A lead-gill binding model to predict acute lead toxicity to rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss, approximately 2 g) were exposed to 0.6-1.0 microM Pb (125-200 microgl(-1)) for 3 h in ion-poor water. Complexing ligands (citrate, ethylenediamine, organic matter (OM)) or competing cations (Ca, Mg, Na) were added to the water. After exposure, trout gills were removed and analyzed for accumulated Pb. From these exposures, a conditional equilibrium binding constant (K) for Pb-gill binding was calculated (log K(Pb-gillPb)=6.0), plus conditional binding constants for cationic competition at the Pb binding sites and for Pb binding to OM in the water. These log K values were entered into the MINEQL+ aquatic chemistry equilibrium program, to calculate binding of Pb by trout gills. Two versions of the Pb-gill binding model were generated, one of which took into account OM quality as indicated by a simple measure of OM aromaticity, the specific absorption coefficient. The two model versions were tested against acute Pb toxicity (as the time to reach 50% fish mortality; LT50) during 1-week exposures of trout to 3.9 microM Pb in water collected from across southern Ontario. Both versions of the model generated highly significant correlations between the LT50 values and gill Pb concentrations calculated from measured exposure water chemistry, with the OM quality version correlating slightly better. Water pH also correlated well with the LT50 values, because the Pb exposures were in the pH range (7-8) where there is a nearly linear relationship between water pH and inorganic complexation of Pb. Advantages of the Pb-gill binding model include its completeness and the flexibility inherent in its conceptual framework, for example the inclusion of competition by Ca and H(+) for Pb binding sites on gills, and inclusion of complexation of Pb in the water column by natural OM and by carbonate.     

Extension of the biotic ligand model of acute toxicity to a physiologically-based model of the survival time of rainbow trout (Oncorhynchus mykiss) exposed to silver

Chemical speciation controls the bioavailability and toxicity of metals in aquatic systems and regulatory agencies are recognizing this as they develop updated water quality criteria (WQC) for metals. The factors that affect bioavailability may be quantitatively evaluated with the biotic ligand model (BLM). Within the context of the BLM framework, the 'biotic ligand' is the site where metal binding results in the manifestation of a toxic effect. While the BLM does account for the speciation and complexation of dissolved metal in solution, and competition among the free metal ion and other cations for binding sites at the biotic ligand, it does not explicitly consider either the physiological effects of metals on aquatic organisms, or the direct effect of water chemistry parameters such as pH, Ca(2+)and Na(+) on the physiological state of the organism. Here, a physiologically-based model of survival time is described. In addition to incorporating the effects of water chemistry on metal availability to the organism, via the BLM, it also considers the interaction of water chemistry on the physiological condition of the organism, independent of its effect on metal availability. At the same time it explicitly considers the degree of interaction of these factors with the organism and how this affects the rate at which cumulative damage occurs. An example application of the model to toxicity data for rainbow trout exposed to silver is presented to illustrate how this framework may be used to predict survival time for alternative exposure durations. The sodium balance model (SBM) that is described herein, a specific application of a more generic ion balance model (IBM) framework, adds a new physiological dimension to the previously developed BLM. As such it also necessarily adds another layer of complexity to this already useful predictive framework. While the demonstrated capability of the SBM to predict effects in relation to exposure duration is a useful feature of this mechanistically-based framework, it is envisioned that, with suitable refinements, it may also have utility in other areas of toxicological and regulatory interest. Such areas include the analysis of time variable exposure conditions, residual after-effects of exposure to metals, acclimation, chronic toxicity and species and genus sensitivity. Each of these is of potential utility to longer-term ongoing efforts to develop and refine WQC for metals.     

