Mechanisms Of Rainbow Trout Research Articles

Characterization of expression response in post-prandial situation of food sensing system in rainbow trout (Oncorhynchus mykiss) fed a plant-based diet: Focus on free fatty acid receptors and their signaling pathways

Recently identified and characterized in rainbow trout (Oncorhynchus mikyss), the main gustatory receptors for fatty acid, known as free fatty acids receptor (FFAR) 1 and 2, may play a crucial role in nutrient detection and regulation of feeding behavior. However, our understanding of their post-prandial roles remains limited. This study investigates the impact of a single meal (acute effect) with an alternative plant-based diet (vegetal diet, V diet devoid of fish meal and fish oil) on the food detection mechanisms in trout. After 30 days of feeding with commercial-like diet (C diet made up of a mix of fish meal, fish oil and plant ingredients) followed 5 days of fasting, we conducted a post-prandial kinetic experiment after single meal with C or V diet. We assessed the expression pattern of ffar genes, their associated signaling pathways (calcium and indolamine pathways) in the tongue along with the expression pattern of appetite-regulating neuropeptides in the hypothalamus. Additionally, we evaluated central activity through brain activity markers in the telencephalon of trout at various post-meal time points, including 20 min, 2, 6, 10 and 24 h. Our results revealed that the diet nature (C vs V) influences the expression of ffar genes, while some paralogs of ffar2 (ffar2a2 and ffar2b1.2) were early up-regulated by V diet, ffar2b1.1 and ffar1 were latter up-regulated by C diet. Differential regulations in the tongues of V diet fish were also observed on calcium signaling and an early alteration in serotonin turnover. The expression increase of neuropeptide npya from 6 to 10 h after meal, could suggest a greater sensation of hunger in fish fed the C diet. Finally, our findings suggest that rainbow trout exhibit a keen ability to detect a novel feed after a single meal with V diet at the brain level, particularly in areas associated with decision-making and behavior control. Altogether, results not only show that the integrative feeding response appears to be dysregulated by V diet, but also highlight the noteworthy sensitivity of nutrient detection mechanisms in rainbow trout to new food sources. FFARs may play a role in finely discerning diets with varying levels of ω-3 LC-PUFAs in rainbow trout. This heightened detection sensitivity holds the potential to significantly influence the regulation of feeding behavior and food detection in farmed fish.

Dynamic and systemic regulatory mechanisms in rainbow trout (Oncorhynchus mykiss) in response to acute hypoxia and reoxygenation stress

Hypoxia frequently occurs in natural aquatic systems and aquaculture environments due to extreme climate fluctuations, high-density farming, environmental pollution and global warming, resulting in serious ecological damage and enormous economic losses. Rainbow trout (Oncorhynchus mykiss), a key economic fish species worldwide, is extremely sensitive to hypoxia. However, the regulatory mechanisms in response to environmental hypoxia and reoxygenation stress remain unknown. Herein, rainbow trout liver transcriptomes and biochemical parameters were investigated in response to hypoxia for different durations (3, 12 and 24 h), reoxygenation (hypoxia for 24 h followed by reoxygenation for 3 h) and normoxia to highlight dynamic changes in molecular regulation and oxidative stress. In RNA-seq analysis, 5906 differentially expressed genes, two significantly expressed gene sets (profiles 10 and 12) and two hypoxia specific modules (MEgreen and MEturquoise) were screened by differential expression analysis, short time-series expression miner (STEM) and weighted gene co-expression network analysis (WGCNA), respectively. The intersection of the above analyses further identified several key hub genes related to hypoxia, including hif-1α, epo, igfbp1, ddit4, pck1, g6pc, cyp1a1 and dusp1. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed different metabolic strategies at different stress stages; signal transmission and protein synthesis process were strongly influenced by hypoxia. The immune system was inhibited, and a switch from programmed to pathological cell death was observed from hypoxia for 12 h to 24 h. After reoxygenation for 3 h, mitochondrial oxidative phosphorylation was strongly activated to provide more cellular ATP. Enzymatic and non-enzymatic antioxidant systems clearly coordinate to alleviate oxidative stress injury caused by hypoxia. This study reveals dynamic and systemic regulatory mechanisms in rainbow trout liver, and lays a foundation for further study on the molecular mechanisms governing responses to environmental hypoxia and reoxygenation stress.

