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.     

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.     

Somatostatin inhibits insulin-like growth factor-I receptor expression in the gill of a teleost fish (Oncorhynchus mykiss)

Rainbow trout gill tissue was used to examine the role of somatostatin (SS) on insulin-like growth factor-I (IGF-I) receptor expression. In vivo implantation of fish with somatostatin-14 (SS-14) reduced expression of IGF-I receptor mRNAs as well as [(125)I]-IGF-I binding. In vitro incubation of gill filaments with SS-14 or various SS isoforms, including SS-28 and [Tyr(7), Gly(10)]-SS-14-containing peptides, directly inhibited IGF-I receptor mRNA expression. SS-14 also inhibited [(125)I]-IGF-I binding in vitro. These data indicate that SSs inhibit the mRNA and functional expression of IGF-I receptors in gill, and suggest that SSs regulate growth in an extrapituitary manner by reducing sensitivity to IGF-I.     

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     

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.     

Long-term primary culture of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver

Summary Long-term primary cultures of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver have been established. Nearly homogenous (>97%) populations of hepatocytes were placed into primary culture and remained viable and proliferative for at least 70 d. In addition to hepatocytes, proliferative biliary cells persisted in the cultures for at least 30 d. Finally, a third type of epithelial cell, which we have termed a “spindle cell,” consistently appeared and proliferated to confluence in these cultures. The confluent cultures of spindle cells were successfully subcultured and passaged. The initial behavior, growth, and optimization of serum and media requirements for these cells is described. All three cell types proliferated as measured by thymidine incorporation, autoradiography, proliferating cellular nuclear antigen analysis, and propidium iodine staining. Further efforts to characterize the cells included western blotting and immunohistochemical staining with antibodies to cytokeratins previously reported in fish liver. From these data, it appears that all three cell populations are epithelial in nature. Furthermore, significant changes in actin organization, often indicative of transformation or pluripotent cells, were observed with increased time in primary culture.     

Lipopolysaccharide O-antigen prevents phagocytosis of Vibrio anguillarum by rainbow trout (Oncorhynchus mykiss) skin epithelial cells

Colonization of host tissues is a first step taken by many pathogens during the initial stages of infection. Despite the impact of bacterial disease on wild and farmed fish, only a few direct studies have characterized bacterial factors required for colonization of fish tissues. In this study, using live-cell and confocal microscopy, rainbow trout skin epithelial cells, the main structural component of the skin epidermis, were demonstrated to phagocytize bacteria. Mutant analyses showed that the fish pathogen Vibrio anguillarum required the lipopolysaccharide O-antigen to evade phagocytosis and that O-antigen transport required the putative wzm-wzt-wbhA operon, which encodes two ABC polysaccharide transporter proteins and a methyltransferase. Pretreatment of the epithelial cells with mannose prevented phagocytosis of V. anguillarum suggesting that a mannose receptor is involved in the uptake process. In addition, the O-antigen transport mutants could not colonize the skin but they did colonize the intestines of rainbow trout. The O-antigen polysaccharides were also shown to aid resistance to the antimicrobial factors, lysozyme and polymyxin B. In summary, rainbow trout skin epithelial cells play a role in the fish innate immunity by clearing bacteria from the skin epidermis. In defense, V. anguillarum utilizes O-antigen polysaccharides to evade phagocytosis by the epithelial cells allowing it to colonize rapidly fish skin tissues.     

Spatial arrangement of fish gill secondary lamellar cells in intact and dissociated tissues from rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

Gill structure of rainbow trout and Atlantic salmon was investigated using cell disaggregation and dry fracture techniques for scanning electron microscopy (SEM), allowing new interpreta-tions of the structure of the secondary lamella. The basement membrane underlying the lamellar epithelium (secondary epithelium) was shown to be a tough sheet with numerous depressions corresponding to underlying pillar cells. This membrane is probably the most important structural element of the secondary lamella, capable of withstanding considerable mechanical stress. For the first time the structure of the apical surface of the secondary lamella was shown by SEM to consist of an outer microridged coat overlying a fibrous coat which appears continuous with the extracellular matrix surrounding the rest of the cell. When cells were detached they rounded up and the external microridged coat became more vesicle like, indicating the labile nature of this coat. In cell suspension preparations, epithelial, mucus and chloride cells are present as well as many blood derived cells such as erythrocytes, presumptive leucocytes and thrombocytes.     

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.     

