Membrane order conservation in raft and non-raft regions of hepatocyte plasma membranes from thermally acclimated rainbow trout

Homeoviscous adaptation (HVA), the thermal conservation of membrane fluidity/order at different body temperatures, has been observed to varying degrees in different membranes. However, HVA has not been studied in raft and non-raft regions of the plasma membrane (PM) separately. Rafts are ordered PM microdomains implicated in signal transduction, membrane traffic and cholesterol homeostasis. Using infrared spectroscopy, we measured order in raft-enriched PM (raft) and raft-depleted PM (RDPM) isolated from hepatocytes of rainbow trout (Oncorhynchus mykiss) acclimated to 5 and 20 degrees C. We found approximately 130% and 90% order compensation in raft and RDPM, respectively, suggesting their independent regulation. Raft was more ordered than RDPM in the warm-acclimated trout, a difference fully explained by a 58% enrichment of cholesterol, compared to RPDM. Unexpectedly, raft and RDPM from cold-acclimated trout did not differ in cholesterol content or order. Freezing the membrane samples during preparation had no effect on order. Treatment with cyclodextrin depleted cholesterol by 36%, 56%, and 55%, producing significant decreases in order in raft and RDPM from warm-acclimated trout and RDPM from cold-acclimated trout, respectively. However, a 69% depletion of cholesterol from raft from cold-acclimated trout had no significant effect on order. This result, and the lack of a difference in order between raft and RDPM, suggests that raft and non-raft PM in cold-acclimated trout are not spatially segregated by phase separation due to cholesterol.     

Proteomic identification of rainbow trout seminal plasma proteins

In the study, the combination of protein fractionation by 1DE and HPLC‐ESI‐MS/MS was used to characterize the rainbow trout seminal plasma proteome. Our results led to the creation of a catalogue of rainbow trout seminal plasma proteins (152 proteins) and significantly contributed to the current knowledge regarding the protein composition of fish seminal plasma. The major proteins of rainbow trout seminal plasma, such as transferrin, apolipoproteins, complement C3, serum albumin, and hemopexin‐, alpha‐1‐antiproteinase‐, and precerebellin‐like protein, were recognized as acute‐phase proteins (proteins that plasma concentration changes in response to inflammation). This study provides the basis for further functional studies of fish seminal plasma proteins, as well as for the identification of novel biomarkers for sperm quality. The MS data have been deposited in the ProteomeXchange with identifier PXD000306 ( http://proteomecentral.proteomexchange.org/dataset/PXD000306 ).     

Cholesterol affects physical properties and (Na+,K+)-ATPase in basolateral membranes of renal and intestinal epithelia from thermally acclimated rainbow trout

Previous work has shown that cholesterol levels are modulated in plasma membranes from some but not all tissues of poikilotherms over the course of temperature change. To gain a better understanding of tissue and membrane domain-specific cholesterol function during thermal adaptation we examined effects of cholesterol on membrane physical properties and (Na⁺,K⁺)-ATPase in native and cholesterol-enriched basolateral membranes from kidney and intestine of thermally acclimated trout (Oncorhynchus mykiss). Membrane order (as indicated by fluorescence depolarization studies) is increased, whereas its thermal sensitivity is decreased by elevated cholesterol levels in mem branes with relatively low endogenous amounts of cholesterol (intestinal membranes and renal membranes from cold-acclimated fish). Thermal sensitivities of membrane order in kidney are 1.5-fold higher in native compared with cholesterol-enriched basolateral membranes. For renal plasma membranes, (Na⁺,K⁺)- ATPase activity is lowest near the transition between native and surpraphysiological cholesterol levels. Endogenous cholesterol levels (relative to phospholipid contents) in intestinal basolateral membranes from cold-acclimated fish vary more than 1.5-fold; membranes with cholesterol/phospholipid molar ratios of 0.3 have activities of (Na⁺,K⁺)-ATPase that are twofold lower than native membranes having a ratio of 0.2. These results suggests that maintenance of cholesterol levels in intestinal basolateral membranes during thermal acclimation may ensure sufficient activity of (Na⁺,K⁺)-ATPase. Membrane function in kidney, with its high native cholesterol content, is less likely to be affected by temperature change. Accepted: 21 January 1997     

A lysozyme isolated from rainbow trout acts on mastitis pathogens

Abstract The antibacterial effects of two lysozymes purified from rainbow trout kidney (type I and II) were tested on eight bacterial strains isolated from cases of clinical mastitis (staphylococci, streptococci and coliforms). Three other lytic agents were included in the experiments as controls: hen egg-white lysozyme, lysostaphin and mutanolysin. Proliferating bacteria were incubated with the various lytic agents, either in hearts infusion broth or in milk. The type II rainbow trout lysozyme decreased the number of live bacteria (colony forming units) of all the strains tested, but was most efficient against staphylococci. The other two lysozymes had little effect.     

