A bioinformatics-based update on microRNAs and their targets in rainbow trout (Oncorhynchus mykiss)

MicroRNAs (miRNAs) participate in various vitally biological processes via controlling target genes activity and thousands of miRNAs have been identified in many species to date, including 18,698 known animal miRNA in miRBase. However, there are only limited studies reported in rainbow trout (Oncorhynchus mykiss) especially via the computational-based approaches. In present study, we systematically investigated the miRNAs in rainbow trout using a well-developed comparative genome-based homologue search. A total of 196 potential miRNAs, belonging to 124 miRNA families, were identified, most of which were firstly reported in rainbow trout. The length of miRNAs ranged from 17 to 24 nt with an average of 20 nt while the length of their precursors varied from 47 to 152 nt with an average of 85 nt. The identified miRNAs were not evenly distributed in each miRNA family, with only one member per family for a majority, and multiple members were also identified for several families. Nucleotide U was dominant in the pre-miRNAs with a percentage of 30.04%. The rainbow trout pre-miRNAs had relatively high negative minimal folding free energy (MFE) and adjusted MFE (AMFE). Not only the mature miRNAs but their precursor sequences are conserved among the living organisms. About 2466 O. mykiss genes were predicted as potential targets for 189 miRNAs. Gene Ontology (GO) analysis showed that nearly 2093, 2107, and 2081 target genes are involved in cellular component, molecular function, and biological processes respectively. KEGG pathway enrichment analysis illuminated that these miRNAs targets might regulate 105 metabolic pathways, including those of purine metabolism, nitrogen metabolism, and oxidative phosphorylation. This study has provided an update on rainbow trout miRNAs and their targets, which represents a foundation for future studies.     

Effect of salinity on flavin-containing monooxygenase expression and activity in rainbow trout (Oncorhynchus mykiss)

In order to obtain more information about the physiological role(s) of flavin-containing monooxygenases (FMOs) in euryhaline teleost fishes, two experimental series were performed using adult and juvenile rainbow trout (Oncorhynchus mykiss). Cannulated adult trout were exposed to freshwater or 21% seawater for 48 h, whereas juvenile trout were acclimated to one of four different salinities: freshwater, 7%, 14%, or 21% during a 2-week period. FMO expression and activity were determined in red blood cells (RBC), liver, gill, kidney, gut, heart and brain. Furthermore, the content of trimethylamine oxide (TMAO; an FMO metabolite and an osmolyte) as well as urea were determined in various tissues. FMO expression and activity increased significantly and in a salinity dependent manner in osmoregulatory organs (gills, kidney and gut) in both juveniles and adult trout and, furthermore, in RBC in adults. No significant changes were observed in liver or heart. Urea content increased significantly and in a salinity dependent manner in all tissues, whereas TMAO was accumulated primarily in muscle tissue. Salinity dependent adjustment of FMO expression and activity primarily in osmoregulatory organs as well as regulation of TMAO content in muscle is consistent with previous studies showing an association of FMO with osmoregulation in euryhaline teleosts. However, the lack of a parallel increase of TMAO with urea in other tissues of fish at high salinity indicates other mechanisms of protection from intracellular urea may exist in non-muscular tissues.     

Modulation of antioxidant defence system in brain of rainbow trout (Oncorhynchus mykiss) after chronic carbamazepine treatment