Integrated analysis of immune parameters, miRNA-mRNA interaction, and immune genes expression in the liver of rainbow trout following infectious hematopoietic necrosis virus infection.

Rainbow trout (Oncorhynchus mykiss) is an important economical cold-water fish worldwide. However, infection with infectious hematopoietic necrosis virus (IHNV) has severely restricted the development of aquaculture and caused huge economic losses. Currently, little is known about the immune defense mechanisms of rainbow trout against IHNV. In this study, we detected the changes of immune parameters over different post-infection periods (6-, 12-, 24-, 48-, 72-, 96-, 120-, and 144 hours post-infection (hpi)), mRNA and miRNA expression profiles under 48 hpi (T48L) compared to control (C48L), and key immune-related genes expression patterns in rainbow trout liver following IHNV challenge through biochemical methods, RNA-seq, and qRT-PCR, and the function of miR-330-y was verified by overexpression and silencing in vitro and in vivo. The results revealed that alkaline phosphatase (AKP), alanine aminotransferase (ALT), catalase (CAT), and total superoxide dismutase (T-SOD) activities, and lysozyme (LZM) content showed significant peaks at 48 hpi, whereas malondialdehyde (MDA) content and aspartate aminotransferase (AST) activity decreased continuously during infection, and acid phosphatase (ACP) activity varied slightly. From RNA-seq, a total of 6844 genes and 86 miRNAs were differentially expressed, and numerous immune-related differentially expressed genes (DEGs) involved in RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, cytokine-cytokine receptor interaction, and antigen processing and presentation were significantly upregulated in T48Lm group, including IFIH1, DHX58, MAVS, TRAF3, IRF3, IRF7, MX1, TLR3, TLR8, MYD88, NOD1, NOD2, IL-8, CXCR1, CD209, CD83, and TAP1. Integrated analysis identified seven miRNAs (miR-425-x, miR-185-x, miR-338-x, miR-330-y, miR-361-x, miR-505-y, and miR-191-x) that target at least three key immune-related DEGs. Expression analysis showed that IFIH1, DHX58, IRF3, IRF7, MX1, TLR3, TLR8, and MYD88 showed a marked increase after 24 hpi during infection. Further research confirmed TAP1 as one of the targets of miR-330-y, overexpression of miR-330-y with mimics or agomir significantly reduced the expression levels of TAP1, IRF3, and IFN, and the opposite effects were obtained by inhibitor. These results facilitate in-depth understanding of the immune mechanisms in rainbow trout against IHNV.

Effects of feeding level and sexual maturation on expression of genes regulating growth mechanisms in rainbow trout (Oncorhynchus mykiss)