Lectin histochemistry of rainbow trout (Oncorhynchus mykiss) gill and skin

In order to characterize the glycoconjugate residues in skin and gills of the adult rainbow trout, the binding pattern of five biotinylated lectins with different carbohydrate specificities was examined. In the skin, mucous cells revealed binding sites for PNA and SBA; filament-containing cells were additionally labelled with Con A. However, the basal cell layer showed no reaction. In the gill, subpopulations of mucous cells reacted with Con A, PNA, SBA and UEA-I. This broader spectrum of glycoconjugates in gill mucous cells compared with the epidermal mucous cells could point to the additional function of gill mucus in ion and osmoregulation. Lectin binding sites were less common in the respiratory epithelial cells of the secondary lamellae than in those of the primary lamellae. Chloride cells revealed mannose, galactose and fucose residues. Immature chloride cells, as indicated by a comparison with Na⁺/K⁺ ATPase immunolabelling, reacted with Con A; subpopulations of them reacted with PNA, SBA and UEA-I. The results form the basis for further investigations in which these cell populations can be analysed under different environmental conditions     

The mucous coat on gill lamellae of rainbow trout (Oncorhynchus mykiss)

The existence of a layer of mucus covering the gill lamellae of healthy rainbow trout (Oncorhynchus mykiss) was investigated. Using cryo-scanning electron microscopy, a smooth, undulating, thin layer was observed which completely covered gill filaments and lamellae, thereby obscuring epithelial microridges. After processing cryopreserved gill arches in glutaraldehyde for conventional scanning electron microscopy, the layer was no longer present and epithelial microridges were clearly visible. The identity of this layer was investigated using cryopreserved gills which were treated in one of two ways. First, gills were incubated with a rabbit antiserum to gill mucus, with normal rabbit serum, or with phosphate-buffered saline. Following fixation in glutaraldehyde and processing, only the gill tissue incubated with the mucus-specific antiserum was still covered with the smooth layer. The layer was also retained on the gills of fish anesthetized in a solution containing mucusspecific antiserum and then processes in glutaraldehyde for conventional scanning electron microscopy. The tenacious nature of the mucous layer was demonstrated by its stability following exposure to formalin and a cationic detergent. Second, the presence of this layer was confirmed on gill tissue which was cryopreserved, followed by freeze-substitution and vapor fixation, and then examined by transmission electron microscopy.     

Lectin histochemistry of rainbow trout (Oncorhynchus mykiss) gill and skin

In order to characterize the glycoconjugate residues in skin and gills of the adult rainbow trout, the binding pattern of five biotinylated lectins with different carbohydrate specificities was examined. In the skin, mucous cells revealed binding sites for PNA and SBA; filament-containing cells were additionally labelled with Con A. However, the basal cell layer showed no reaction. In the gill, subpopulations of mucous cells reacted with Con A, PNA, SBA and UEA-I. This broader spectrum of glycoconjugates in gill mucous cells compared with the epidermal mucous cells could point to the additional function of gill mucus in ion and osmoregulation. Lectin binding sites were less common in the respiratory epithelial cells of the secondary lamellae than in those of the primary lamellae. Chloride cells revealed mannose, galactose and fucose residues. Immature chloride cells, as indicated by a comparison with Na⁺/K⁺ ATPase immunolabelling, reacted with Con A; subpopulations of them reacted with PNA, SBA and UEA-I. The results form the basis for further investigations in which these cell populations can be analysed under different environmental conditions This revised version was published online in November 2006 with corrections to the Cover Date.     

Metal-ion-mediated oxidative stress in the gill homogenate of rainbow trout (Oncorhynchus mykiss)

Human activities play a major role in toxic and carcinogenic metal pollution of the environment. This study was undertaken to evaluate the effects of copper and mercury at the 400-to 1000-μM concentration range on some biochemical markers of oxidative stress, such as lipid peroxidation (LPO), glutathione-S-transferase (GST) activity, and reduced glutathione (GSH) content in the rainbow trout gill homogenates with or without supplementation of manganese, selenium, and bovine serum albumin (BSA). The integrity of DNA was also measured to assess metal ion toxicity. The results showed that the LPO and specific activity of GST were elevated. This indicated that cell-protecting antioxidant mechanisms were overtaxed and could not prevent membrane peroxidation. Following the addition of metals, the GSH content was also significantly reduced in a concentration-dependent manner. Mercury was found to be more effective than copper. The application of antioxidants proved beneficial in inhibiting LPO, reducing GST activity, and elevating the GSH levels in the gill samples. Manganese was more effective than selenium and BSA. Surprisingly, when BSA (1.0%) was added to the gill homogenates treated with a 1000-μM concentration of metal ions, instead of alleviating malondialdehyde (MDA) generation, a drastic elevation in the MDA levels, alleviation in GST activity, and a further decrease in glutathione (GSH) levels were observed, which were most likely the result of pro-oxidant activity of BSA. The results also indicated that mercury and copper functioned as genotoxic pollutants, which altered the DNA integrity by inducing the single and double-stranded DNA breaks in the gill cell nuclei. Collectively, toxicity of metal ions is related to the depletion of GSH content and inhibition of antioxidant enzyme GST, resulting in the propagation of LPO and DNA damage.     

