RESUMEN
The production and release of cortisol during stress responses are key regulators of growth in teleosts. Understanding the molecular responses to cortisol is crucial for the sustainable farming of rainbow trout (Oncorhynchus mykiss) and other salmonid species. While several studies have explored the genomic and non-genomic impacts of cortisol on fish growth and skeletal muscle development, the long-term effects driven by epigenetic mechanisms, such as cortisol-induced DNA methylation, remain unexplored. In this study, we analyzed the transcriptome and genome-wide DNA methylation in the skeletal muscle of rainbow trout seven days after cortisol administration. We identified 550 differentially expressed genes (DEGs) by RNA-seq and 9059 differentially methylated genes (DMGs) via whole-genome bisulfite sequencing (WGBS) analysis. KEGG enrichment analysis showed that cortisol modulates the differential expression of genes associated with nucleotide metabolism, ECM-receptor interaction, and the regulation of actin cytoskeleton pathways. Similarly, cortisol induced the differential methylation of genes associated with focal adhesion, adrenergic signaling in cardiomyocytes, and Wnt signaling. Through integrative analyses, we determined that 126 genes showed a negative correlation between up-regulated expression and down-regulated methylation. KEGG enrichment analysis of these genes indicated participation in ECM-receptor interaction, regulation of actin cytoskeleton, and focal adhesion. Using RT-qPCR, we confirmed the differential expression of lamb3, itga6, limk2, itgb4, capn2, and thbs1. This study revealed for the first time the molecular responses of skeletal muscle to cortisol at the transcriptomic and whole-genome DNA methylation levels in rainbow trout.
Asunto(s)
Metilación de ADN , Hidrocortisona , Músculo Esquelético , Oncorhynchus mykiss , Estrés Fisiológico , Transcriptoma , Animales , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/metabolismo , Hidrocortisona/metabolismo , Hidrocortisona/farmacología , Músculo Esquelético/metabolismo , Músculo Esquelético/efectos de los fármacos , Estrés Fisiológico/genética , Epigénesis Genética , Epigenómica/métodos , Perfilación de la Expresión Génica , Proteínas de Peces/genética , Proteínas de Peces/metabolismoRESUMEN
In the context of harmful algal blooms, fish can be exposed to the combined effects of more than one toxin. We studied the effects of consecutive exposure to Microcystin-LR (MCLR) in vivo and paralytic shellfish toxins (PST) ex vivo/in vitro (MCLR+PST) in the rainbow trout Oncorhynchus mykiss's middle intestine. We fed juvenile fish with MCLR incorporated in the feed every 12 h and euthanized them 48 h after the first feeding. Immediately, we removed the middle intestine to make ex vivo and in vitro preparations and exposed them to PST for one hour. We analyzed glutathione (GSH) and glutathione disulfide (GSSG) contents, glutathione S-transferase (GST), glutathione reductase (GR), catalase (CAT), and protein phosphatase 1 (PP1) activities in ex vivo intestinal strips; apical and basolateral ATP-biding cassette subfamily C (Abcc)-mediated transport in ex vivo everted and non- everted sacs; and reactive oxygen species (ROS) production in isolated enterocytes in vitro. MCLR+PST treatment decreased the GSH content, GSH/GSSG ratio, GST activity, and increased ROS production. GR activity remained unchanged, while CAT activity only increased in response to PST. MCLR inhibited PP1 activity and activated Abcc-mediated transport only at the basolateral side of the intestine. Our results show a combined effect of MCLR+PST on the oxidative balance in the O. mykiss middle intestine, which is not affected by the two toxins groups when applied individually. Basolateral Abcc transporters activation by MCLR treatment could lead to an increase in the absorption of toxicants (including MCLR) into the organism. Therefore, MCLR makes the O. mykiss middle intestine more sensitive to possibly co-occurring cyanotoxins like PST.