We investigated the effect of long-term exposure to CBZ on the antioxidant system in brain tissue of rainbow trout. Fish were exposed to sublethal concentrations of CBZ (1.0 μg/L, 0.2 mg/L or 2.0 mg/L) for 7, 21, and 42 days. Oxidative stress indices (LPO and CP) and activities of antioxidant enzymes (SOD, CAT, GPx and GR) in fish brain were measured. In addition, non-enzymatic antioxidant (GSH) was determined after 42 days exposure. Carbamazepine exposure at 0.2 mg/L led to significant increases (p < 0.05) of LPO and CP after 42 days and, at 2.0 mg/L, after 21 days. Activities of the antioxidant enzymes SOD, CAT, and GPx in CBZ-treated groups slightly increased during the first period (7 days). However, activities of all measured antioxidant enzymes were significantly inhibited (p < 0.05) at 0.2 mg/L exposure after 42 days and after 21 days at 2.0 mg/L. After 42 days, the content of GSH in fish brain was significantly lower (p < 0.05) in groups exposed to CBZ at 0.2 mg/L and 2.0 mg/L than in other groups. Prolonged exposure to CBZ resulted in excess reactive oxygen species formation, finally resulting in oxidative damage to lipids and proteins and inhibited antioxidant capacities in fish brain. In short, a low level of oxidative stress could induce the adaptive responses of antioxidant enzymes, but long-term exposure to CBZ could lead to serious oxidative damage in fish brain.     

Ecotoxocological effects of short-term exposure to a human pharmaceutical Verapamil in juvenile rainbow trout (Oncorhynchus mykiss)

Verapamil (VRP) is a calcium channel blocker that is a highly prescribed compound and commonly present in aquatic environment, but the ecotoxicological effects of this pharmaceutical in fish have not been fully documented. In this study, the toxic effects of VRP were studied in juvenile rainbow trout, Oncorhynchus mykiss, by acute static bioassay. In the acute test, the median lethal concentration (LC50, 2.72 mg/L) was evaluated and the behavioral changes were obviously intensified with increasing VRP concentrations. Compared to the control, oxidative stress was observed in fish tissues with different levels after short-term exposure to sublethal concentrations (0.27 and 1.35 mg/L) of VRP. Activities of SOD and GPx in fish brain were induced at 0.27 mg/L VRP, but all the antioxidant enzymes (SOD, GPx and GR) in fish brain were decreased at 1.35 mg/L VRP. When compared to the control, all the antioxidant enzymes in gill were decreased in both treated groups, but there was no significant change in muscle. Additional, muscle DNA/RNA ratio in fish exposed at 1.35 mg/L VRP was significantly lower than that in the control. Furthermore, through chemometrics of all parameters measured in fish exposed to sublethal VRP concentrations using principal component analysis, two groups with 89.8% of total accumulated variance were distinguished. In short, the physiological and biochemical responses in of fish indicated that VRP-induced environmental stress; but according to VRP residual status in the natural environment, more long-term experiments at lower concentrations will be necessary in the future.     

Reduction in swimming performance in juvenile rainbow trout (Oncorhynchus mykiss) following sublethal exposure to pyrethroid insecticides

While the lethal toxicity of pyrethroid insecticides to fish is well documented, their sublethal physio-behavioral effects remain poorly characterized. Known pyrethroid-associated changes to insect neuromuscular function may translate into similar effects in fish, thereby altering swimming ability and affecting foraging, predator avoidance, and migration. Three experiments were conducted using critical (U_crit) and burst (U_max) swimming speeds to assess the sublethal effects of the pyrethroids permethrin and deltamethrin in juvenile rainbow trout (Oncorhynchus mykiss). Fish were exposed to deltamethrin (100, 200, or 300 ng/L) or permethrin (1, 2, or 3 μg/L) in water for 4 d, and assessed for swimming performance. Deltamethrin (200 and 300 ng/L) reduced U_crit, but not U_max, while both swim performance measurements were unaffected by permethrin. Subsequent experiments used only U_crit to assess deltamethrin exposure. In a time course experiment, deltamethrin (300 ng/L) reduced U_crit after 1 and 4 d of exposure, but after 7 d of exposure U_crit was fully recovered. Finally, deltamethrin (1, 2, or 3 μg/L) reduced U_crit after 1 h bath exposures similar to recommended protocols for deltamethrin based sea-lice treatment in aquaculture. The real-world implications of the revealed pyrethroid-associated swimming ability reductions in salmon may be important in areas close to aquaculture facilities.     