Differentially expressed proteins in rainbow trout adipocytes isolated from visceral and subcutaneous tissues

In rainbow trout, subcutaneous (in dorsal and ventral positions) and visceral fat deposits are known to influence the yield of edible flesh, whilst their respective roles in metabolism, storage and release of fatty acids have not, so far, been directly studied. The present work aimed to identify, by using 2D electrophoresis, proteins differentially expressed in isolated mature adipocytes originating from these various localizations in prepubescent females. A total of nine proteins were estimated to be differentially expressed according to the localisation of the adipocytes. Seven protein spots were considered to be present in the three fat deposits at differing abundances, and among them only six were estimated as being specific to fat tissues. Among these, five were more abundant in subcutaneous adipocytes of both sites compared to perivisceral adipocytes. Four were identified: three as H-FABP, ATP synthase, serum deprivation-response protein, indicating higher metabolic activity in subcutaneous adipocytes, while the latter, annexin, indicative of a higher proportion of less mature adipocytes, as also suggested by their smaller mean diameter. The more abundant protein in visceral isolated adipocytes is actin, known to be involved in cytoskeleton structure and to increase during adipogenesis. This allows us to suggest their more mature stage of development, in relation with their higher mean diameter.     

Copper exposure and the degeneration of olfactory receptors in rainbow trout (Oncorhynchus mykiss)

Abstract

Sublethal concentrations of copper in water cause the degeneration of olfactory receptors in rainbow trout (Oncorhynchus mykiss). Receptor cell loss has been correlated to the loss of olfaction in fish and may cause difficulties in olfactory mediated behaviors such as migration. This study investigated the effects of three levels of copper (100, 75 and 50 mg L^?1) on the olfactory epithelium of rainbow trout. Twenty fish randomly allocated between three exposure groups and one control were exposed for 24 hours under static renewal conditions. Light and scanning electron microscopic observations of olfactory tissue were taken to determine the extent of degeneration of receptors. In addition, levels of copper and zinc in the brain tissues were analyzed to determine if the olfactory route was a significant route of copper exposure and transfer to fish brain tissue. Results indicate that degeneration of receptors is related to the concentration of copper. Levels of copper in brain were found to be below detection of the instrument. Levels of zinc were extremely variable ranging from 52 to 132 ng zinc g^?1 brain tissue.     

A novel antimicrobial function for a ribosomal peptide from rainbow trout skin

An antimicrobial peptide was purified from skin secretions and epithelial cells of rainbow trout by cation exchange and reversed phase chromatography. Partial N-terminal amino acid sequence of the purified peptide revealed 100% identity with the first 11 residues of a 40S ribosomal peptide from medaka fish. Its molecular mass, determined by matrix-associated laser desorption/ionisation mass spectrometry, was found to be 6676.6Da. These results indicate that this antimicrobial peptide is likely to be the 40S ribosomal protein S30. It is active at submicromolar concentrations, with an effective 50% reduction concentration of 0.02-0.04 microM against Planococcus citreus. Thus, in addition to its conventional function in the cell as part of the small ribosomal subunit, this peptide may play a role in protection against intracellular or extracellular pathogens.     