Calcium signalling in isolated single chromaffin cells of the rainbow trout (Oncorhynchus mykiss)

Chromaffin cells were isolated from the posterior cardinal vein of rainbow trout (Oncorhynchus mykiss) to assess their suitability as a model system for studying mechanisms of catecholamine secretion in fish and to evaluate intracellular calcium changes associated with cholinoreceptor stimulation. Immunocytochemistry in concert with fluorescence microscopy was employed to identify characteristic chromaffin cell proteins and thus to confirm the presence of these specific cells in suspensions and cultures. Dopamine-β-hydroxylase, an enzyme of the catecholamine-synthesising Blaschko pathway, was identified in cytoplasmic vesicles of the isolated chromaffin cells. The actin filament-severing protein, scinderin, was co-localized with actin in the sub-plasmalemmal membrane of these chromaffin cells. Intracellular calcium [Ca²⁺]_i was measured in single chromaffin cells by microspectrofluorometry using the fluorescent dye Fura-2. Significant increases in [Ca²⁺]_i were observed in chromaffin cells in response to depolarisation of the cell membrane by high concentrations of K⁺ or by the stimulation of the cell by the cholinergic receptor agonists, nicotine, acetylcholine or carbachol. The response to the reversible agonist, nicotine, was attenuated following addition of the nicotinic receptor blocker hexamethonium. Such attenuation, however, did not occur when hexamethonium was added after stimulation with the non-specific irreversible cholinergic agonist, carbachol. These results demonstrate the presence of functional cholinoreceptors, linked to intracellular calcium signalling, on isolated trout chromaffin cells and reveal the potential of these cells as a model system for studying aspects of catecholamine secretion in fish.     

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.     

The expression of immune-regulatory genes in rainbow trout, Oncorhynchus mykiss, during amoebic gill disease (AGD)

Amoebic gill disease (AGD) is an ectoparasitic disease caused by infection with the protozoan Neoparamoeba sp. and is characterised by epithelial hyperplasia that manifests as gill lesions. In order to examine the nature of the immune response to AGD, the expression of a range of immune-regulatory genes was examined in naïve uninfected rainbow trout, Oncorhynchus mykiss, and naïve rainbow trout subjected to a laboratory-induced AGD infection. The immune-regulatory genes examined were interleukin-1 beta isoform 1 (IL-1β1), tumour necrosis factor alpha isoforms 1 and 2 (TNF-α1, TNF-α2), interleukin-8 (IL-8), transforming growth factor beta isoform 1 (TGF-β1), inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), major histocompatibility complex beta chain (MHC-II β-chain) and T-cell receptor beta chain (TCR β-chain). Immune-regulatory genes that were up/down-regulated in AGD-infected trout compared to uninfected controls at 0, 7, and 14 days post-inoculation (p.i.) in gill, liver and anterior kidney tissue were initially identified by means of semi-quantitative RT–PCR. Up/down-regulated immune-regulatory genes were subsequently quantitated and validated by real-time RT–PCR (qRT–PCR). The extent of AGD-associated pathology was consistent amongst all AGD-infected trout at 7 days p.i. and increased considerably by 14 days p.i. At both 7 and 14 days p.i. IL-1β1 and iNOS gene expression was significantly up-regulated in the gills, and IL-8 was significantly up-regulated in the liver of AGD-infected trout at 7 days p.i. These data demonstrate the involvement of the immune response to AGD at the molecular level, and indicate the importance of this response at the site of infection and the possible involvement of a systemic immune response.     

Characterization of probiotic carnobacteria isolated from rainbow trout (Oncorhynchus mykiss) intestine

Aims:  To characterize two probiotic carnobacterial isolates, Carnobacterium maltaromaticum (B26) and C. divergens (B33), derived from rainbow trout ( Oncorhynchus mykiss ) intestine.
Methods and Results:  Both cultures, which were able to colonize the fish gut mucosal layer, comprised nonsporogenous, nonmotile, Gram-positive, catalase and oxidase-negative rods. The growth of both carnobacteria occurred between 0 and 37°C, in 0–10% (w/v) NaCl and at pH 5–10. Specifically, strain B26 grew in nutrient broth supplemented with 15% (w/v) NaCl. The most abundant cellular fatty acid of both cultures was 9-octadecenoic acid (18 : 1 n -9) (B26 = 52·6%; B33 = 40·6%), which was characteristic of Carnobacterium . Both cultures were inhibitory to Aeromonas salmonicida , Aer. hydrophila , Streptococcus iniae and Vibrio anguillarum , and strain B33 inhibited Listeria monocytogenes . Both carnobacteria, which did not contain plasmids, produced inhibitory compounds against Gram-positive and Gram-negative bacteria.
Conclusions:  Both probiotic cultures, B26 and B33, had unique phenotypic characteristics and showed a broad spectrum of antibiotic resistance against varying pathogenic bacteria.
Significance and Impact of the Study:  The results of this study contribute to new information and significance of carnobacterial species.     