Transfection efficiency and cytotoxicity of cationic liposomes in primary cultures of rainbow trout (Oncorhynchus mykiss) gill cells

Immunisation of fish by immersion has been applied for inactivated, whole cell bacterins, where the gill epithelial cells are considered as one of the prime uptake sites. Antigen entry is a critical factor for delivery of vaccine antigens through the immersion route, also for DNA vaccines, and delivery systems like cationic liposomes may enhance uptake. In this study, the aim was to examine the efficiency of cationic liposomes as a means to transfect primary cultures of rainbow trout gill cells with plasmids encoding viral or reporter proteins. Furthermore, the effects of the concentration and composition of liposomes/lipoplex on the viability of the cells were evaluated. Transfection of the gill cells was possible with both plasmids following transfection with lipoplexes of a neutral charge. Low concentrations and neutral/negatively charged formulations were favourable with respect to the toxicity of the formulations. Given that the mucous barrier covering the gills is overcome, this system might be useful for the priming of the local immunity in the fish gills.     

Histopathological changes induced by maneb and carbaryl on some tissues of rainbow trout, Oncorhynchus mykiss

Acute toxicity of the pesticides, maneb and carbaryl, to juvenile rainbow trout were evaluated under static-renewal test conditions. Actual concentrations of maneb ranged from 0.10 mg/L to 2.00 mg/L and carbaryl ranged from 0.20 mg/L to 3.90 mg/L. The concentrations of maneb that killed 50% of the rainbow trout (3.27 ± 0.9 g) within 24-h (24-h; LC₅₀), 48-h, 72-h and 96-h were 1.19 ± 0.12, 1.04 ± 0.11, 0.92 ± 0.12 and 0.81 ± 0.14 mg/L (95% confidence limits), respectively. LC₅₀ values of carbaryl for 24-h, 48-h, 72-h and 96-h were 2.52 ± 0.71, 2.16 ± 0.63, 1.71 ± 0.46 and 1.39 ± 0.15 mg/L, respectively. None of the unexposed control fish died and the first fish died 6 h after exposure to maneb (≥1.30 mg/L), and carbaryl (≥2.60 mg/L). Lamellar edema, separation of epithelium from lamellae, lamellar fusion, swelling of the epithelial cells and epithelial cell necrosis were observed on maneb and carbaryl exposed fish. Gills also had scattered areas of focal lamellar hyperplasia. Fish exposed to pesticides had inflammation and focal necrosis in liver, trunk kidney and spleen. Maneb and carbaryl had similar histopathological lesions. In order, the most affected organs were gill, trunk kidney and liver.     

Disparate infection patterns of Ceratomyxa shasta (Myxozoa) in rainbow trout (Oncorhynchus mykiss) and Chinook salmon (Oncorhynchus tshawytscha) correlate with internal transcribed spacer-1 sequence variation in the parasite

Ceratomyxa shasta is a virulent myxosporean parasite of salmon and trout in the Pacific Northwest of North America. The parasite is endemic in the Klamath River, Oregon/California, where a series of dams prevent movement of fish hosts between the upper and lower parts of the basin. Ceratomyxa shasta exhibits a range of infection patterns in different fish species above and below the dams. We hypothesised that the variations in infection and disease are indicators that different strains of the parasite exist, each with distinct host associations. Accordingly, we sought to identify strain-specific genetic markers in the ssrRNA and internal transcribed spacer region 1 (ITS-1). We examined 46 C. shasta isolates from water samples and two fish hosts, from June 2007 field exposures at upper and lower Klamath River sites with similarly high parasite densities. We found 100% of non-native rainbow trout became infected and died at both locations. In contrast, mortality in native Chinook salmon was <10% in the upper basin, compared with up to 40% in the lower basin. Parasite ssrRNA sequences were identical from all fish. However, ITS-1 sequences contained multiple polymorphic loci and a trinucleotide repeat (ATC)_0-3 from which we defined four genotypes: 0, I, II and III. Non-native rainbow trout at both sites were infected with genotype II and with a low level of genotype III. Chinook salmon in the upper basin had genotypes II and III, whereas in the lower basin genotype I predominated. Genotype I was not detected in water from the upper basin, a finding consistent with the lack of anadromous Chinook salmon there. Genotype O was only detected in water from the upper basin. Resolution of C. shasta into sympatric, host-specific genotypes has implications for taxonomy, monitoring and management of this significant parasite.     