Environmental influences on cardiovascular variables in rainbow trout, Oncorhynchus mykiss (Walbaum)

Two groups of wild (lakedwelling and anadromous), and a group of hatchery-reared Oncorhynchus mykiss (Walbaum) (rainbow trout) were sampled in order to measure cardiac morphometrics, haemoglobin concentration, and the DNA and protein concentration in cardiac muscle. A combination of these variables was used to distinguish wild fish from domestic ones.
The wild fish had significantly higher levels of haemoglobin (for male fish, 10.10 and 10.07 g 100 ml⁻¹ vs. 7.69 g d⁻¹) and larger relative ventricle mass (females, 0.091 and 0.099% ofbody mass vs. 0.073%; males, 0.108 and 0.134% vs. 0.102%; immatures, 0.086 and 0.094% vs. 0, 072%, respectively) than the domestic fish. The anadromous and domestic fish had significantly higher amounts of compact tissue when compared with lake fish (females, 43 and 47% of ventricle mass vs. 34%, respectively). Ventricle size distinguished wild fish from domestic fish, except male anadromous and male domestic fish which were distinguished only by haemoglobin and compact tissue values. Immature fishes from all groups had lower total protein levels in the ventricle, lower compact tissue levels, and less haemoglobin. Points regarding the potential environmental influences in determining these cardiovascular trends are discussed.     

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.     

Effects of hypoxia on the venous circulation in rainbow trout (Oncorhynchus mykiss)

Hypoxia in fish is generally associated with bradycardia while cardiac output (Q) remains unaltered or slightly increased due to a compensatory increase in stroke volume (SV). Rainbow trout (Oncorhynchus mykiss) were subjected to severe (P(W)O2=7.3+/-0.2 kPa) or mild (P(W)O2=11.5+/-0.2 kPa) hypoxia. Central venous pressure (P(ven)), dorsal aortic pressure (P(da)), heart rate (f(H)) and Q, were recorded in vivo. Both levels of hypoxia triggered a significant increase in P(ven). Severe hypoxia was associated with bradycardia and unaltered Q, whereas mild hypoxia was associated with a small but significant increase in Q and no bradycardia. These findings indicate that an increase in P(ven) promotes an increase in SV during hypoxia. Since mild hypoxia increased P(ven), Q and SV without bradycardia or reduced systemic resistance (R(sys)), we hypothesize that an active increase in venous tone serving to mobilize blood to the central venous compartment in order to increase cardiac preload and consequently SV, is an important cardiovascular trait associated with hypoxia. Pharmacological pre-treatment with prazosin (1 mg kg(-1)) did not conclusively reveal the underlying mechanisms to the observed changes in P(ven). This study discusses the influence of venous pooling, reduced R(sys) and altered venous tone on changes in P(ven) observed during hypoxia.     

A consolidated linkage map for rainbow trout (Oncorhynchus mykiss)

Androgenetic doubled haploid progeny produced from a cross between the Oregon State University and Arlee clonal rainbow trout (Oncorhynchus mykiss) lines, used for a previous published rainbow trout map, were used to update the map with the addition of more amplified fragment length polymorphic (AFLP) markers, microsatellites, type I and allozyme markers. We have added more than 900 markers, bringing the total number to 1359 genetic markers and the sex phenotype including 799 EcoRI AFLPs, 174 PstI AFLPs, 226 microsatellites, 72 VNTR, 38 SINE markers, 29 known genes, 12 minisatellites, five RAPDs, and four allozymes. Thirty major linkage groups were identified. Synteny of linkage groups in our map with the outcrossed microsatellite map has been established for all except one linkage group in this doubled haploid cross. Putative homeologous relationships among linkage groups, resulting from the autotetraploid nature of the salmonid genome, have been revealed based on the placement of duplicated microsatellites and type I loci.     