The energy and nutrient demands of sexual maturation in salmonids requires a shift from somatic growth to gonadal development, although a loss of skeletal muscle can be avoided with adequate nutrition. How nutrient availability or ration affects the endocrine and growth factor systems that integrate nutrient partitioning, muscle metabolism, and growth during sexual maturation is poorly understood. Primary objectives of the current study were to determine how sexual maturation and ration affect expression of estrogen receptors, components of the GH/IGF axis and TGFβ superfamily system, and muscle specific regulatory factors during gonadal development in female rainbow trout (Oncorhynchus mykiss). Female diploid rainbow trout approaching ovulation were fed for 12 weeks at 0.25% and 0.50% tank biomass, and to apparent satiation. Controls for the effects of gonadal development included sterile triploid trout and two diploid females that failed to undergo gonadal development. Expression of era1 was increased in muscle of maturing diploids suggesting an increase in the catabolic effects of 17β-estradiol (E2) on muscle. In the liver, expression of era2 increased with maturation, but expression of all estrogen receptors were increased with higher rations suggesting improved E2 sensitivity for vitellogenin production. Hepatic expression of ghrs and igfs were generally reduced with maturation suggesting a reduction in systemic growth promotion. Increased ration improved capacity for IGF signaling in muscle via increased igf1 and igfr1b expression, potentially lessening effects of reduced circulating IGF-I on muscle growth. The primary carrier of IGFs, hepatic igfbp2b1, increased with maturation likely reducing systemic IGF activity, and was also elevated with higher rations. All other hepatic IGF binding proteins, except igfbp1, showed some reduction with maturation and several increased with increased ration. Increased muscle igfbp6b1 suggests restricted myogenesis with maturation. Reductions in expression of muscle regulatory factors with maturation including pax7a, myog and myf5, support reduced myogenesis with maturation but were unaffected by ration. In addition, although mstn2a was the only myostatin gene affected by maturation or ration, altered expression of components of the TGFβ superfamily suggest increased ration may inhibit myostatin signaling. In summary, moderate feeding appears to counter many changes in endocrine and growth factor system gene expression associated with growth reduction during maturation, perhaps altering endocrine and growth factor regulation to spare muscle atrophy and better prepare the fish for post-spawning recovery.

Variable impacts of L-arginine or L-NAME during early life on molecular and cellular markers of muscle growth mechanisms in rainbow trout

Two experiments were conducted to test if manipulations of the Arginine-Nitric oxide pathway during the early life of rainbow trout would act on its early myogenic process. In experiment 1, trout embryos were immersed at 72° days post-fertilization (°dpf) or 104°dpf in water alone (control treatment, C) or containing 2 mM/L L-Arg (treatment A) or 1 mM/L of L-NAME, a NOS inhibitor (treatment N). We observed the beginning of expression of myf5 and fmhc genes at 72°dpf and 96°dpf, respectively. “A” treatment doubled the free Arg content of eggs but did not affect either the pattern of expression of myf5 and fmhc, nor white muscle cross-sectional area and number of white muscle fibres at hatching, nor embryo survival and fry growth. “N” treatment also did not affect these markers. In experiment 2, trout fry were fed from first feeding onwards and during 20 days either a control diet (C) or the same diet supplemented with L-NAME (0.1 g/100 g diet, N-diet). In C-fed fry, distribution of a single meal after overnight fasting induced changes in pcna, myod1, myog, fmhc, inos, nnos and ctsd gene expressions. N-feeding decreased fry growth but did not change their growth trajectory or survival. Twenty days of N-feeding led, compared to C-feeding, to changes in kinetics of transcription of pcna, myod1, myog, fmhc, inos, nnos, ctsd genes and to decreased white muscle cross-sectional area, total number of white muscle fibres, and number of large muscle fibres. L-NAME feeding thus decreased fry muscle growth by altering both hyperplasia and hypertrophy.

Identification and characterization of microRNAs in the liver of rainbow trout in response to heat stress by high-throughput sequencing

MicroRNAs (miRNAs) play key roles in the stress response in animals. The rainbow trout is a widely cultivated cold-water fish species that usually encounters heat stress, which affects its growth and may cause diseases. However, there is limited information on the miRNAs involved in heat stress in this species. To identify the heat-responsive miRNAs at the genome level in rainbow trout, we conducted high-throughput sequencing of 6 small RNA libraries from the liver tissues of rainbow trout specimens under control and heat-treated conditions. A total of 363 conserved miRNAs and 638 novel miRNAs were identified, among which 39 (21 conserved and 18 novel) miRNAs were differentially expressed in response to heat stress. Eight important miRNAs were further validated using quantitative real-time PCR. GO enrichment and KEGG pathway analyses of the potential target genes of the DE miRNAs, revealed DNA replication and protein folding and unfolding were affected by heat stress. Several key pathways involved in the heat stress response were characterized, such as protein processing in the endoplasmic reticulum, NOD-like receptor signaling pathway, and progesterone-mediated oocyte maturation. In addition, 20 important functional interaction pairs among the 65 negatively correlated miRNA-mRNA pairs were identified using integrated miRNA-mRNA analysis and miRNA prediction algorithms, which including potential important miRNAs and target genes such as miR-301a, miR-301d, let-7c, novel_1181 and HSP90BA, HSP90BB, HSP40, PDIA3. This study showed that the identified miRNAs probably play important roles in the heat stress response and lay a foundation for further studies on the thermal adaptation mechanisms in rainbow trout.