Asunto(s)
Mucosa Intestinal , Toxinas Marinas , Microcistinas , Oncorhynchus mykiss , Estrés Oxidativo , Especies Reactivas de Oxígeno , Animales , Microcistinas/toxicidad , Toxinas Marinas/toxicidad , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Estrés Oxidativo/efectos de los fármacos , Oncorhynchus mykiss/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Glutatión/metabolismo , Saxitoxina/toxicidadRESUMEN
Global climate change favors explosive population growth events (blooms) of phytoplanktonic species, often producing toxic products, e.g., several genera of cyanobacteria synthesize a family of cyanotoxins called microcystins (MCs). Freshwater fish such as the rainbow trout Oncorhynchus mykiss can uptake MCs accumulated in the food chain. We studied the toxic effects and modulation of the activity and expression of multixenobiotic resistance proteins (ABCC transporters and the enzyme glutathione S-transferase (GST) in the O. mykiss middle intestine by microcystin-LR (MCLR). Juvenile fish were fed with MCLR incorporated in the food every 12 h and euthanized at 12, 24, or 48 h. We estimated the ABCC-mediated transport in ex vivo intestinal strips to estimate ABCC-mediated transport activity. We measured total and reduced (GSH) glutathione contents and GST and glutathione reductase (GR) activities. We studied MCLR cytotoxicity by measuring protein phosphatase 1 (PP1) activity and lysosomal membrane stability. Finally, we examined the relationship between ROS production and lysosomal membrane stability through in vitro experiments. Dietary MCLR had a time-dependent effect on ABCC-mediated transport, from inhibition at 12 h to a significant increase after 48 h. GST activity decreased only at 12 h, and GR activity only increased at 48 h. There were no effects on GSH or total glutathione contents. MCLR inhibited PP1 activity and diminished the lysosomal membrane stability at the three experimental times. In the in vitro study, the lysosomal membrane stability decreased in a concentration-dependent fashion from 0 to 5 µmol L - 1 MCLR, while ROS production increased only at 5 µmol L - 1 MCLR. MCLR did not affect mRNA expression of abcc2 or gst-π. We conclude that MCLR modulates ABCC-mediated transport activity in O. mykiss's middle intestine in a time-dependent manner. The transport rate increase does not impair MCLR cytotoxic effects.
Asunto(s)
Oncorhynchus mykiss , Contaminantes Químicos del Agua , Animales , Microcistinas/toxicidad , Microcistinas/metabolismo , Oncorhynchus mykiss/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Contaminantes Químicos del Agua/toxicidad , Intestinos , Glutatión Transferasa/metabolismo , Glutatión/metabolismoRESUMEN
The organophosphorus pesticide chlorpyrifos, detected in water and food worldwide, has also been found in the Río Negro and Neuquén Valley, North Patagonia, Argentina, where the rainbow trout, Oncorhynchus mykiss, is one of the most abundant fish species. We analyzed whether chlorpyrifos affects the transport activity of the ATP-binding cassette protein transporters from the subfamily C (ABCC), which are critical components of multixenobiotic resistance. We exposed ex vivo O. mykiss middle intestine strips (non-polarized) and segments (polarized) for one hour to 0 (solvent control), 3, 10, and 20 µg L-1 and to 0, 10, and 20 µg L-1 chlorpyrifos, respectively. We estimated the Abcc-mediated transport rate by measuring the transport rate of the specific Abcc substrate 2,4-dinitrophenyl-S-glutathione (DNP-SG). In addition, we measured the enzymatic activity of cholinesterase, carboxylesterase, glutathione-S-transferase, and 7-ethoxyresorufin-O-deethylase (EROD, indicative of the activity of cytochrome P450 monooxygenase 1A, CYP1A). We also measured lipid peroxidation using the thiobarbituric acid reactive substances method and the gene expression of Abcc2 and genes of the AhR pathway, AhR, ARNT, and cyp1a, by qRT-PCR. Chlorpyrifos induced the DNP-SG transport rate in middle intestine strips in a concentration-dependent manner (49-71%). In polarized preparations, the induction of the DNP-SG transport rate was observed only in everted segments exposed to 20 µg L-1 chlorpyrifos (40%), indicating that CPF only stimulated the apical (luminal) transport flux. Exposure to chlorpyrifos increased GST activity by 42% in intestine strips and inhibited EROD activity (47.5%). In addition, chlorpyrifos exposure inhibited cholinesterase (34-55%) and carboxylesterase (33-42.5%) activities at all the concentrations assayed and increased TBARS levels in a concentration-dependent manner (71-123%). Exposure to 20 µgL-1 chlorpyrifos did not affect the mRNA expression of the studied genes. The lack of inhibition of DNP-SG transport suggests that chlorpyrifos is not an Abcc substrate. Instead, CPF induces the activity of Abcc proteins in the apical membrane of enterocytes, likely through a post-translational pathway.
Asunto(s)
Cloropirifos , Oncorhynchus mykiss , Plaguicidas , Contaminantes Químicos del Agua , Transportadoras de Casetes de Unión a ATP , Adenosina Trifosfato/metabolismo , Animales , Hidrolasas de Éster Carboxílico/metabolismo , Cloropirifos/farmacología , Colinesterasas , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Glutatión/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Intestinos , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/metabolismo , Compuestos Organofosforados/metabolismo , Plaguicidas/metabolismo , ARN Mensajero/metabolismo , Solventes , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Agua/metabolismo , Contaminantes Químicos del Agua/toxicidadRESUMEN
Feeding and digestion are metabolically demanding causing a rise on metabolic rate called Specific Dynamic Action (SDA). Although SDA has been vastly reported in fish, its potential consequences on the oxidative-antioxidant balance has not been evaluated to date in fish, a model with a long alkaline tide associated with feeding as well. Using rainbow trout (Oncorhynchus mykiss) as a model species, the aims of the present study were to: (1) assess potential oxidative damages and changes in oxidative defences after feeding on a single meal, and (2) identify the timescale of such changes over a 96 h post-feeding period. Oxidative damage in proteins and lipids and the activities of four enzymatic antioxidant defences: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were measured in gill, stomach, intestine and liver. DNA damage was measured in red blood cells. Fish were sampled before and after 1.5, 6, 24, 48, 72 and 96 h of ingestion of a 3% body mass ration. Trends of post-prandial damage were present in all tissues, but only protein oxidation varied significatively during digestion in the stomach. The intestine and stomach presented the highest enzymatic activities, likely due to the high metabolic action that these tissues have during digestion, with peaks during post-feeding: at 24 h of SOD in stomach and at 48 h of CAT in intestine. Observed GPx peaks during post-feeding in gills are likely due to the exacerbated demands for ion fluxes and/or oxygen during feeding. The differential response of the antioxidant system observed in tissues of rainbow trout during digestion indicates a coordinated and tissue-specific antioxidant defence.