Characterization of Ni transport into brush border membrane vesicles (BBMVs) isolated from the kidney of the freshwater rainbow trout (Oncorhynchus mykiss)

The transport of nickel (Ni) across the renal brush border membrane of the rainbow trout was examined in vitro using brush border membrane vesicles (BBMVs). Both transmembrane transport of Ni into an osmotically active intravesicular space, and binding of Ni to the brush border membrane itself, were confirmed. Nickel (Ni) uptake fitted a two component kinetic model. Saturable, temperature-dependent transport dominated at lower Ni concentrations, with a moderate linear diffusive component of Ni transport apparent at higher Ni concentrations. An affinity constant (K_m) for Ni transport within the specifically described vesicular media was calculated as 17.9 ± 1.9 μM, the maximal rate of transport (J_max) was calculated as 108.3 ± 3.7 nmol mg protein⁻¹ min⁻¹, and the slope of the linear diffusive component was calculated as 0.049 ± 0.005 nmol mg protein⁻¹ min⁻¹ per μM of Ni. Efflux of Ni from BBMVs was fitted to an exponential decay curve with a half-time (T_1/2) of 125.2 ± 7.3 s. Ni uptake into renal BBMVs was inhibited by magnesium at a 100:1 Mg to Ni molar ratio, and by magnesium and calcium at a 1000:1 molar ratio. In the presence of histidine at a 100:1 histidine to Ni ratio, Ni uptake was almost completely abolished. At a 1:1 molar ratio, histidine inhibited Ni uptake by approximately 50%. Ni-histidine complexation was rapid, with a T_1/2 of 12.2 s describing the Ni-histidine equilibration time needed to inhibit Ni uptake into renal BBMVs by 50%. Characterization of Ni transport across cellular membranes is an important step in understanding both the processes underlying homeostatic regulation of Ni, and the toxicological implications of excessive Ni exposure in aquatic ecosystems.     

Effects of body size on biochemical characteristics of trabecular cardiac muscle and plasma of rainbow trout (Oncorhynchus mykiss)

The objective of this study was to characterize biochemical indices of oxidative metabolism in trabecular cardiac muscle of female rainbow trout over a 200-fold size range. We also examined scaling effects on plasma concentrations of protein, albumin, and non-esterified fatty acids (NEFA). Animals were sampled from four size categories (<20 g, 100–200 g, 300–400 g and >2 kg). Ventricle mass relative to body mass was size-dependent, with the smallest trout having smaller hearts. Total protein in cardiac tissue was 20% higher in animals weighing over 300 g compared to fish less than 200 g. Plasma albumin and protein were increased 50–70% in trout over 100 g compared to fish smaller than 20 g. NEFA in plasma were similar for all animals. Activities of carnitine palmitoyltransferase and cytochrome oxidase were elevated 53% and 38%, respectively, in trabecular cardiac muscle of the largest trout compared to the smallest animals. Citrate synthase activity was independent of heart size, suggesting that the increase in oxidative capacity was not due to an increase in mitochondrial density. The increased capacity to bind and transport fatty acids in blood and the higher oxidative capacity of cardiac muscle may reflect a metabolic adaptation for increased oxidation of fatty acids and ventricular performance in larger female trout. These findings are not consistent with the scaling paradigm of oxidative metabolism and may reflect changes in ventricular architecture with ontogeny.     