Purification and characterization of calpain and calpastatin from rainbow trout, Oncorhynchus mykiss

Although the calpain system has been studied extensively in mammalian animals, much less is known about the properties of μ-calpain, m-calpain, and calpastatin in lower vertebrates such as fish. These three proteins were isolated and partly characterized from rainbow trout, Oncorhynchus mykiss, muscle. Trout m-calpain contains an 80-kDa large subunit, but the  26-kDa small subunit from trout m-calpain is smaller than the 28-kDa small subunit from mammalian calpains. Trout μ-calpain and calpastatin were only partly purified; identity of trout μ-calpain was confirmed by labeling with antibodies to bovine skeletal muscle μ-calpain, and identity of trout calpastatin was confirmed by specific inhibition of bovine skeletal muscle μ- and m-calpain. Trout μ-calpain requires 4.4 ± 2.8 μM and trout m-calpain requires 585 ± 51 μM Ca²⁺ for half-maximal activity, similar to the Ca²⁺ requirements of μ- and m-calpain from mammalian tissues. Sequencing tryptic peptides indicated that the amino acid sequence of trout calpastatin shares little homology with the amino acid sequences of mammalian calpastatins. Screening a rainbow trout cDNA library identified three cDNAs encoding for the large subunit of a putative m-calpain. The amino acid sequence predicted by trout m-calpain cDNA was 65% identical to the human 80-kDa m-calpain sequence. Gene duplication and polyploidy occur in fish, and the amino acid sequence of the trout m-calpain 80-kDa subunit identified in this study was 83% identical to the sequence of a trout m-calpain 80-kDa subunit described earlier. This is the first report of two isoforms of m-calpain in a single species.     

Cardio-respiratory effects of chloramine-T exposure in rainbow trout

In order to establish whether the blood gas respiratory disturbances noted with exposure to chloramine-T are due to differences in the rates of uptake of O₂ and excretion of CO₂ or gill blood flow, adult rainbow trout (Oncorhynchus mykiss) were fitted with dorsal aorta and bulbus arteriosus catheters to facilitate blood pressure recordings, an ultrasonic blood flow probe and opercular impedance electrodes. Fish received either a 45-min static exposure to 9 mg l⁻¹ chloramine-T or tap water (control) and continuous recordings of blood pressure, and ventilation frequency and amplitude were made. Pre- and post-exposure arterial and venous blood samples were taken and analyzed for O₂ and CO₂ content, hemoglobin concentration and hematocrit. Chloramine-T exposure had no effect on any of the continuously recorded parameters. However, individual measurements (made immediately prior to and following exposure) of cardiac output and O₂ uptake rates increased significantly following exposure to chloramine-T compared to before exposure. CO₂ excretion rates were unaffected by chloramine-T exposure. Calculation of the perfusion convection requirement showed a significant increase for CO₂ but not for O₂. It was concluded that increases in O₂ uptake resulted from increased cardiac output but that CO₂ excretion, a diffusion-limited process, was not increased due to additional diffusive limitations caused by the irritant effect of chloramine-T.     

Organization of the histone genes in the rainbow trout (Salmo gairdnerii)

Summary Twelve clones containing histone genes were isolated from a genomic trout library constructed in the vector Charon 4A. Each of the clones was found to contain a conserved 10.2-kb Eco RI fragment that contained one copy of each of the histones in the order H4-H2B-H1-H2A-H3, all of which are transcribed from the same strand. Genomic Southern blots indicate that these clusters are representative of the vast majority of the histone genes in the trout. Tandemly linked clusters were not found. Approximately 145 copies of this cluster are present in a trout sperm cell. Sequence analysis has shown the genes to be without introns and to show strong selection for codons ending in C or G. Consensus signals similar to those found in other histone genes are present in the flanking regions.     

Serotonin, but not melatonin, plays a role in shaping dominant-subordinate relationships and aggression in rainbow trout

The aim of this study was to clarify to what extent the effects of elevated dietary L-tryptophan (Trp) on aggressive behavior and stress responsiveness in rainbow trout are mediated by circulating melatonin and central serotonin (5-HT), respectively. Isolated rainbow trout were paired for 1h a day for 7 days in order to create fish with experience of being dominant and subordinate. Following this week, the fish were tested for aggressive behavior using a resident-intruder test after which they were subjected to one of four treatments: (1) tryptophan, (2) the selective serotonin reuptake inhibitor (SSRI) citalopram, (3) melatonin, and (4) no treatment (controls). After 7 days of treatment, the fish were subjected to a second resident-intruder test. Trp-supplemented feed resulted in a suppression of aggressive behavior in fish with experience of being dominant. Moreover, fish fed Trp-supplemented feed, regardless of social experience, also displayed lower plasma cortisol levels than controls. These effects of elevated dietary Trp were closely mimicked by citalopram treatment, whereas exogenous melatonin had no effect on either aggressive behavior or plasma cortisol. Thus, the effect of elevated dietary Trp on aggressive behavior and stress responses does not appear to be mediated by melatonin even though elevated dietary intake of Trp resulted in an increase in plasma melatonin concentrations.     

