Cotinus coggyria) on rainbow trout (Oncorhynchus mykiss) by dietary intake were investigated. Fish were fed daily ad libitum with diets containing 0.5% and 1.0% tetra for 3 weeks. After this period, fish were switched back to the basal diet for additional 6 weeks. Extracellular and intracellular respiratory burst activities, phagocytosis in blood leukocytes, lysozyme activities, and total plasma protein levels were evaluated at the end of the tetra feeding period and every 3 weeks during the basal diet period. Extracellular and intracellular respiratory burst activities, phagocytic activity, lysozyme activity and total protein level parameters of the groups containing 0.5% and 1.0% tetra were higher than the control group at the end of the 3rd, 6th and 9th weeks, respectively (P < 0.05). The highest values of the non-specific immune parameters were observed in the group fed with 1.0% tetra. Tetra groups did not show any significant difference (P > 0.05) in terms of specific growth rate and average weight of the fish.