Differential Bioaccumulation of Mercury and Selenium in Stomach Contents and Tissues of Three Colorado, USA, Cutthroat Trout Populations.

Total mercury (THg) and selenium (TSe) levels were measured in stomach contents (SC) and twelve tissues of cutthroat trout (Oncorhynchus clarkii) occurring in three high-elevation lakes of Colorado, USA, inhabiting watersheds absent past and current mining activities. For 32 of 36 tissues, including muscle, mean THg wet weight (ww) concentrations were greater than in the diet (SC) for all sites, indicating biomagnification. Ranges of THg (µg/kg ww) for SC and stomach tissue (ST) were 1.23-73.54 and 14.55-61.35, respectively. Selenium concentrations in fish muscle were not greater than in the SC indicating a trophic transfer factor < 1.0. However, in several other tissues, mean Se dry weight (dw) levels were greater than in SC for all three lakes. Ranges of TSe for SC and ST were 166-7544 and 797-7523 (µg/kg dw), respectively. The muscle to egg/ovary ratio for Se averaged 2.30, 4.60, and 2.68 for the three populations. The variability of SC (planktonic vs. benthic) and differential distributions of THg and TSe in SC and organ-tissues generated questions focusing on the seasonal, physiological, and genetic drivers of these organometal(loid)s in subalpine trout.

Determination of the healthiness of aquaculture fish by enzymes and histopathological methods.

The health of fishes from select aquacultures was investigated by conducting histopathologic and enzymatic analyses, as well as by examining pollutant accumulation rates in fish tissues ranging in age from juvenile to two years old. Histopathologic examinations demonstrated that the fishes had some abnormalities in their livers, spleens, intestines and reproduction systems, such as lipidation, ovotestis formation, lysis and enlargements of the tissues. The occurrence rate of these abnormalities was not very frequent but also not negligible. Statistical analysis demonstrated that enzyme activity (i.e. CAT, EROD, SOD) and protein concentration fluctuated predominantly by age and season. These parameters were not found to be related to the fish farm or other spatial changes, when their existing environmental conditions were not extreme (i.e. polluted or otherwise unsuitable). Metal concentrations (i.e. Ni, Cu, Zn, As, Cd, Pb and Hg) were never found to be higher than national or international regulatory limits. The quality of the fishes caught from optimal farm conditions may be evaluated as good quality for human consumption.

Effects of traditional Chinese medicines on immunity and culturable gut microflora to Oncorhynchus masou.

The present study was conducted to evaluate the effect of dietary traditional Chinese medicines on the growth, immunity, and composition of culturable gut microflora in Oncorhynchus masou. Diets were formulated to contain no medicine (control), antitoxic decoction (A), general antiphlogistic decoction (B), or Herbae Artemisiae Capillariae decoction (C). Fish were manually fed twice daily till apparent satiation for 30 days. Compared with that in the control group, supplementation with the three kinds of Chinese herbal medicine enhanced fish growth significantly (P < 0.05). The activities of liver superoxide dismutase and glutathione peroxidase in the treatment groups were significantly higher compared with those in the control group (P < 0.05). The quantity of intestinal microflora was higher in the treatment groups compared with that in the control group. Moreover, there were some effects of dietary Chinese herbal medicine on the composition of intestinal microflora. Microflora of Pseudomonas sp., Psychrobacter sp., Microbacterium sp., Macrococcus sp., Burkholderia sp., and Arthrobacter sp. were found in the treatment groups, whereas there were none in the control group. There was a significant increase in their amounts in the treatment groups (P < 0.05). The three kinds of traditional Chinese medicines can improve the growth and immunity of Oncorhynchus masou and affect the quantity and composition of intestinal microflora.

Food chain model based on field data to predict westslope cutthroat trout (Oncorhynchus clarkii lewisi) ovary selenium concentrations from water selenium concentrations in the Elk Valley, British Columbia.

Previous studies conducted in the Elk River watershed showed that selenium concentrations are higher in aquatic biota in lentic compared to lotic habitats of the system having similar water selenium concentrations. Studies have also shown that water selenium concentrations have increased over time (~10% per year) and recent annual average concentrations have ranged up to 0.044 mg/L in areas downstream from mine discharges. For the present study, trophic transfer of selenium was characterized in lotic versus lentic habitats using concentrations measured in field-collected samples and assuming a three-step food chain of water to the base of the food web (biofilm), to benthic invertebrates, and then to westslope cutthroat trout (WCT) ovaries. Food chain models were developed for each habitat type (lotic and lentic) by combining linear regression equations for the three transfer relationships, allowing for prediction of fish ovary concentrations from water concentrations. Greater accumulation of selenium in lentic areas was mostly attributable to greater uptake at the base of the food chain compared to lotic areas. Enrichment/trophic transfer factors for selenium at all levels of the lotic and lentic food chains decreased and then became near constant as exposure concentrations increased. The lotic model predicted little increase in WCT ovary selenium concentrations over an eightfold increase in water concentrations (~0.005-0.040 mg/L), accounting for the lack of observed increase in within-area fish tissue concentrations over time despite increasing trends in water concentrations.