The energetic cost of protein synthesis in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss)

Summary To establish the energetic cost of protein synthesis, isolated trout hepatocytes were used to measure protein synthesis and respiration simultaneously at a variety of temperatures. The presence of bovine serum albumin was essential for the viability of isolated hepatocytes during isolation, but, in order to measure protein synthesis rates, oxygen consumption rates and RNA-to-protein ratios, BSA had to be washed from the cells. Isolated hepatocytes were found to be capable of protein synthesis and oxygen consumption at constant rates over a wide range of oxygen tension. Cycloheximide was used to inhibit protein synthesis. Isolated hepatocytes used on average 79.7±9.5% of their total oxygen consumption on cycloheximide-sensitive protein synthesis and 2.8±2.8% on maintaining ouabain-sensitive Na⁺/K⁺-ATPase activity. The energetic cost of protein synthesis in terms of moles of adenosine triphosphate per gram of protein synthesis decreased with increasing rates of protein synthesis at higher temperatures. It is suggested that the energetic cost consists of a fixed (independent of synthesis rate) and a variable component (dependent on synthesis rate).Abbreviations BSA bovine serum albumin - dpm disintegrations per min - k _s fractional rate of protein synthesis - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid - PHE phenylalanine; PO₂ oxygen tension - PCA perchloric acid     

Comparative metabolic profiling of chloramphenicol by isolated hepatocytes from rat and trout (Oncorhynchus mykiss)

1. Isolated liver cells from rat and rainbow trout were used to study the biotransformation of labelled chloramphenicol (CP).2. The ³H-CP metabolic rates were 1.7 nmol/hr/10⁶ rat hepatocytes and 0.2nmol/hr/10⁶ trout hepatocytes.3. In rat, as in trout liver cells, the major metabolite was CP-glucuronide. In addition to this conjugate, CP-base, CP-alcohol and CP-oxamic acid were detected in significant amounts.4. These results are compared with in vivo data on CP metabolism previously obtained in our laboratory in fish and mammals.     

Adhesion of high and low virulence Flavobacterium psychrophilum strains to isolated gill arches of rainbow trout Oncorhynchus mykiss

The ability of Flavobacterium psychrophilum to adhere to the gill tissue of rainbow trout Oncorhynchus mykiss was evaluated. A gill perfusion model was adopted, offering a number of advantages compared to other in vitro as well as in vivo models. A comparison between the adhesion capacity of a high and low virulence F. psychrophilum strain was made. Experiments were additionally carried out to assess the influence of water quality (organic material, nitrite) and temperature on the adhesion process of the bacterial cells. The high virulence strain attached more readily to the gill tissue than did the low virulence strain. Moreover, the adherence of the high virulence strain of F. psychrophilum was influenced by a number of factors. These were immersion of the gill arches in water to which organic material or nitrite were added, and elevated temperature. The former 2 increased the adhesion ability, while the latter had a negative influence on the adherence process.     

Culturing and characterization of astrocytes isolated from juvenile rainbow trout (Oncorhynchus mykiss)

An access to brain cell cultures from fish would enable screening of possible neurotoxic chemicals contaminating the aquatic environment. In the present study, a protocol for a successful routine isolation and culturing of brain cells from juvenile rainbow trout was worked out. The coating material was shown to be of importance for cell proliferation. Cells grow better on a surface coated with laminin than on those coated with poly-L-lysine (PLL), poly-D-lysin (PDL) or poly-L-ornithine (PLO). The best cell growth was obtained on double-coated surfaces (PLL, PDL or PLO plus laminin). On such a culture substrate and with a seeding density of 1 x 10(7) cells/cm(2) confluence was obtained within 3-4 weeks at an incubation temperature of 18 degrees C. Approximately 95% of the cells were identified as astrocytes on the basis of a positive staining with antibodies against the astrocyte specific glial protein (GFAP). No oligodendrocytes or fibroblasts were identified in the cultures, and despite several efforts, neurons did not grow under the culture conditions used. When challenged with ligands known to awake a calcium transient in mammalian astrocytes, 44% of the cells responded to ATP with an increase in [Ca 2+](i), 38% to norepinephrine, 27% to 5-hydroxytryptamine, 7% to histamine and 6% to glutamate. Kainate, quisqualate and gamma-aminobutyric acid did not awake a calcium transient in the cells. Using a proper protocol, it is thus quite easy to get an almost pure culture of astrocyte, whereas neurones proved to very difficult to culture.     

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.