Effects of sex steroids on expression of genes regulating growth-related mechanisms in rainbow trout (Oncorhynchus mykiss)

Effects of a single injection of 17β-estradiol (E2), testosterone (T), or 5β-dihydrotestosterone (DHT) on expression of genes central to the growth hormone (GH)/insulin-like growth factor (IGF) axis, muscle-regulatory factors, transforming growth factor-beta (TGFβ) superfamily signaling cascade, and estrogen receptors were determined in rainbow trout (Oncorhynchus mykiss) liver and white muscle tissue. In liver in addition to regulating GH sensitivity and IGF production, sex steroids also affected expression of IGF binding proteins, as E2, T, and DHT increased expression of igfbp2b and E2 also increased expression of igfbp2 and igfbp4. Regulation of this system also occurred in white muscle in which E2 increased expression of igf1, igf2, and igfbp5b1, suggesting anabolic capacity may be maintained in white muscle in the presence of E2. In contrast, DHT decreased expression of igfbp5b1. DHT and T decreased expression of myogenin, while other muscle regulatory factors were either not affected or responded similarly for all steroid treatments. Genes within the TGFβ superfamily signaling cascade responded to steroid treatment in both liver and muscle, suggesting a regulatory role for sex steroids in the ability to transmit signals initiated by TGFβ superfamily ligands, with a greater number of genes responding in liver than in muscle. Estrogen receptors were also regulated by sex steroids, with era1 expression increasing for all treatments in muscle, but only E2- and T-treatment in liver. E2 reduced expression of erb2 in liver. Collectively, these data identify how physiological mechanisms are regulated by sex steroids in a manner that promotes the disparate effects of androgens and estrogens on growth in salmonids.

Different immunization routes appear to induce protection against Aeromonas salmonicida through different immune mechanisms in rainbow trout

Different immunization routes appear to induce protection against Aeromonas salmonicida through different immune mechanisms in rainbow trout

Stress alters food intake and glucosensing response in hypothalamus, hindbrain, liver, and Brockmann bodies of rainbow trout

In fish food intake is altered under stress conditions, and in a fish teleost model like rainbow trout food intake is associated with the activity of the glucosensor systems. Thus, we aimed to evaluate the possible interaction of stress with the response of glucosensor mechanisms in rainbow trout. Thus, we subjected rainbow trout (via intraperitoneal injections) to normoglycaemic (control), hypoglycaemic (4 mg.kg -1 bovine insulin) or hyperglycaemic (500 mg.kg -1 glucose body mass) conditions for 5 days under normal stocking density (NSD, 10 kg fish mass·m -3) or stress conditions induced by high stocking density (HSD, 70 kg fish mass·m -3). The experimental design was appropriate since hypoglycemia and hyperglycemia were observed in fish under NSD whereas in normoglycaemic fish HSD induced changes in stress-related parameters similar to those reported in fish literature, such as increased levels of cortisol and glucose in plasma and decreased levels of glycogen in liver. Food intake did not respond to changes in plasma glucose levels in fish under HSD conditions, in contrast with the decreased food intake observed when glucose levels increased in fish under NSD conditions. Moreover, the changes with the increase in plasma glucose levels in parameters involved in glucosensing in liver, Brockmann bodies (BB), hypothalamus, and hindbrain of fish in NSD either disappeared (DHAP and GAP levels, and GK, PK, and GPase activities in liver; glucose, DHAP and GAP levels in BB; glucose and DHAP levels, and GK and PK activities in hypothalamus; glycogen and DHAP levels, and GSase activity in hindbrain) or changed (cortisol levels in plasma; glycogen and GAP levels, and GSase and FBPase activities in liver; GK and PK activities in BB; GK and PK activities in hindbrain) in fish under HSD. Those changes suggest for the first time in fish the existence of an interaction between glucosensing capacity and stress. The readjustment in the activity of glucosensor systems is also associated with changes in food intake resulting in an inability of the fish to compensate with changes in food intake those of circulating glucose levels as observed in fish under non-stressed conditions.