Developmental toxicity of selenium to Dolly Varden char (Salvelinus malma).

Gametes were collected from Dolly Varden char (Salvelinus malma) from waterbodies in a region exposed to mining-related selenium (Se) releases in British Columbia, Canada. Fertilized eggs were incubated in a laboratory and deformities were assessed on newly-hatched alevins using a graduated severity index. No effects were observed on egg or alevin survival or larval weight across the studied exposure range of 5.4 to 66 mg/kg dry weight in egg. Length of some larvae was reduced at the highest egg Se concentrations and a clear residue-response relationship was observed for larval deformity. The egg concentration corresponding to a 10% increase in the frequency of deformity (EC10) was 54 mg/kg dry weight, which is substantially higher than reported for other cold-water fish species.

Effects of dietary selenomethionine on cutthroat trout (Oncorhynchus clarki bouvieri) growth and reproductive performance over a life cycle.

A 2.5-year feeding trial was conducted in which cutthroat trout (Oncorhynchus clarki bouvieri) were fed either a basal diet (1.2 microg Se/g diet) or the basal diet supplemented with 2, 4, 6, 8, or 10 microg Se/g diet as selenomethionine from 1 g weight to maturation [corrected]. After 44 weeks of feeding, a subsample of fish was removed from dietary treatment groups and fed the basal diet for an additional 32 weeks. Concentrations of Se in whole fish and eggs increased in proportion to dietary Se intake, but no differences in growth, feed intake, survival, or egg hatchability were observed among dietary groups. Cranial-facial deformities in second-generation offspring were less than 6% in all treatment groups except for fish fed the diet supplemented with 4 microg Se/g diet as selenomethionine [corrected], where a 9.2% incidence was observed. Fish switched from selenomethionine-supplemented diets to the basal diet lost Se, calculated as microg Se lost/g weight gain, at 1.01, 2.84, 4.42, and 4.42 for dietary treatment groups 3, 4, 5, and 6, respectively. Results suggest no toxicity of dietary selenomethionine up to 10 microg/g supplemented diet and that with total life-cycle exposure, cutthroat trout increase Se excretion to maintain whole-body concentrations below toxic levels.

Masou salmon (Oncorhynchus masou) ethanol extract decreases 3-hydroxy-3-methylglutaryl coenzyme A reductase expression in diet-induced obese mice.

This study was designed to evaluate the hypocholesterolemic effects of masou salmon 70% ethanol extract (MSE) and to determine the molecular mechanism by which MSE exerts its effects in high-fat (HF) diet-induced obese mice. We hypothesize that the MSE may contain abundant n-3 fatty acids, so a diet containing MSE may also have hypolipidemic effects by assessing several key gene expressions in cholesterol metabolism such as the low-density lipoprotein (LDL) receptor, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and cholesterol 7alpha-hydroxylase (CYP7A1). To test this hypothesis, C57BL/6J mice were fed a 40% HF diet for 5 weeks, after which time the animals were fed an HF diet containing 0 mg/kg, 75 mg/kg, or 150 mg/kg MSE (HF, HF + MSE 1, and HF + MSE 2 groups, respectively) for an additional 4 weeks (n = 8 in each group, for a total of 24 mice). We found that feeding MSE with an HF diet prevented hypercholesterolemia in diet-induced obese mice; daily MSE feeding reduced total cholesterol levels in plasma and liver by 12.3% and 16.2%, respectively. Furthermore, we examined the expression of key cholesterol metabolism genes by reverse transcription-polymerase chain reaction and found that messenger RNA levels of HMG-CoA reductase were decreased by up to 5-fold, but the expression of both LDL receptor and CYP7A1 did not change. Thus, MSE may exert its hypocholesterolemic effect by altering the expression of HMG-CoA reductase.

Reproductive success, early life stage development, and survival of westslope cutthroat trout (Oncorhynchus clarki lewisi) exposed to elevated selenium in an area of active coal mining.

