Parvalbumin expression in trout swimming muscle correlates with relaxation rate

Rainbow trout (Oncorhynchus mykiss) display longitudinal and developmental shifts in muscle relaxation rate. This study aimed to determine the role of variations in parvalbumin content in modulating muscle relaxation. Parvalbumin is a low molecular weight protein that buffers myoplasmic Ca2+ and enhances muscle relaxation. In some fish, longitudinal variations in muscle relaxation have been linked to variations in the total amount of parvalbumin present in muscle and in the relative expression of two parvalbumin isoforms. We have demonstrated previously that anterior slow-twitch or red myotomal muscle relaxes more rapidly than that from the posterior for both rainbow and brook trout. Further, younger rainbow trout parr have faster red muscle relaxation rates than older smolts. Here we report similar results for fast-twitch or white muscle. We quantified the parvalbumin expression in red and white muscle from different body positions of rainbow trout parr and smolts and for brook trout (Salvelinus fontinalis) adults. There was a significant shift in total parvalbumin content of muscle: the faster muscle from the anterior myotome contained greater amounts of parvalbumin. For brook trout, longitudinal variation in relaxation rate was also associated with shifts in the relative expression of the two parvalbumin isoforms. The faster muscle of parr contained more parvalbumin. Lastly, trout white muscle tended to have higher levels of parvalbumin and greater levels of the Parv2 (relative to Parv1) isoform as compared to red muscle. Parvalbumin expression correlated with muscle relaxation rate in trout, although there were species-specific differences in the importance of altering total parvalbumin content versus shifts in relative parvalbumin isoform expression.     

Gender related and dexamethasone induced differences in the mRNA levels of the MRF genes in rat anterior tibial skeletal muscle

Muscle formation and postnatal growth is under the control of the muscle regulatory factors (MRF) gene family, consisting of four genes: MyoD1, myogenin, myf-5, and myf-6. Muscle mass is also known to be affected by specific drugs, like glucocorticoids. Glucocorticoids have also been characterized as muscle atrophying agents. However, glucocorticoids are also the only drugs reported to have a beneficial effect on the treatment of muscle degenerative disorders. Since muscle mass relates to gender, this may be partially caused by gender. The aim of this study is to investigate gender-related basal and dexamethasone-induced expression of the MRF genes. Gender-specific MRF mRNA levels were investigated in anterior tibial muscles of the rat. Myogenin, myf-5, and myf-6 mRNA level was significantly higher in female rats than in male rats. Since muscle mass is usually higher in males, we conclude that the development of gender-related differences in muscle mass is not primarily under the control of the mRNA levels of the MRF genes. Male rats treated with dexamethasone for 14 days (1 mg per kg body weight) showed increased levels of MyoD1, myogenin and myf-5 compared to control male rats. Female rats treated with dexamethasone showed decreased expression of myf-6 compared to control female rats. These results suggest that dexamethasone increase satellite cell-specific MRF activity in male muscle tissue, which is suggested to be associated with muscle hypertrophy, while maintenance of muscle tissue is affected in female muscle tissue. Therefore, we conclude that both basal and dexamethasone-induced MRF gene mRNA levels are regulated gender-specific.     

Swimming performance and morphology of juvenile sockeye salmon, <Emphasis Type="Italic">Oncorhynchus nerka</Emphasis>: comparison of inlet and outlet fry populations

We raised two populations of sockeye salmon fry from fertilized eggs in the laboratory and tested the hypothesis that outlet fry populations, fish which must migrate upstream to reach rearing lakes after yolk-sac absorption, have better swimming ability and morphological characteristics conducive to enhanced swimming performance than inlet fry populations, fish which migrate downstream to rearing lakes. Despite being of identical age, fry from the outlet population were larger (approx. 6.7% longer, ~5 mm on average) and more laterally compressed than inlet fry at the time of our initial experiments. Using an open-top box flume, we found that the burst-swimming performance (in cm s⁻¹) of the outlet population was 31% better. We found no differences between populations in prolonged-swimming performance. We were unable to find any direct relationships between measures of swimming performance and size or shape variables, suggesting that the larger, more robust morphology of outlet fry was not responsible for the superior burst ability. Recent biochemical studies indicate outlet fry may be metabolically better provisioned for burst swimming than inlet fry. It is possible that the morphological differences between the populations of fry reflect adaptations needed by adults during their migration and spawning.     