Identification and expression analysis of rainbow trout pumilio-1 and pumilio-2

Pumilio is a sequence-specific RNA-binding protein that regulates translation from the relevant mRNA. The PUF-domain, the RNA-binding motif of Pumilio, is highly conserved across species. In the present study, we have identified two pumilio genes (pumilio-1 and pumilio-2) in rainbow trout and analyzed their expression patterns in its tissues. Pumilio-1 mRNA and pumilio-2A mRNA code for typical full length Pumilio proteins that contain a PUF-domain, whereas pumilio-2B mRNA is a splice variant of pumilio-2 and encodes a protein that lacks the PUF-domain. We have also identified a novel 72-bp exon that has not been reported in other animal species but is conserved in fish species. The insertion of this novel exon leads to the expression of an isoform of the Pumilio-2 protein with a slightly altered conformation of the PUF-domain. Pumilio-1 mRNA and pumilio-2A mRNA (irrespective of the presence of the 72-bp exon) are expressed in both the brain and ovaries at high levels, whereas pumilio-2B mRNA is expressed at low levels in all the rainbow trout tissues examined. Western blot analysis also indicates that the full length Pumilio proteins are expressed predominantly in the brain and ovaries. These data suggest that the Pumilio proteins have physiological roles and are involved in regulatory mechanisms in rainbow trout.

Enhancement of innate immunity in rainbow trout ( Oncorhynchus mykiss Walbaum) associated with dietary intake of carotenoids from natural products

The effects of orally administered carotenoids from natural sources on the non-specific defense mechanisms of rainbow trout were evaluated in a nine-week feeding trial. Fish were fed four diets containing either β-carotene or astaxanthin at 100 and 200 mg kg −1 from the marine algae Dunaliella salina and red yeast Phaffia rhodozyma, respectively, and a control diet containing no supplemented carotenoids. Specific growth rate and feed:gain ratio were not affected by dietary carotenoid supplementation. Among the humoral factors, serum alternative complement activity increased significantly in all carotenoid supplemented groups when compared to the control. On the other hand, serum lysozyme activity increased in the Dunaliella group but not in the Phaffia group, whereas plasma total immunoglobulin levels were not altered by the feeding treatments. As for the cellular responses, the superoxide anion production from the head kidney remained unchanged while the phagocytic rate and index in all supplemented groups were significantly higher than those of the control. These findings demonstrate that dietary carotenoids from both D. salina and P. rhodozyma can modulate some of the innate defense mechanisms in rainbow trout.

Influence of various dietary synthetic carotenoids on bio-defence mechanisms in rainbow trout,Oncorhynchus mykiss(Walbaum)