The effects of accumulated Se on the reproductive success and larval development of cutthroat trout (Oncorhynchus clarki lewis,) collected from a site of active coal mining in British Columbia were assessed. Eggs from 12 fish from an exposed site (Clode Pond) and 16 from a reference site (O'Rourke Lake) were field-collected and reared in the laboratory. Egg Se concentrations ranged from 12.3 to 16.7 and 11.8 to 140.0 microg/g dry weight (dw) from fish collected at the reference and exposed sites, respectively. Other studies, including those with this species, have not shown Se to affect egg viability; however, in the present study, eggs with Se concentrations > 86.3 microg/g dw were not successfully fertilized or were nonviable at fertilization, while eggs with concentrations > 46.8 and < 75.4 microg/g dw were fertilized (96% reached the eyed stage) but did not produce viable fry. A significant positive relationship between egg Se concentration and alevin mortality was observed. Deformities were analyzed in surviving fry which developed from eggs with Se concentrations between 11.8 and 20.6 microg/g dw. No relationship between Se concentration in eggs and deformities or edema was found in this range, suggesting that the no-effect threshold for deformity is > 20.6 microg/g dw. The present data, in conjunction with the data from several other studies in temperate fish, suggest that current Se thresholds are conservative for cold-water fish.

Expression of masu salmon delta5-desaturase-like gene elevated EPA and DHA biosynthesis in zebrafish.

Farmed fish could substitute for marine capture fish as a source of fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) beneficial for human health; however, they require these compounds in their diets. In the present study on a model fish species, we modified the EPA/DHA biosynthesis pathway by overexpression of masu salmon Delta5-desaturase-like gene in zebrafish to increase its ability to synthesize EPA and DHA. Expression of this gene in transgenic fish fed a commercial diet and Artemia helped to improve their EPA content by 1.21-fold and DHA by 1.24-fold. In similar fish that were fed only Artemia the increments were 1.14-fold for EPA and 1.13-fold for DHA, compared with nontransgenic fish. In contrast, eicosatetraenoic acid content decreased, as it is a substrate of Delta5-desaturase, while the total lipid remained constant. The results demonstrated that masu salmon Delta5-desaturase is functional in zebrafish and can modify its fatty acid metabolic pathway. The technique could be applied to farmed fish to generate a nutritionally richer product for human consumption.

Disturbance of plasma melatonin profile by high dose melatonin administration inhibits testicular maturation of precocious male masu salmon.

We have previously shown that the testicular development of underyearling male masu salmon Oncorhynchus masou reared under a long photoperiod was accelerated by oral melatonin treatment (0.5 mg melatonin/kg body weight/day), suggesting that melatonin mediates photoperiodic signaling. In this study, we further examined the effects of a disturbance in the plasma melatonin profile on gonadal development in underyearling male masu salmon by administering a higher dose of melatonin. Fish randomly selected in June were divided into two groups. They were reared under a light:dark (LD) cycle of 16:8 (lights on 04:00-20:00 hr) and fed with pellets sprayed with melatonin or vehicle twice a day at 08:30 and at 15:30 hr (7.5 mg melatonin/kg body weight/day) until October. Fish were sampled on Day 0, 25, 60, 90 and 120. The plasma melatonin levels were high in the dark phase and low in the light phase in the control group, while they were constantly high with no significant change in the melatonin-treated group. Melatonin treatment had inhibitory effects on the gonadosomatic index and plasma testosterone levels. Pituitary salmon gonadotropin-releasing hormone content and luteinizing hormone content were significantly lower in the melatonin-treated group on Day 60 and 90, respectively. These results indicate that the plasma melatonin profile is important for mediating photoperiodic signals that regulate brain-pituitary-gonadal axis in underyearling precocious male masu salmon.

Salt water-acclimated pink salmon fry (Oncorhynchus gorbuscha) develop stress-related visceral lesions after 10-day exposure to sublethal concentrations of the water-soluble fraction of North Slope crude oil.

Pink salmon fry. Oncorhynchus gorbuscha, after a 10-day exposure to one of two sublethal concentrations (25-54 microg x L(-1) or 178-348 microg x L(-1)) of the water-soluble fractions from Alaska North Slope crude oil, possessed morphologic and stress induced lesions in their hepatic, head kidney and gill tissues. Analysis of livers from oil-exposed fry revealed a variety of hepatocellular changes, including steatosis, nuclear pleomorphism. megalocytosis and necrosis. Epithelial proliferation of the bile ducts also occurred. An increase in the head kidney's interrenal cell nuclear diameter, a biomarker for stress responses, was correlated with hydrocarbon exposure. Gill abnormalities such as eqithelial lifting. fusion, mucous cell hyperplasia and vascular constriction were found in all test groups, but were more severe in fry given the high water soluble fraction of crude oil. The study demonstrated that sublethal exposure to the water-soluble fraction of crude oil results in multiple microscopic lesions (in several viscera) that are consistent with a pronounced response to environmental stress.

The effect of bioaccumulated selenium on mortalities and deformities in the eggs, larvae, and fry of a wild population of cutthroat trout (Oncorhynchus clarki lewisi).