Plasma ghrelin levels in rainbow trout in response to fasting, feeding and food composition, and effects of ghrelin on voluntary food intake

Ghrelin, a peptide hormone which stimulates growth hormone (GH) release, appetite and adiposity in mammals, was recently identified in fish. In this study, the roles of ghrelin in regulating food intake and the growth hormone (GH)-insulin-like growth factor I (IGF-I) system of rainbow trout (Oncorhynchus mykiss) were investigated in three experiments: 1) Pre- and postprandial plasma levels of ghrelin were measured in relation to dietary composition and food intake through dietary inclusion of radio-dense lead-glass beads, 2) the effect of a single intraperitoneal (i.p.) injection with rainbow trout ghrelin on short-term voluntary food intake was examined and 3) the effect of one to three weeks fasting on circulating ghrelin levels and the correlation with plasma GH and IGF-I levels, growth and lipid content in the liver and muscle was studied. There was no postprandial change in plasma ghrelin levels. Fish fed a normal-protein/high-lipid (31.4%) diet tended to have higher plasma ghrelin levels than those fed a high-protein/low-lipid (14.1%) diet. Plasma ghrelin levels decreased during fasting and correlated positively with specific growth rates, condition factor, liver and muscle lipid content, and negatively with plasma GH and IGF-I levels. An i.p. ghrelin injection did not affect food intake during 12-hours post-injection. It is concluded that ghrelin release in rainbow trout may be influenced by long-term energy status, and possibly by diet composition. Further, in rainbow trout, ghrelin seems to be linked to growth and metabolism, but does not seem to stimulate short-term appetite through a peripheral action.     

Alterations in expression of genes associated with muscle metabolism and growth during nutritional restriction and refeeding in rainbow trout

Rainbow trout, as well as many other species of fish, demonstrate the ability to survive starvation for long periods of time. During starvation, growth rate is decreased and muscle exhibits signs of wasting. However, upon resumption of feeding, accelerated growth is often observed. Alterations in muscle metabolism occur during feed restriction and refeeding, although the ways in which these alterations affect the molecular pathways that control muscle growth have not been fully determined. To analyze changes in muscle metabolism and growth during starvation and refeeding, real-time PCR was used to test the expression of six metabolic-related genes and eight muscle-specific genes in rainbow trout white muscle prior to and after 30 days of starvation, and after 4 and 14 days of refeeding. The six metabolic-related genes chosen are indicative of specific metabolic pathways: glycolysis, glycogenesis, gluconeogenesis, the pentose phosphate pathway, and fatty acid formation. The eight muscle specific genes chosen are key components in muscle growth and structural integrity, i.e., MRFs, MEFs, myostatins, and myosin. Alterations in expression of the tested metabolic-related genes and muscle-specific genes suggest that during both starvation and refeeding, changes in specific metabolic pathways initiate shifts in muscle that result mainly in the modification of myotube hypertrophy. The expression levels of many of the metabolic-related genes were altered during the refeeding period compared to those observed before the starvation period began. However, the accelerated growth often observed during refeeding is likely driven by changes in normal muscle metabolism, and the altered expression observed here may be a demonstration of those changes.     

Molecular characterization of the MuRF genes in rainbow trout: Potential role in muscle degradation