Hormonal and environmental regulation of epithelial calcium channel in gill of rainbow trout (Oncorhynchus mykiss)

We indirectly tested the idea that the epithelial Ca2+ channel (ECaC) of the trout gill is regulated in an appropriate manner to adjust rates of Ca2+ uptake. This was accomplished by assessing the levels of gill ECaC mRNA and protein in fish exposed to treatments known to increase or decrease Ca2+ uptake capacity. Exposure of trout to soft water ([Ca2+]=20-30 nmol/l) for 5 days (a treatment known to increase Ca2+ uptake capacity) caused a significant increase in ECaC mRNA levels and an increase in ECaC protein expression. The inducement of hypercalcemia by infusing fish with CaCl2 (a treatment known to reduce Ca2+ uptake) was associated with a significant decrease in ECaC mRNA levels, yet protein levels were unaltered. ECaC mRNA and protein expression were increased in fish treated with the hypercalcemic hormone cortisol. Finally, exposure of trout to 48 h of hypercapnia (approximately 7.5 mmHg, a treatment known to increase Ca2+ uptake capacity) elicited an approximately 100-fold increase in the levels of ECaC mRNA and a significant increase in protein expression. Immunocytochemical analysis of the gills from hypercapnic fish suggested a marked increase in the apical expression of ECaC on pavement cells and a subpopulation of mitochondria-rich cells. The results of this study provide evidence that Ca2+ uptake rates are, in part, regulated by the numbers of apical membrane Ca2+ channels that, in turn, modulate the inward flux of Ca2+ into gill epithelial cells.     

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%).     

Effects of cadmium chloride on the development of rainbow trout Oncorhynchus mykiss early life stages

The sub-chronic (28–56 days) effects of exposure to low concentrations of cadmium (Cd; 0·05, 0·25, 0·50 and 2·50 μg l⁻¹) shortly following fertilization on embryos, larvae and juvenile rainbow trout Oncorhynchus mykiss were examined. Premature hatching occurred at lower concentrations (0·05 and 0·25 μg l⁻¹ Cd), however, delayed hatching was seen in the 2·50 μg l⁻¹ Cd group, with >90% of hatching occurring on the last day of the hatching period. Larval growth was negatively affected by Cd exposure in a concentration-dependent manner. Larvae exposed to 2·50 μg l⁻¹ Cd were 13·9 ± 0·8% shorter in total length ( L _T) and weighed 22·4 ± 3·5% (mean ± s . e .) less than controls at the end of the exposure period. Plasma sex steroid concentrations (oestradiol in juvenile females and 11-ketotestosterone in juvenile males) were elevated (four- to 10-fold over controls) in exposed fish in both males and females, following 28 days of exposure to 0·05, 0·25 and 0·50 μg l⁻¹ Cd, respectively. These results suggest that environmentally realistic concentrations (in the μg l⁻¹ range) of Cd can affect the development of O. mykiss impacting embryos, larvae and juvenile fish.     

Effects of acute iron overload on Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)

Distribution of radioiron to various tissues after intraperitoneal injections was examined in Atlantic salmon and rainbow trout. Liver and spleen were found to be the major iron storage tissues. Injections of 1 or 5 mg iron as ferric ammonium citrate led to a fall in hemoglobin levels in both species after 2 d. Hemoglobin levels returned to normal levels in rainbow trout after 8 d, but Atlantic salmon had not recovered, and Hb levels fell below 3 g/100 mL. In both species, the fall in Hb was associated with a raise in iron levels in spleen and liver, suggesting damage to erythrocytes. Atlantic salmon liver ferritin showed a two- to threefold increase, while rainbow trout showed a sixfold increase, and a more rapid response. The toxic effect of iron in fish appears to be different from the effect in other vertebrates.     