To determine if elevated concentrations of waterborne selenium (Se), caused by coal mining, in the Elk River in southeastern British Columbia, may be causing reproductive or teratogenic effects in wild cutthroat trout (Oncorhynchus clarki lewisi), fertilized eggs from exposed and reference fish were raised in the laboratory. Eggs from each female were reared separately and the percent mortalities and deformities were related to the selenium content of the eggs. Selenium concentrations in females from the exposed site were highest in the liver (36.6 +/- 22.5 microg/g dry weight, range: 18.3 to 114), followed by the eggs (21.0 +/- 18.3 microg/g, range: 8.7 to 81.3) and the muscle (12.5 +/- 7.7 microg/g, range: 6.7 to 41). Despite these elevated egg Se concentrations, there was no significant effect on fertilization; time to hatch; percent hatch; or egg, larvae, and fry deformities or mortalities. Reproductive failure and embryonic terata have been reported at much lower egg Se concentrations in other fish species. The lack of any toxic response in this study may be due to an evolved tolerance to higher tissue Se concentrations in a population of fish living in a seleniferous river system.

Molecular cloning and evolution of transferrin cDNAs in salmonids.

Transferrin complementary DNAs were cloned from the livers of seven species in three genera of salmonids (kokanee salmon Oncorhynchus nerka, amago salmon Oncorhynchus masou ishikawa, masu salmon Oncorhynchus masou masou, Japanese char Salvelinus pluvius, brook trout Salvelinus fontinalis, lake trout Salvelinus namaycush, and brown trout Salmo trutta) subsequent to polymerase chain reaction amplification with primers derived from conserved regions of transferrin cDNA sequences. The transferrin cDNAs of the seven species of salmonids had sizes of 2.2 to 2.4 kb and encoded an open reading frame consisting of 691 amino acids with a putative signal peptide of 18 amino acids. The alignment of salmonid transferrin cDNAs showed a duplicated structure and conserved anion-binding residues, iron-binding residues, and cysteine residues for disulfide bridges. The deduced amino acid sequences of the seven salmonid transferrin cDNAs share 85% to 99% homology. A phylogenetic tree of amino acid sequences of transferrin cDNAs from salmonids showed that the relationship among the three genera of salmonids (Oncorhynchus, Salvelinus, and Salmo) is well correlated with that derived from classic morphologic and genetic analyses.

Ontogenetic development of gonadotropin-releasing hormone-like immunoreactive neurons in the brain of the chum salmon, Oncorhynchus keta.

In the chum salmon, gonadotropin-releasing hormone-immunoreactive (GnRH-ir) cells were first detected in the olfactory placode of embryos at 16 days after fertilization, and then appeared sequentially in the olfactory nerve and the distal part of the forebrain by hatching. Four months after hatching, the terminal nerve was differentiated along the olfactory nerve, and GnRH-ir fibers extended to the hypothalamus and hypophysis. GnRH-ir cells occurred later in the preoptic area in about 1-year-old juveniles. These data suggest that GnRH neurons originate in the olfactory placode and then migrate into the brain along the olfactory nerve.

Specific binding of [3H]pGlu-3-Me-His-Pro-NH2 ([3H]MeTRH) to hypothalamic membranes of juvenile rainbow trout, Oncorhynchus mykiss.

We investigated the existence and nature of specific [3H]pGlu-3-Me-His-Pro-NH2 ([3H]MeTRH) binding sites in juvenile rainbow trout (Oncorhynchus mykiss) hypothalamus. Washed hypothalamic membranes were incubated with [3H]MeTRH in the absence (B0) or presence of pGlu-His-Pro-NH2 (TRH) or MeTRH under various experimental paradigms; incubations were terminated by filtration and bound radioactivity was determined by liquid scintillation spectroscopy. Specific binding (Bsp) was tissue dependent, associable, dissociable, and thermolabile. Estimated rates of association (k+1) and dissociation (k-1) were 1.64 x 10(7) M-1 min-1 and 1.98 x 10(-2) min-1, respectively, providing a kinetically derived dissociation rate constant (Kd) of 1.21 x 10(-9) M. [3H]MeTRH binding was displaceable; LIGAND-analysis of three independent homologous displacement experiments consistently indicated a single class of binding sites with an average Kd = 6.91 (+/- 4.32) x 10(-9) M and average maximum binding capacity (Bmax) of 8.84 (+/- 2.72) x 10(-15) mol/mg protein. Native TRH also displaced the radiolabel in a dose dependent manner; LIGAND-estimates for Kd and Bmax were 1.52 (+/- 0.12) x 10(-9) M and 3.79 (+/- 0.99) x 10(-15) mol/mg protein (n = 3 experiments), respectively. Our data indicate that presence of a single class of specific high-affinity TRH-binding sites in the rainbow trout hypothalamus; these findings suggest a role for TRH in regulating the release of hypophysiotrophic factors in the teleost hypothalamus.