Muscle growth is determined primarily by the balance between protein synthesis and degradation. When rates of protein synthesis are similar between individuals, protein degradation is critical in explaining differences in growth efficiency. Studies in mammals showed that muscle atrophy results from increased protein breakdown, and is associated with activation of the ubiquitin proteasome pathway, including induction of the muscle-specific ubiquitin protein ligase, MuRF1. Animals lacking MuRF1 are resistant to muscle atrophy. In fish, little is known about the role of the proteasome/MuRF pathway in muscle degradation. The objectives of this study were to: 1) clone and characterize MuRF genes in rainbow trout; and 2) determine expression of MuRF genes in association with starvation- and vitellogenesis-induced muscle atrophy in rainbow trout. We have identified full-length cDNA sequences for three MuRF genes (MuRF1, MuRF2, and MuRF3). These genes encode proteins with typical MuRF structural domains, including a RING-finger, a B-box and a Leucine-rich coiled-coil domain. RT-PCR analysis showed that MuRF genes are predominantly expressed in muscle and heart tissues. Real time PCR analysis revealed that expression of all MuRF genes is up-regulated during starvation and MuRF3 is up-regulated in vitellogenesis-associated muscle degradation. These results suggest that MuRF genes have an important role in fish muscle protein degradation. Further studies are warranted to assess the potential use of MuRF genes as tools to monitor fish muscle growth and degradation.     

Effects of vitamin B1 (thiamine) deficiency in lake trout (Salvelinus namaycush) alevins at hatching stage

The objectives of this study were to examine the relationship between thiamine concentrations in unfertilized eggs and yolksac individuals of lake trout (Salvelinus namaycush), along with any associated histopathological changes in the tissues of alevins at the hatching stage. We address these questions in a lake trout population from different spawning grounds of Lake Michigan (North and South), known for compromised survival due to early mortality syndrome (EMS). However, a dichotomous forage base of lake trout spawning stocks, with a dietary thiaminase-rich alewife in the North, and dietary low-thiaminase round goby in the South, provides the basis for the assumption that different diets may lead to differences in severity of EMS between different stocks. Lake trout eggs of 18 females were collected and fertilized individually with the sperm of several males. The eggs, eyed embryos and newly-hatched alevins were sampled to examine thiamine utilization during embryogenesis. Progenies of females with low (< 0.73 nmol/g) and high (> 0.85 nmol/g) levels of thiamine were chosen for histological studies. The obtained results showed that total thiamine levels in the body and yolk of eyed embryos and alevins at hatching were influenced by thiamine levels of unfertilized eggs and it decreased during embryogenesis (to 51% in eyed embryos and 28% in newly-hatched alevins in comparison to unfertilized eggs). The survival of lake trout until hatching stage does not correlate with the thiamine level, however it was affected by collection site and was significantly higher in fish from the South site (Julian's Reef). At the hatching stage, no pathological changes were observed in the brain, olfactory lobe, retina or liver in embryos regardless of thiamine concentrations in unfertilized eggs. It has been concluded that an enhanced thiamine requirement for the fast muscle mass growth near the swim-up stage is responsible for overt and histopathological signs of EMS. Current study confirms earlier findings that lake trout suffering from EMS can be successfully treated by immersion in thiamine solution as late as at the swim-up stage.     

Innate immune gene expression in individual zebrafish after Listonella anguillarum inoculation

Zebrafish were intraperitoneally injected with 10(6)CFU (LD50) Listonella anguillarum. Three inoculated and control fish were collected at 1, 2, 4, 6 and 22h post infection (hpi) and the expression of genes related to the immune response (il1b, cebpb, tfa, mpx, tnfa, nitr9, tlr22, hsc70, cp, mrlp1, c3b and lyz) in each fish was monitored by means of real-time RT-PCR. A similar experiment was performed considering an intermediate time point at 15 hpi. Different relative levels of expression were found among genes. Also, wide interindividual variation in gene expression for most genes was detected among fish, inoculated or not. A steady increase of expression starting from the initial stages of the interaction was found for interleukin-1beta. An initial increase in levels of gene expression was found for the genes coding for the CCAAT/Enhancer Binding Protein subunit beta and the Novel Immune-Type Receptor 9, although their levels decreased later on and were indistinguishable from the controls at 22 hpi. Finally, some genes (Transferrin, Myeloid-specific Peroxidase and Tumour Necrosis Factor alpha) were upregulated at 22 hpi. Taken together, our results show an induction in gene expression of genes involved in the inflammatory and immune response upon L. anguillarum infection but also reveal the existence of a wide variation in the levels of expression of the studied genes in the zebrafish population.     