Gastro-intestinal transport of calcium and cadmium in fresh water and seawater acclimated trout (Oncorhynchus mykiss)

Transport of calcium (Ca) and cadmium (Cd) was examined along the gastro-intestinal tract (GIT) of freshwater and seawater Oncorhynchus mykiss irideus (FWT and SWTies respectively) using in vitro and in vivo experiments. Based on known physiological differences between FWT and SWT which aid in regulating ion levels and osmolarity, we hypothesized that SWT would have lower rates of Ca uptake. Also, we predicted that Cd rates would also be lower because Cd is known to share a common transport mechanism with Ca. Kinetics of Ca and Cd transport were determined using mucosal salines of varying concentrations [1, 10, 30, 60, and 100 (mmol L^− 1 for Ca, μmol L^− 1 for Cd)]. Linear and saturating relationships were found for Ca for FWT and SWT, but overall SWT had lower rates. Linear and/or saturating relationships were also found for Cd uptake, but rates varied little between fish types. Elevated Ca had no inhibitory effect on Cd transport, and Ca channel blockers nifedipine and verapamil had little effect on Ca or Cd uptake. However, lanthanum reduced Ca transport into some compartments. A 21 day in vivo feeding experiment was also performed where FWT and SWT were exposed to control diets or Cd-spiked diets (552 μg Cd g^− 1 food). Whole body Cd uptake between fish types was similar, but the majority of Cd in SWT remained in the posterior intestine tissue, while FWT transported more Cd through their gut wall. Overall it appears that large differences in Ca and Cd uptake between FWT and SWT exist, with SWT generally having lower rates.     

A putative serine protease,SpSsp1, from Saprolegnia parasitica is recognised by sera of rainbow trout, Oncorhynchus mykiss

Saprolegniosis, the disease caused by Saprolegnia sp., results in considerable economic losses in aquaculture. Current control methods are inadequate, as they are either largely ineffective or present environmental and fish health concerns. Vaccination of fish presents an attractive alternative to these control methods. Therefore we set out to identify suitable antigens that could help generate a fish vaccine against Saprolegnia parasitica. Unexpectedly, antibodies against S. parasitica were found in serum from healthy rainbow trout, Oncorhynchus mykiss. The antibodies detected a single band in secreted proteins that were run on a one-dimensional SDS-polyacrylamide gel, which corresponded to two protein spots on a two-dimensional gel. The proteins were analysed by liquid chromatography tandem mass spectrometry. Mascot and bioinformatic analysis resulted in the identification of a single secreted protein, SpSsp1, of 481 amino acid residues, containing a subtilisin domain. Expression analysis demonstrated that SpSsp1 is highly expressed in all tested mycelial stages of S. parasitica. Investigation of other non-infected trout from several fish farms in the United Kingdom showed similar activity in their sera towards SpSsp1. Several fish that had no visible saprolegniosis showed an antibody response towards SpSsp1 suggesting that SpSsp1 might be a useful candidate for future vaccination trial experiments.     

Comparative genome mapping reveals evidence of gene conversion between Sox9 paralogs of rainbow trout (Oncorhynchus mykiss)

Considerable evidence suggests that one genome duplication event preceded the divergence of teleost fishes and a second genome duplication event occurred before the radiation of teleosts of the family Salmonidae. Two Sox9 genes have been isolated from a number of teleosts and are called Sox9a and Sox9b. Two Sox9 gene copies have also been isolated from rainbow trout, a salmonid fish and are called Sox9 and Sox9α2. Previous evaluations of the evolutionary history of rainbow trout Sox9 gene copies using phylogenetic reconstructions of their coding regions indicated that they both belong to the Sox9b clade. In this study, we determine the true evolutionary history of Sox9 gene copies in rainbow trout. We show that the locus referred to as Sox9 in rainbow trout is itself duplicated. Mapping of the duplicated Sox9 gene copies indicates that they are co-orthologs of Sox9b while mapping of Sox9α2 indicates that it is an ortholog of Sox9a. This relationship is supported by phylogenetic reconstruction of Sox9 gene copies in teleosts using their 3′ untranslated regions. The conflicting phylogenetic topology of Sox9 genes in rainbow trout indicates the occurrence of gene conversion events between Sox9 and Sox9α2 which is supported by a number of recombination analyses.     