Asunto(s)
Oncorhynchus mykiss , Animales , Antioxidantes/metabolismo , Catalasa/metabolismo , Glutatión Peroxidasa/metabolismo , Glutatión Transferasa/metabolismo , Oncorhynchus mykiss/metabolismo , Estrés Oxidativo , Superóxido Dismutasa/metabolismoRESUMEN
Lactic acid bacteria are a powerful vehicle for releasing of cytokines and immunostimulant peptides at the gastrointestinal level after oral administration. However, its therapeutic application against pathogens that affect rainbow trout and Atlantic salmon has been little explored. Type II interferon in Atlantic salmon activates the antiviral response, protecting against viral infection, but its role against bacterial infection has not been tested in vivo. In this work, through the design of a recombinant lactic acid bacterium capable of producing Interferon gamma from Atlantic salmon, we explore its role against bacterial infection and the ability to stimulate systemic immune response after oral administration of the recombinant probiotic. Recombinant interferon was active in vitro, mainly stimulating IL-6 expression in SHK-1 cells. In vivo, oral administration of the recombinant probiotic produced an increase in IL-6, IFNγ and IL-12 in the spleen and kidney, in addition to stimulating the activity of lysozyme in serum. The challenge trials indicated that the administration of the IFNγ-producing probiotic doubled the survival in fish infected with F. psychrophilum. In conclusion, our results showed that the oral administration of lactic acid bacteria producing IFNγ managed to stimulate the immune response at a systemic level, conferring protection against pathogens, showing a biotechnological potential for its application in aquaculture.
Asunto(s)
Proteínas de Peces/metabolismo , Infecciones por Flavobacteriaceae/prevención & control , Flavobacterium/patogenicidad , Interferón gamma/metabolismo , Lactococcus lactis/metabolismo , Oncorhynchus mykiss/microbiología , Probióticos/administración & dosificación , Administración Oral , Animales , Línea Celular , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Infecciones por Flavobacteriaceae/inmunología , Infecciones por Flavobacteriaceae/metabolismo , Infecciones por Flavobacteriaceae/microbiología , Flavobacterium/inmunología , Interacciones Huésped-Patógeno , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/inmunología , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/inmunología , Oncorhynchus mykiss/metabolismo , FilogeniaRESUMEN
We studied the absorption, cytotoxicity and oxidative stress markers of Paralytic Shellfish Toxins (PST) from three extracts from Alexandrium catenella and A. ostenfeldii, in middle Oncorhynchus mykiss intestine in vitro and ex vivo preparations. We measured glutathione (GSH) content, glutathione-S transferase (GST), glutathione reductase (GR) and catalase (CAT) enzymatic activity, and lipid peroxidation in isolated epithelium exposed to 0.13 and 1.3 µM PST. ROS production and lysosomal membrane stability (as neutral red retention time 50%, NRRT50) were analyzed in isolated enterocytes exposed to PST alone or plus 3 µM of the ABCC transport inhibitor MK571. In addition, the concentration-dependent effects of PST on NRRT50 were assayed in a concentration range from 0 to 1.3 µM PST. We studied the effects of three different PST extracts on the transport rate of the ABCC substrate DNP-SG by isolated epithelium. The extract with highest inhibition capacity was selected for studying polarized DNP-SG transport in everted and non-everted intestinal segments. We registered lower GSH content and GST activity, and higher GR activity, with no significant changes in CAT activity, lipid peroxidation or ROS level. PST exposure decreased NRRT50 in a concentration-depend manner (IC50 = 0.0045 µM), but PST effects were not augmented by addition of MK571. All the three PST extracts inhibited ABCC transport activity, but this inhibition was effective only when the toxins were applied to the apical side of the intestine and DNP-SG transport was measured at the basolateral side. Our results indicate that PST are absorbed by the enterocytes from the intestine lumen. Inside the enterocytes, these toxins decrease GSH content and inhibit the basolateral ABCC transporters affecting the normal functions of the cell. Furthermore, PST produce a strong cytotoxic effect to the enterocytes by damaging the lysosomal membrane, even at low, non-neurotoxic concentrations.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Glutatión/análogos & derivados , Mucosa Intestinal/efectos de los fármacos , Lisosomas/efectos de los fármacos , Oncorhynchus mykiss/metabolismo , Estrés Oxidativo/efectos de los fármacos , Saxitoxina/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Catalasa/metabolismo , Dinoflagelados/metabolismo , Enterocitos/efectos de los fármacos , Enterocitos/metabolismo , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Mucosa Intestinal/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Lisosomas/metabolismo , MariscosRESUMEN