Divergent evolution of the vertebrate polysialyltransferase Stx and Pst genes revealed by fish-to-mammal comparison

Polysialic acid (PSA) is a developmentally regulated carbohydrate attached to the neural cell adhesion molecule (NCAM). PSA is involved in dynamic processes like cell migration, neurite outgrowth and neuronal plasticity. In mammals, polysialylation of NCAM is catalyzed independently by two polysialyltransferases, STX (ST8Sia II) and PST (ST8Sia IV), with STX mainly acting during early development and PST at later stages and into adulthood. Here, we functionally characterize zebrafish Stx and Pst homolog genes during fish development and evaluate their catalytic affinity for NCAM in vitro. Both genes have the typical gene architecture and share conserved synteny with their mammalian homologues. Expression analysis, gene-targeted knockdown experiments and in vitro catalytic assays indicate that zebrafish Stx is the principal--if not unique--polysialyltransferase performing NCAM-PSA modifications in both developing and adult fish. The knockdown of Stx exclusively affects PSA synthesis, producing defects in axonal growth and guidance. Zebrafish Pst is in principle capable of synthesizing PSA, however, our data argue against a fundamental function of the enzyme during development. Our findings reveal an important divergence of Stx and Pst enzymes in vertebrates, which is also characterized by a differential gene loss and rapid evolution of Pst genes within the bony-fish class.     

鳜早期发育阶段骨骼肌纤维的增生与肥大生长

骨骼肌是鱼体的主要组成部分,也是衡量鱼体生长发育的重要指标.为了解鳜(Siniperca chuatsi)早期发育阶段骨骼肌纤维的生长发育特征,通过制作孵化后1 ～41日龄个体骨骼肌背右侧第一肌节石蜡切片,利用图像分析软件统计该肌节中肌纤维的数目和面积,分析了鳜骨骼肌纤维的增生和肥大生长特征.结果表明,鳜早期发育阶段骨...     

Mercury accumulation in five species of freshwater fish in Lake Tyrifjorden,south-east Norway,with emphasis on their suitability as test organisms

Synopsis Mercury concentration in axial muscle of brown trout, Salmo trutta, whitefish, Coregonus lavaretus, Arctic charr, Salvelinus alpinus, smelt, Osmerus eperlanus and pike, Esox lucius, were studied in Lake Tyrifjorden during 1978–1982. Our data demonstrate that older and bigger fish on an average have higher mercury concentration than smaller and younger. Further, complex life histories as in brown trout influence the pattern of mercury accumulation. During young stages accumulation in brown trout is moderate, while accumulation in older stages is highly correlated to lake residency time. Based on our data we suggest the following requirements for a test organism and the collecting procedure; (1) life history should be simple with small sexual differences, (2) ageing should be easy and reliable, and (3) large representative samples should be easily obtained during (4) a fixed biological period i.e. the spawning period. We consider smelt as an appropriate test organism based on these criteria.     

Growth hormone differentially regulates growth and growth-related gene expression in closely related fish species

Zebrafish (Danio rerio) have become an important model organism for developmental biology and human health studies. We recently demonstrated differential growth patterns between the zebrafish and a close relative the giant danio (Danio aequipinnatus), where the giant danio appears to exhibit indeterminate growth similar to most fish species important for commercial production, while zebrafish exhibit determinate growth more similar to mammalian growth. This study focused on evaluating muscle growth regulation differences in adult zebrafish and giant danio utilizing growth hormone treatment as a mode of growth manipulation. Growth hormone treatment resulted in increased overall growth in giant danio, but failed to increase growth in the zebrafish. Growth hormone treatment increased muscle IGF-I and GHrI gene expression in both species, but to a larger degree in the giant danio. In contrast, zebrafish exhibited a larger increase in IrA and IGF-IrB gene expression in muscle in response to GH treatment. In addition muscle myostatin levels were differentially regulated between the two species, with a down-regulation observed in rapidly growing, GH-treated giant danio and an up-regulation in zebrafish not actively growing in response to GH. This is the first report of differential expression of growth-regulating genes in closely related fish species exhibiting opposing growth paradigms. These results further support the role that the zebrafish and giant danio can play important model organisms for determinate and indeterminate growth.