Effects of Ichthyophonus hoferi on condition indices and blood chemistry of experimentally infected rainbow trout (Oncorhynchus mykiss)

Body condition, hepatosomatic index and blood chemistry of Oncorhynchus mykiss experimentally infected with a tissue dwelling fish pathogenic fungus, Ichthyophonus hoferi, were monitored over a 6 wk period. This was to determine whether the infection constituted a stress manifest by changes in the hypothalamic-pituitary interrenal axis, and especially plasma cortisol levels. Infection caused anaemia and leucopenia but did not change the condition, hepatosomatic indices, or plasma chloride, cholesterol, cortisol, creatinine, glucose, osmolarity, potassium, total protein, sodium and T4. It is suggested that increased cortisol levels may not be a normal component of the stress response of fish to disease caused by invasive infectious agents.     

Effects of acute and prolonged naphthalene exposure on brain monoaminergic neurotransmitters in rainbow trout (Oncorhynchus mykiss)

We have shown previously that acute (1 to 6 h) and prolonged (1 to 5 days) exposure of rainbow trout to naphthalene resulted in decreased plasmatic cortisol and 17-beta-estradiol levels. In order to elucidate the mechanisms through which naphthalene might disrupt endocrine regulation, the present study investigated whether brain monoaminergic neurotransmitters are altered by the action of this polycyclic aromatic hydrocarbon. In a first experiment, immature rainbow trout were injected with vegetable oil alone or containing naphthalene (10 and 50 mg/kg, i.p.), and sacrificed 1, 3 and 6 h after treatment. In a second experiment, slow-coconut oil implants alone or containing naphthalene (doses of 10 and 50 mg/kg) were i.p. located and fish sacrificed 1, 3 and 5 days after treatment. Levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and noradrenaline (NA) were measured in several brain regions by HPLC. The results show that short-term naphthalene increases DA and 5-HT contents in hypothalamus and telencephalon, but differentially alter contents of the acid metabolites. Implants with naphthalene reduced DA content in hypothalamus and preoptic region but increased in telencephalon. 5-HT metabolism was decreased in hypothalamus, preoptic region, pituitary and brain stem after 3 to 6 days of treatment. In addition, the levels of NA were increased in hypothalamus and telencephalon after acute treatment and in hypothalamus and preoptic area after several days of exposure to naphthalene. These data suggest that brain neurotransmitter systems are sensitive to polycyclic aromatic hydrocarbons and could represent a target of the naphthalene-induced neuroendocrine disruption.     

Effects of gastric distension on the cardiovascular system in rainbow trout (Oncorhynchus mykiss)

When animals feed, blood flow to the gastrointestinal tract increases to ensure an adequate oxygen supply to the gastrointestinal tissue and an effective absorption of nutrients. In mammals, this increase depends on the chemical properties of the food, as well as, to some extent, on the mechanical distension of the stomach wall. By using an inflatable nitrile balloon positioned in the stomach, we investigated the cardiovascular responses to mechanical stretch of the stomach wall in rainbow trout (Oncorhynchus mykiss). Distension with a volume equivalent to a meal of 2% of the body mass increased dorsal aortic blood pressure by up to 29%, and central venous blood pressure increased transiently nearly fivefold. The increase in arterial pressure was mediated by an increased vascular resistance of both the systemic and the intestinal circulation. Cardiac output, heart rate, and stroke volume (SV) did not change, and only transient changes in gut blood flow were observed. The increase in arterial pressure was abolished by the alpha-adrenergic antagonist prazosin, indicating an active adrenergic vasoconstriction, whereas the venous pressor response could be the consequence of a passive increase in intraperitoneal pressure. Our results show that mechanical distension of the stomach causes an instantaneous increase in general vascular resistance, which may facilitate a redistribution of blood to the gastrointestinal tract when chemical stimuli from a meal induce vasodilation in the gut circulation. The normal postprandial increase in gut blood flow in teleosts is, therefore, most likely partly dependent on mechanical stimuli, as well as on chemical stimuli.