BACKGROUND: Skeletal muscle is one of the tissues most affected by stress conditions. The protein degradation in this tissue is vital for the supply of energy mediated by different proteolytic pathways such as the ubiquitin-proteasome (UPS), autophagy-lysosome (ALS) and the calpain/calpastatin system (CCS). Nevertheless, the regulation of this proteolytic axis under stress conditions is not yet completely clear. Chile is the main producer of rainbow trout (Oncorhynchus mykiss) in the world. This intensive fish farming has resulted in growing problems as crowding and stress are one of the major problems in the freshwater stage. In this context, we evaluated the crowding effect in juvenile rainbow trout kept in high stocking density (30 kg/m3) for 15, 45 and 60 days, using a control group of fish (10 kg/m3). RESULTS: Plasmatic cortisol and glucose were evaluated by enzyme immunoassay. The mRNA levels of stress-related genes (gr1, gr2, mr, hsp70, klf15 and redd1), markers of the UPS (atrogin1 and murf1) and CCS (capn1, capn1, cast-l and cast-s) were evaluated using qPCR. ALS (LC3-I/II and P62/SQSTM1) and growth markers (4E-BP1 and ERK) were measured by Western blot analysis. The cortisol levels increased concomitantly with weight loss at 45 days of crowding. The UPS alone was upregulated at 15 days of high stocking density, while ALS activation was observed at 60 days. However, the CCS was inactivated during the entire trial. CONCLUSION: All these data suggest that stress conditions, such as crowding, promote muscle degradation in a time-dependent manner through the upregulation of the UPS at early stages of chronic stress and activation of the ALS in long-term stress, while the CCS is strongly inhibited by stress conditions in the rainbow trout muscle farmed during freshwater stage. Our descriptive study will allow perform functional analysis to determine, in a more detailed way, the effect of stress on skeletal muscle physiology as well as in the animal welfare in rainbow trout. Moreover, it is the first step to elucidate the optimal crop density in the freshwater stage and improve the standards of Chilean aquaculture.
Asunto(s)
Aglomeración , Músculo Esquelético/metabolismo , Oncorhynchus mykiss/metabolismo , Proteolisis , Animales , Acuicultura/métodos , Autofagia , Peso Corporal , Proteínas de Unión al Calcio/metabolismo , Calpaína/metabolismo , Hidrocortisona/sangre , Lisosomas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Mensajero , Estrés Fisiológico/genética , Ubiquitina/metabolismoRESUMEN
Triploid fish are usually sterile. Thus, the energy and nutrients intended for sexual maturation may be available to enhance flesh quality and physical growth. The present study aimed to investigate differences in the metabolic substrates, lipids and proteins, between storage tissues from diploid and triploid female rainbow trout. Monthly, metabolic substrates were quantified in liver, muscle, and ovaries, which were collected during the first reproductive cycle. In general, it was possible to identify a seasonal and similar deposition of metabolites in different tissues of 2n and 3n females, mainly at early stages of gonadal maturation. However, from the stages 5-6, the ovaries showed great differences between ploidies, with higher concentration of lipids and protein in 2n females. This result reflects the incorporation of vitellogenin in oocytes, which is a process that does not occur in 3n females. It was possible to observe seasonal hepato-somatic index changes in 2n females, with higher values observed in the post-ovulatory stage, and the triploid animals showed lower values compared to 2n, with no seasonal difference. Viscero-somatic index can reflect the mobilization of substrates, with higher values found for 2n females in stage 5-6, which is the period of active mobilization of tissue substrates.