虹鳟对时间和地点的学习记忆(英文)

为确定虹鳟(Oncorhynchus mykiss)是否能将日投放两次食物的不同时间和地点联系起来,在30天内,于09:00-10:00将食饵投放在水族箱一侧,于15:00-16:00将食饵投放在水族箱另一侧.在第21天和28天,不放食饵以确定虹鳟对时间-地点的学习记忆.结果表明,虹鳟将不同时间和地点联系起来以获取食物,并且在水族箱两个投放食饵之处均表现出预取食活动,提示该物种具有控制时间-地点学习任务的内在机制.     

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.     

The effects of chronic immune stimulation on muscle growth in rainbow trout

Successful production of aquaculture species depends on efficient growth with low susceptibility to disease. Therefore, selection programs have focused on rapid growth combined with disease resistance. However, chronic immune stimulation diminishes muscle growth (a syndrome referred to as cachexia), and decreases growth efficiency in production animals, including rainbow trout. In mammals, recent results show that increased levels of pro-inflammatory cytokines, such as those seen during an immune assault, specifically target myosin and MyoD and inhibit muscle growth. This suggests that increased disease resistance in fish, a desired trait for production, may actually decrease the growth of muscle, the main aquacultural commodity. To test this possibility, a rainbow trout model of cachexia was developed and characterized. A six-week study was conducted in which rainbow trout were chronically immune stimulated by repeated injections of LPS. Growth indices were monitored, and whole body and muscle proximate analyses, real-time PCR, and Western blotting were conducted to examine the resulting cachectic phenotype. Muscle ratio was decreased in fish chronically immunostimulated, however expression levels of MyoD2 and myosin were not decreased compared to fish that were not immunostimulated, indicating that while muscle accretion was altered, the mechanism by which it occurred was somewhat different than that characterized in mammals. Microarray analysis was used to compare gene expression in fish that had been chronically immunostimulated versus those that had not to identify possible alternative mechanisms of cachexia in fish.     

Histopathology changes in the rainbow trout (Onchorhyncus mykiss) consuming boric acid supplemented fish fodder

ObjectiveEven though boron (B), as a trace micronutrient, occurs in natural waters and organisms, its high concentration could cause harmful and even toxic for organisms. The aim of present study was to investigate the effects of boric acid (BA) added to feed (0.01%, 0.05%, 0.10%, and 0.20% of B in feed) on Rainbow Trout (Oncorhynchus mykiss) by histopathological methods and compared to the control feed as without B.MethodologyAt the end of the feeding, after weighing the live weight of 9 rainbow trout from each group, tissue fragments were taken from the liver, gill, kidney, skeletal muscle, spleen and brains of fish, which systemic necropsies was done, and were fixed in 10 % buffered formalin solution.ResultsHistopathological examinations revealed degenerative and necrotic changes in the liver, gill, kidney, skeletal muscle, spleen and brain. Hydropic and vacuolar degenerations in liver parenchyma, lamellar edema in the gills, hyaline accumulation in the kidneys, degenerations in the muscles, necrosis in the spleen and hyperemia in the brain were observed in all groups except control group. It was observed that the destruction of boric acid on fish increased depending on the amount of boron supplemented to the feed, and the most effect was in the group fed with 0.20 % boron supplemented feed.ConclusionsThe findings aside from causing pathological changes in all organs in terms of histopathological findings of Boric Acid (BA), the most severe lesions were observed in the liver.     

Induced gynogenesis in the rainbow trout: Sex and survival of progenies production of all-triploid populations

Summary Gynogenesis could be an efficient way for producing inbred lines in commercial fish species. Gamma-irradiation of sperm gives haploid embryos that all die without hatching; in the present study, we optimized heat treatment of the eggs, in order to produce high rates of diploid gynogenetics. When the eggs are heated to 26 °C for 20 min after 25 min of development, 80% of the embryos hatch, and all the resulting fry are diploid; nevertheless, high mortalities are recorded until feeding start. The monosex female constitution of gynogenetic offspring confirms the female homogamety in the rainbow trout.When the eggs are treated with the same heat shock 25 min after a fertilization with functional sperm, alltriploid populations are obtained; their survival until feeding start is not different from the control.