Acid-base disequilibrium in the venous blood of rainbow trout (Oncorhynchus mykiss)

Acid-base equilibria/disequilibria were evaluated in vivo in post-branchial arterial blood and pre-branchial venous blood of freshwater rainbow trout (Oncorhynchus mykiss). This was accomplished using arterial and venous extracorporeal circuits in conjunction with a stopped-flow apparatus. After the abrupt stoppage of circulating post-branchial blood within the stopped-flow apparatus, pH increased slowly ([Delta]pH = +0.032 ± 0.004 pH units; n = 15), thus confirming the existence of an acid-base disequilibrium state in the arterial blood of rainbow trout. The slow downstream pH changes were unaffected by prior treatment of fish with the carbonic anhydrase inhibitor benzolamide (1.2 mg kg^-1; [Delta]pH = +0.032 ± 0.01 pH units; n = 5) but were eliminated after intra-vascular injection of 10 mg kg^-1 bovine carbonic anhydrase ([Delta]pH = -0.011 ± 0.003 pH units; n = 8). These results demonstrate that the acid-base disequilibrium in the arterial blood reflects a total absence of extracellular carbonic anhydrase activity. Similar stopped-flow experiments revealed the existence of a reduced, yet significant, acid-base disequilibrium in the venous blood circulating within the caudal vein ([Delta]pH = +0.004 ± 0.003 pH units; n = 15). Selective inhibition of extracellular carbonic anhydrase using benzolamide did not significantly influence the magnitude of the venous pH disequilibrium ([Delta]pH = +0.007 ± 0.007 pH units; n = 8) whereas intra-vascular injection of carbonic anhydrase eliminated the pH disequilibrium. These results demonstrate that extracellular carbonic anhydrase, although reported to be present within the skeletal muscle of rainbow trout, does not accelerate post-capillary pH changes in the venous circulation.     

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.     

Blood oxygenation and cardiorespiratory function in steelhead trout (Oncorhynchus mykiss) challenged with an acute temperature increase and zatebradine-induced bradycardia

To explore whether temperature-dependent increases in cardiac output (Q) are mediated solely through heart rate (f_H) in fish to ensure adequate/efficient blood oxygenation, we injected steelhead trout with saline (control) or zatebradine hydrochloride (1.0 mg kg⁻¹), and measured blood oxygen status, cardiorespiratory variables and cardiorespiratory synchrony during a critical thermal maximum (CT_Max) test. The increasing temperature regimen itself (from 12 °C to CT_Max) resulted in large decreases in arterial oxygen partial pressure (P_aO₂) and content (C_aO₂) (by ∼35% and 25%, respectively). Further, there was little evidence of cardiorespiratory synchrony at 12 °C, and the number of fish that showed synchrony at high temperatures only increased marginally (to 3 out of 7) despite the large decrease in P_aO₂. These results: (1) indicate that in some situations (e.g. when ventilation is exclusively/predominantly dependent on buccal–opercular pumping) the upper thermal tolerance of fish may be constrained by both cardiovascular and ventilatory performance; and (2) question the importance of cardiorespiratory synchrony (ventilation–perfusion matching) for gas exchange in salmonids, and fishes, in general.     

Hypothermic storage of isolated spermatogonia and oogonia from rainbow trout (Oncorhynchus mykiss)

A growing number of fish species are endangered due to human activities. A short- or long-time preservation of gametes could conserve genetic resources of threatened fish species. The aim of this study was to evaluate a hypothermic condition for short-term preservation of spermatogonia and oogonia cells isolated from immature transgenic rainbow trout, Oncorhynchus mykiss, and to determine the maximum time point for further transplantation. Viability rate of germ cells was investigated after isolation and during storage at 4 °C up to 24 h. Dulbecco's modification of Eagle's medium supplemented with Hepes fetal bovine serum and l-glutamine was used as hypothermic storage media. The results showed that while viability decreased following 24 h storage, the remaining viable cells did not vary morphologically as well as GFP intensity retained similar to those observed in freshly isolated cells. The hypothermal storage study indicated that culture medium is suitable for preserving germ cells in the short periods of time. Simplicity, easily available culture media and low cost provide new insight into hypothermic conditions for preserving and transporting of germ cells for next applied and basic studies.