Asunto(s)
Animales , Oncorhynchus mykiss/fisiología , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/metabolismo , TriploidíaRESUMEN
Triploid fish are usually sterile. Thus, the energy and nutrients intended for sexual maturation may be available to enhance flesh quality and physical growth. The present study aimed to investigate differences in the metabolic substrates, lipids and proteins, between storage tissues from diploid and triploid female rainbow trout. Monthly, metabolic substrates were quantified in liver, muscle, and ovaries, which were collected during the first reproductive cycle. In general, it was possible to identify a seasonal and similar deposition of metabolites in different tissues of 2n and 3n females, mainly at early stages of gonadal maturation. However, from the stages 5-6, the ovaries showed great differences between ploidies, with higher concentration of lipids and protein in 2n females. This result reflects the incorporation of vitellogenin in oocytes, which is a process that does not occur in 3n females. It was possible to observe seasonal hepato-somatic index changes in 2n females, with higher values observed in the post-ovulatory stage, and the triploid animals showed lower values compared to 2n, with no seasonal difference. Viscero-somatic index can reflect the mobilization of substrates, with higher values found for 2n females in stage 5-6, which is the period of active mobilization of tissue substrates.(AU)
Asunto(s)
Animales , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/metabolismo , Oncorhynchus mykiss/fisiología , TriploidíaRESUMEN
Cortisol modulates energy metabolism promoting the mobilization of glucose and increasing proteolysis to overcome stressful situations in teleost. The cortisol metabolic effects are attributed to genomic mechanisms that involve the interaction of cortisol with its glucocorticoid intracellular receptor. Furthermore, cortisol can also interact with plasma membrane glucocorticoid receptors activating a rapid nongenomic signaling; however, its contribution during the early acute phase stress response in fish is unknown. In the present work, we evaluated the effects of membrane-initiated cortisol actions in vivo in the proteome of rainbow trout (Oncorhynchus mykiss) skeletal muscle. Quantitative iTRAQ analyses were performed to examine proteomic changes in rainbow trout stimulated with physiological concentrations of cortisol and cortisol-BSA, a membrane-impermeable cortisol conjugate. A total of 873 proteins were identified, among which 61 and 47 proteins were differentially expressed under cortisol and cortisol-BSA treatments, respectively. Functional clustering analysis revealed an upregulation of proteins associated with mitochondria and oxidative phosphorylation. These results were validated by Western blot analysis. Additionally, using rainbow trout myotubes, the participation of membrane glucocorticoid receptors in gene expression was evaluated. The results obtained suggest that cortisol acts through a membrane canonical glucocorticoid receptor and mediates the expression of proteins associated with mitochondrial oxidative phosphorylation.
Asunto(s)
Proteínas de Peces/metabolismo , Músculo Esquelético/metabolismo , Oncorhynchus mykiss/metabolismo , Proteómica , Receptores de Glucocorticoides/metabolismo , Animales , Hidrocortisona/administración & dosificación , Metales/metabolismo , Proteínas Mitocondriales/metabolismo , Músculo Esquelético/efectos de los fármacos , Fosforilación Oxidativa , Albúmina Sérica Bovina/administración & dosificaciónRESUMEN
Cu, Mn, Fe, Zn, Cd and Pb levels were measured in liver and muscle samples of Rainbow Trout Oncorhynchus mykiss collected from three watersheds with different land-uses: native forest, exotic plantation, and agriculture in Chile, during January, April, July, and October 2012. Cd and Pb levels were not detected in the liver and muscle, probably since they are under the detection limits. Higher metal concentrations (liver-muscle tissues) were detected in samples from agriculture and exotic plantation streams, whereas trout from native forest streams had lower metal concentrations. Higher metal concentrations were detected in liver tissue compared to muscle tissue, and both negatively correlated to the length and weight of the fish. This suggest the liver had higher ability to accumulate Cu, Mn, Fe and Zn compared to muscle tissue. The concentration range of Fe and Zn recorded in the muscle are within the range reported by other authors, whereas Mn and Cu concentrations are higher than reported in the literature. However, at all sites the concentration of selected metals were below the limits permitted by current legislation (FAO), and therefore did not put the human population at risk, suggesting that is eating wild rainbow trout safe in Chile.