This study examined the influence of different carotenoids on growth and some immune indices in rainbow trout. Six semipurified casein-based diets were formulated to contain one of three different carotenoids: astaxanthin, canthaxanthin and β-carotene, at 100 mg kg−1, each of them with vitamins A, C and E either added or omitted. The two control diets contained no carotenoids and were either with or without the vitamins. Rainbow trout weighing about 140 g were fed the diets for 9 weeks. Specific growth rate, feed:gain ratio and nonspecific immune parameters were determined. Growth and feed conversion were similar among the groups. Immune parameters like production of reactive oxygen species by head kidney leukocytes and plasma total immunoglobulin levels did not vary with the treatment. Serum complement activity in both β-carotene groups and the vitamin-containing astaxanthin group were significantly higher than both the control fish. Serum lysozyme activity in the vitamin-containing β-carotene and astaxanthin groups were significantly different from both control groups. Phagocytic activity was also high in the vitamin-containing β-carotene and astaxanthin groups compared with the controls. For phagocytic index, in addition to the foregoing groups, the vitamin-containing canthaxanthin group gave better results compared with the controls. The vitamin-containing astaxanthin and β-carotene groups also exhibited better nonspecific cytotoxicity for the peripheral blood lymphocytes at all effector-to-target ratios. Thus, among the carotenoids studied, β-carotene and astaxanthin elevated humoral factors such as serum complement and lysozyme activity, as well as cellular factors such as phagocytosis and nonspecific cytotoxicity. In the presence of the vitamins the carotenoids exerted a greater influence on the bio-defense mechanisms of rainbow trout.

Prevention of stress in rainbow trout ( Oncorhynchus mykiss) fed diets containing different doses of glucan

An experiment was performed to determine the effects of stress on non-specific defence mechanisms in rainbow trout fed diets containing different doses of glucan. Fish were fed with 0, 0.1, 0.5 and 1.0% glucan concentration in food. After 4 weeks of feeding, fish were stressed by 2 h transportation. The effect of stress was investigated by measuring changes in blood levels of cortisol, glucose, total protein and composition of their leukocyte population, as well as by changes in non-specific defence mechanisms of the fish. After 4 weeks feeding with glucan, elevated phagocytosis and oxidative radical production were observed in treated fish, but the levels did not correlate with the different doses of glucan. Stress induced by 2 h transportation caused high cortisol levels in plasma and hyperglycaemia in all groups, but the lowest level of glucose was measured in the group fed the low (0.1%) dose of glucan. Respiratory burst activity, phagocytosis, serum protein and lyzosyme levels were found to be significantly reduced by stress. The most dramatic reduction was observed in the control group, but the changes were not affected by glucan doses. One week post stress, hyperglycaemia was still observed in control and fish fed medium and high doses of glucan. Further reductions of total protein and intracellular oxidative radical production, were measured in all groups, but in fish fed with low dose of glucan the changes were less dramatic. The phagocytosis ratio increased in all groups, but did not attain the levels measured before stress in control and in the group fed the high concentration of glucan. A spontaneous infection with Flexibacter columnaris caused mortality in all groups except the group fed the low level of glucan. The results of the present study show that feeding of glucan in low doses several weeks before transportation can help to prevent negative effects of stress.

Glucan injection or bath exposure given alone or in combination with a bacterin enhance the non-specific defence mechanisms in rainbow trout ( Oncorhynchus mykiss)

Elevations in the non-specific defence mechanisms were noted after trout were injected or bathed in glucan solutions or in solutions containing the glucan combined with a bacterin, Y. ruckeri O-antigen. Periodic non-lethal blood samples showed that neutrophil activity as evaluated by the ability of the cells to stick to glass and produce oxidative radicals as detected by the nitroblue tetrazolium (NBT) assay, rose after treatments within 2 days. Elevated phagocytosis, assessed by increased uptake of glutaraldehyde-treated sheep red blood cells, also confirmed these kinetics. The numbers of circulatory glass-adherent cells from fish given the glucans by injection or bath was twice the level of the sham-treated controls; likewise the phagocytic ratio also was two-fold higher. In following the kinetics of the non-specific defensive responses, the injection of the glucan caused an immediate, slight reduction of NBT staining cells and numbers of leukocytes before the 2–3 day rise; this was not obvious in the fish given the glucan by bath. The glucans could play an important role in the prevention of diseases in fish culture.