Asunto(s)
Metales Pesados/metabolismo , Oncorhynchus mykiss/metabolismo , Contaminantes Químicos del Agua/metabolismo , Agricultura , Animales , Chile , Humanos , Hígado/química , Metales Pesados/análisis , Músculos/química , Ríos , Alimentos Marinos , Contaminantes Químicos del Agua/análisisRESUMEN
In fish of freshwaters environments, the accumulation and toxic effects of arsenite (AsIII) can be attenuated by detoxification proteins such as GST and ABCC transporters. We studied the effects of AsIII on the middle intestine of O. mykiss in ex-vivo and in vivo/ex vivo assays. For the ex vivo assays, we measured the transport rate of the ABCC substrate DNP-SG and GST activity in intestinal strips and everted sacs. AsIII inhibited DNP-SG transport in a concentration-dependent manner, specifically when we applied it on the basolateral side. GST activity increased when we applied a maximum concentration of AsIII. For the in vivo/ex vivo assays, we kept fish in water with or without 7.7⯵molâ¯L-1 of AsIII for 48â¯h. Then, we measured DNP-SG transport rate, GST activity, and PP1 activity in intestine strips during one hour. For PP1 activity, we incubated the strips with or without microcystin-LR (MCLR), a toxin excreted through ABCC2 proteins. We also analyzed Abcc2 and Gst-π mRNA expression in intestine and liver tissue. In the group exposed in vivo to AsIII, DNP-SG transport rate and GST activity were higher and the effect of MCLR over PP1 activity was attenuated. AsIII significantly induced only Abcc2 mRNA expression in both middle intestine and liver. Our results suggest that, in the middle intestine of O. mykiss, AsIII is absorbed mainly at the basolateral side of the enterocytes, excreted to the lumen by ABCC2 transporters, and is capable of modulating Abcc2 mRNA expression by a transcriptional mechanism.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Arsenitos , Gutatión-S-Transferasa pi/metabolismo , Intestinos/enzimología , Hígado/metabolismo , Oncorhynchus mykiss/metabolismo , Animales , Arsenitos/metabolismo , Arsenitos/farmacocinética , Arsenitos/toxicidad , Proteínas de Peces/metabolismo , Regulación de la Expresión Génica , ARN Mensajero , Xenobióticos/metabolismo , Xenobióticos/farmacocinética , Xenobióticos/toxicidadRESUMEN
Growth is one of the most important traits from both a physiological and economic perspective in aquaculture species. Thus, identifying the genomic regions and genes underpinning genetic variation for this trait is of particular interest in several fish species, including rainbow trout. In this work, we perform a genome-wide association study (GWAS) to identify the genomic regions associated with body weight at tagging (BWT) and at 18 months (BW18M) using a dense SNP panel (57 k) and 4596 genotyped rainbow trout from 105 full-sib families belonging to a Chilean breeding population. Analysis was performed by means of single-step GBLUP approach. Genetic variance explained by 20 adjacent SNP windows across the whole genome is reported. To further explore candidate genes, we focused on windows that explained the highest proportion of genetic variance in the top 10 chromosomes for each trait. The main window from the top 10 chromosomes was explored by BLAST using the first and last SNP position of each window to determine the target nucleotide sequence. As expected, the percentage of genetic variance explained by windows was relatively low, due to the polygenic nature of body weight. The most important genomic region for BWT and BW18M were located on chromosomes 15 and 24 and they explained 2.14% and 3.02% of the genetic variance for each trait, respectively. Candidate genes including several growth factors, genes involved in development of skeletal muscle and bone tissue and nutrient metabolism were identified within the associated regions for both traits BWT and BW18M. These results indicate that body weight is polygenic in nature in rainbow trout, with the most important loci explaining as much as 3% of the genetic variance for the trait. The genes identified here represent good candidates for further functional validation to uncover biological mechanisms underlying variation for growth in rainbow trout.
Asunto(s)
Peso Corporal/genética , Proteínas de Peces/genética , Estudio de Asociación del Genoma Completo/métodos , Oncorhynchus mykiss/genética , Animales , Mapeo Cromosómico , Proteínas de Peces/metabolismo , Genómica/métodos , Genotipo , Herencia Multifactorial/genética , Oncorhynchus mykiss/crecimiento & desarrollo , Oncorhynchus mykiss/metabolismo , Polimorfismo de Nucleótido SimpleRESUMEN
Probiotics are being used in biological control of bacterial pathogens, as an alternative to antibiotics, to improve health and production parameters in fish farming. Fish farming production is severely affected by aflatoxins (AFs), which are a significant problem in aquaculture systems. Aflatoxins exert substantial impact on production, causing disease with high mortality and a gradual decline of reared fish stock quality. Some aspects of aflatoxicosis in fish, particularly its effects on the gastrointestinal tract, have not been well documented. The aim of the present study was to evaluate probiotic properties of lactic acid bacterial (LAB) strains isolated from rainbow trout intestine and feed. Moreover, AFB1-binding and/or degrading abilities were also evaluated to assess their use in the formulation of feed additives. Growth at pH 2, the ability to co-aggregate with bacterial pathogens, inhibition of bacterial pathogens, and determination of the inhibitory mechanism were tested. Aflatoxin B1 (AFB1) adsorption and degradation ability were also tested. All strains were able to maintain viable (107 cells ml-1) at pH 2. Pediococcus acidilactici RC001 and RC008 showed the strongest antimicrobial activity, inhibiting all the pathogens tested. The strains produced antimicrobial compounds of different nature, being affected by different treatments (catalase, NaOH and heating), which indicated that they could be H2O2, organic acids or proteins. All LAB strains tested showed the ability to coaggregate pathogenic bacteria, showing inhibition percentages above 40%. Pediococcus acidilactici RC003 was the one with the highest adsorption capacity and all LAB strains were able to degrade AFB1 with percentages higher than 15%, showing significant differences with respect to the control. The ability of some of the LAB strains isolated in the present work to compete with pathogens, together with stability against bile and gastric pH, reduction of bioavailability and degradation of AFB1, may indicate the potential of LAB for use in rainbow trout culture.
Asunto(s)
Aflatoxina B1/metabolismo , Ecosistema , Oncorhynchus mykiss/metabolismo , Oncorhynchus mykiss/microbiología , Pediococcus pentosaceus/metabolismo , Pediococcus/metabolismo , Probióticos/metabolismo , Adsorción , Animales , Disponibilidad Biológica , Pediococcus/aislamiento & purificación , Pediococcus pentosaceus/aislamiento & purificaciónRESUMEN
The advent of functional genomics has sparked the interest in inferring the function of non-coding regions from the transcriptome in non-model species. However, numerous biological processes remain understudied from this perspective, including intestinal immunity in farmed fish. The aim of this study was to infer long non-coding RNA (lncRNAs) expression profiles in rainbow trout (Oncorhynchus mykiss) fed for 30 days with functional diets based on pre- and probiotics. For this, whole transcriptome sequencing was conducted through Illumina technology, and lncRNAs were mined to evaluate transcriptional activity in conjunction with known protein sequences. To detect differentially expressed transcripts, 880 novels and 9067 previously described O. mykiss lncRNAs were used. Expression levels and genome co-localization correlations with coding genes were also analyzed. Significant differences in gene expression were primarily found in the probiotic diet, which had a twofold downregulation of lncRNAs compared to other treatments. Notable differences by diet were also evidenced between the coding genes of distinct metabolic processes. In contrast, genome co-localization of lncRNAs with coding genes was similar for all diets. This study contributes novel knowledge regarding lncRNAs in fish, suggesting key roles in salmons fed with in-feed additives with the capacity to modulate the intestinal homeostasis and host health.
Asunto(s)
Oncorhynchus mykiss/genética , Prebióticos/administración & dosificación , Probióticos/administración & dosificación , ARN Largo no Codificante/genética , Alimentación Animal/análisis , Animales , Acuicultura , Dieta/veterinaria , Genoma , Mucosa Intestinal/metabolismo , Oncorhynchus mykiss/metabolismo , ARN Largo no Codificante/metabolismo , TranscriptomaRESUMEN
The authors examined the potential of pulp mill effluent from pulp-producing countries (Canada, Brazil, New Zealand) to affect fish reproduction. Specifically, the estrogenic effects in juvenile rainbow trout (Oncorhynchus mykiss) pulse-exposed to 11 different mill effluent extracts (intraperitoneal injections of solid-phase extraction-dichloromethane nonpolar fraction). The results indicated that effluent extracts were estrogenic in juvenile trout irrespective of the gender, as reflected by increasing level of plasma vitellogenin (VTG; Brazil > New Zealand > Canada). Despite the high variability observed among mills, differences in VTG levels were related to the type of mill process (kraft > elementary chlorine-free kraft > thermomechanical pulping). Moreover, effluent treatments did not appear to significantly decrease VTG induction. A consistent estrogenic effect was observed in those mills that process a combination of feedstocks (softwood and hardwood), with the highest increase in VTG related to eucalyptus feedstock. The results demonstrate significant estrogenic effects of pulp mill effluents on chronically exposed juvenile trout, suggesting that in vivo metabolic activation of precursors is necessary to cause the observed increases in VTG levels. This molecular estrogenic response provides a useful starting point for predicting population-level impacts through the adverse outcome pathway methodology. Environ Toxicol Chem 2017;36:1547-1555. © 2016 SETAC.
Asunto(s)
Residuos Industriales/análisis , Oncorhynchus mykiss , Papel , Reproducción/efectos de los fármacos , Contaminantes Químicos del Agua/química , Contaminantes Químicos del Agua/toxicidad , Animales , Brasil , Canadá , Estrógenos/metabolismo , Nueva Zelanda , Oncorhynchus mykiss/metabolismo , Extracción en Fase Sólida , Vitelogeninas/metabolismoRESUMEN
Cortisol is an essential regulator of neuroendocrine stress responses in teleosts. Cortisol predominantly affects target tissues through the genomic pathway, which involves interacting with cytoplasmic glucocorticoid receptors, and thereby, modulating stress-response gene expressions. Cortisol also produces rapid effects via non-genomic pathways, which do not involve gene transcription. Although cortisol-mediated genomic pathways are well documented in teleosts, non-genomic pathways are not fully understood. Moreover, no studies have focused on the contribution of non-genomic cortisol pathways in compensatory stress responses in fish. In this study, rainbow trout (Oncorhynchus mykiss) skeletal myotubes were stimulated with physiological concentrations of cortisol and cortisol-BSA, a membrane-impermeable agent, resulting in an early induction of reactive oxygen species (ROS). This production was not suppressed by transcription or translation inhibitors, suggesting non-genomic pathway involvement. Moreover, myotube preincubation with RU486 and NAC completely suppressed cortisol- and cortisol-BSA-induced ROS production. Subcellular fractionation analysis revealed the presence of cell membrane glucocorticoid receptors. Finally, cortisol-BSA induced a significant increase in ERK1/2 and CREB phosphorylation, as well as in CREB-dependent transcriptional activation of the pgc1a gene expression. The obtained results strongly suggest that cortisol acts through a non-genomic glucocorticoid receptor-mediated pathway to induce ROS production and contribute to ERK/CREB/PGC1-α signaling pathway activation as stress compensation mechanisms. J. Cell. Biochem. 118: 718-725, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Proteínas de Peces/metabolismo , Hidrocortisona/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Oncorhynchus mykiss/metabolismo , Receptores de Glucocorticoides/metabolismo , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas de Peces/antagonistas & inhibidores , Antagonistas de Hormonas/farmacología , Hidrocortisona/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mifepristona/farmacología , Modelos Biológicos , Fibras Musculares Esqueléticas/efectos de los fármacos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Glucocorticoides/antagonistas & inhibidores , Espironolactona/análogos & derivados , Espironolactona/farmacología , Estrés FisiológicoRESUMEN
Biomixtures are used for the removal of pesticides from agricultural wastewater. As biomixtures employ high content of lignocellulosic substrates, their bioaugmentation with ligninolytic fungi represents a novel approach for their enhancement. Nonetheless, the decrease in the concentration of the pesticide may result in sublethal concentrations that still affect ecosystems. Two matrices, a microcosm of rice husk (lignocellulosic substrate) bioaugmented with the fungus Trametes versicolor and a biomixture that contained fungally colonized rice husk were used in the degradation of the insecticide/nematicide carbofuran (CFN). Elutriates simulating lixiviates from these matrices were used to assay the ecotoxicological effects at sublethal level over Daphnia magna (Straus) and the fish Oreochromis aureus (Steindachner) and Oncorhynchus mykiss (Walbaum). Elutriates obtained after 30 d of treatment in the rice husk microcosms at dilutions over 2.5% increased the offspring of D. magna as a trade-off stress response, and produced mortality of neonates at dilutions over 5%. Elutriates (dilution 1:200) obtained during a 30 d period did not produce alterations on the oxygen consumption and ammonium excretion of O. mykiss, however these physiological parameters were affected in O. aureus at every time point of treatment, irrespective of the decrease in CFN concentration. When the fungally colonized rice husk was used to prepare a biomixture, where more accelerated degradation is expected, similar alterations on the responses by O. aureus were achieved. Results suggest that despite the good removal of the pesticide, it is necessary to optimize biomixtures to minimize their residual toxicity and potential chronic effects on aquatic life.
Asunto(s)
Carbofurano/aislamiento & purificación , Ecotoxicología/métodos , Plaguicidas/aislamiento & purificación , Trametes/crecimiento & desarrollo , Contaminantes Químicos del Agua/aislamiento & purificación , Purificación del Agua/métodos , Agricultura , Animales , Biodegradación Ambiental , Carbofurano/toxicidad , Cíclidos/crecimiento & desarrollo , Cíclidos/metabolismo , Daphnia/efectos de los fármacos , Daphnia/metabolismo , Oncorhynchus mykiss/crecimiento & desarrollo , Oncorhynchus mykiss/metabolismo , Oryza/microbiología , Plaguicidas/toxicidad , Trametes/metabolismo , Aguas Residuales/química , Contaminantes Químicos del Agua/toxicidadRESUMEN
Flagellin is the main protein component of flagellum in Gram negative and positive bacteria, and it is also the ligand that activates the Toll-like receptor 5 (TLR5) in fish and mammals. In higher vertebrates, flagellin induces the activation of the membrane-bound TLR5 (TLR5M), which promotes the expression of proinflammatory cytokines and chemokines, and other immunological functions. We have previously reported that recombinant flagellin from Vibrio anguillarum and its ND1 domain are able to upregulate the expression of genes encoding major the proinflammatory mediators in gilthead seabream and rainbow trout macrophages. Considering the key role of D1 domain of flagellin for binding to TLR5M and its immunostimulatory activity, we designed and chemically synthesized a peptide derived of this region. The effects of the synthetic peptide were evaluated in vitro using head kidney macrophages from gilthead seabream (Sparus aurata L., Perciformes, Sparidae) and rainbow trout (Oncorhynchus mykiss W., Salmoniformes, Salmonidae). In both species the expression of genes encoding the proinflammatory cytokines interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α), and the chemokine IL-8, was induced upon stimulation of macrophages with the D1 domain synthetic peptide. IL-1ß and IL-8 were the most upregulated genes and to a lesser extent TNF-α. Interestingly, however, the induction activity of the synthetic peptide was higher in gilthead seabream than in rainbow trout macrophages. The results were confirmed at the protein levels for IL-8. Collectively, these results suggest that synthetic peptide derived from flagelling could be a promising approach for the immunostimulation and vaccination of farmed fish.