Dynamic and systemic regulation occurs in the liver of rainbow trout (Oncorhynchus mykiss) in response to environmental hypoxia
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ABSTRACT: The hypoxia frequently occurs in natural aquatic systems and aquaculture environment due to the natural reasons and human factors such as extreme climate, high density farming, environmental pollution and global warming, which have gradually become a huge threat to aquatic ecosystem functions and aquatic organism survival, causing serious ecological damage and enormous economic losses. Rainbow trout (Oncorhynchus mykiss), as a hypoxia-sensitive fish species, is a good model to study hypoxia stress. The molecular regulation and oxidative stress of rainbow trout still remains unknown in response to environmental hypoxia and reoxygenation stress. In this study, the transcriptome and biochemical indexes of rainbow trout liver in response to hypoxia for different durations were analyzed to highlight the changes in the molecular regulation and oxidative stress.
Project description:Hypoxia is a tricky and unavoidable environmental stressor for aquaculture with ongoing ecological deterioration and increasing stocking density, resulting in serious economic losses. Rainbow trout (Oncorhynchus mykiss), a commercially important fish species worldwide, exhibits extraordinarily poor tolerance to hypoxia; however, the regulatory mechanisms under hypoxia stress have not been elucidated. In this study, rainbow trout were separately subjected to moderate (Tm12L) and acute hypoxia for 12 h (Ts12L) and 12 h reoxygenation to investigate the changes of histology, biochemical parameters and transcriptomes (mRNA, miRNA and lncRNA) in liver, and interactions between LOC110519952-novel-m0023-5p-glut1a were further verified. The results showed that acute hypoxia resulted in more severe liver oxidative injury than moderate hypoxia, and anaerobic metabolism and lipid and protein metabolism were enhanced to some extent, especially in Ts12L. We also found that innate immune response was strengthened in Tm12L, while it was inhibited in Ts12L. In addition, dozens of hypoxia-related ceRNA networks derived from 1824 differentially expressed (DE) mRNAs, 427 DEmiRNAs and 545 DElncRNAs were identified, including LOC110519952-novel-m0023-5p-glut1a, LOC110520012-miR-206-y-vegfaa, LOC118965299-novel-m0179-3p-epoa and MSTRG.7382.2-miR-184-y-hmox. Further studies validated that LOC110519952 acted as a ceRNA that can positively regulate glut1a expression by adsorbing novel-m0023-5p. Overexpression of novel-m0023-5p could promote liver cell viability and proliferation, and introduction of LOC110519952 attenuated the effects. The findings contribute to deepen our understanding of the adaptive mechanisms used by rainbow trout in response to hypoxia stress, and provide valuable information for hypoxia-tolerant varieties breeding.
Project description:Hypoxia is a tricky and unavoidable environmental stressor for aquaculture with ongoing ecological deterioration and increasing stocking density, resulting in serious economic losses. Rainbow trout (Oncorhynchus mykiss), a commercially important fish species worldwide, exhibits extraordinarily poor tolerance to hypoxia; however, the regulatory mechanisms under hypoxia stress have not been elucidated. In this study, rainbow trout were separately subjected to moderate (Tm12L) and acute hypoxia for 12 h (Ts12L) and 12 h reoxygenation to investigate the changes of histology, biochemical parameters and transcriptomes (mRNA, miRNA and lncRNA) in liver, and interactions between LOC110519952-novel-m0023-5p-glut1a were further verified. The results showed that acute hypoxia resulted in more severe liver oxidative injury than moderate hypoxia, and anaerobic metabolism and lipid and protein metabolism were enhanced to some extent, especially in Ts12L. We also found that innate immune response was strengthened in Tm12L, while it was inhibited in Ts12L. In addition, dozens of hypoxia-related ceRNA networks derived from 1824 differentially expressed (DE) mRNAs, 427 DEmiRNAs and 545 DElncRNAs were identified, including LOC110519952-novel-m0023-5p-glut1a, LOC110520012-miR-206-y-vegfaa, LOC118965299-novel-m0179-3p-epoa and MSTRG.7382.2-miR-184-y-hmox. Further studies validated that LOC110519952 acted as a ceRNA that can positively regulate glut1a expression by adsorbing novel-m0023-5p. Overexpression of novel-m0023-5p could promote liver cell viability and proliferation, and introduction of LOC110519952 attenuated the effects. The findings contribute to deepen our understanding of the adaptive mechanisms used by rainbow trout in response to hypoxia stress, and provide valuable information for hypoxia-tolerant varieties breeding.
Project description:Hypoxia negatively affects the behavior, growth, reproduction, and survival of fish, causing serious economic losses to aquaculture. Rainbow trout (Oncorhynchus mykiss), an important economic fish worldwide, belongs to a hypoxia-sensitive fish species. However, the regulatory mechanisms of miRNAs under hypoxia stress response of rainbow trout remains unclear. In this study, rainbow trout were subjected to hypoxia stress (DO: 3.5 mg/L) for 3 h (H3h_L), 12 h (H12h_L), 24 h (H24h_L) and 3 h reoxygenation (R3h_L) to systemically evaluate the changes of miRNA expression profiles in liver, and the functions of sha-miR-92a_L+2R+4 were investigated. We found 17, 144, 57 and 55 differentially expressed (DE) miRNAs in H3h_L vs. control (N_L), H12h_L vs. N_L, H24h_L vs. N_L and R3h_L vs. N_L comparisons, respectively. Enrichment analysis revealed that the targets of these DE miRNAs were significantly enriched in HIF signaling pathway, VEGF signaling pathway, FoxO signaling pathway and glycolysis/gluconeogenesis. Through miRNA-mRNA nteraction and weighted gene co-expression network analysis (WGCNA), five key DE miRNAs (sha-miR-92a_L+2R+4, ssa-miR-128-3p, ssa-miR-101b-3p_R+1, ola-miR-199a-5p_R+2 and tni-miR-199_1ss18CG) were identified, which can target at least two hypoxia-responsive genes, such as vegfaa, ho, glut1a and junb. Functional analysis found that sha-miR-92a_L+2R+4 directly regulated vegfaa expression by targeting its 3′-UTR, overexpression of sha-miR-92a_L+2R+4 significantly decreased vegfaa expression in rainbow trout liver cells, while opposite results were obtained after transfection of sha-miR-92a_L+2R+4 inhibitor. Furthermore, overexpression of sha-miR-92a_L+2R+4 promoted rainbow trout liver cell proliferation and inhibited apoptosis. These results deepen our understanding of the crucial roles of miRNAs under hypoxia stress in rainbow trout, and provide valuable information for further studying the regulatory mechanisms of key hypoxia-responsive miRNAs and breeding hypoxia-tolerant rainbow trout species.
Project description:Rainbow trout (Oncorhynchus mykiss), an important economically fish with high food value in China, is very sensitive to hypoxia. The gills are the first tissue to be attacked after hypoxia stress. However, the regulatory mechanisms in gills of rainbow trout to moderate and severe hypoxia stress are unclear. In this study, we determined the changes of biochemical parameters and gene expression in gills of rainbow trout during different periods of hypoxia (4, 8, 12, 24 h, and 1 month) and reoxygenation for 24 h, and analyzed the tissue and transcriptomic changes under moderate hypoxia for 12 h (Tm12G) and severe hypoxia for 12 h (Ts12G) compared with control (CG). The results showed that gill lamellae were bent and ILCM was reduced in the Tm12G group, while this phenomenon was more pronounced in the Ts12G group. At one month of hypoxia, we found that the gill lamellae epithelium was detached. Based on biochemical parameters and transcriptomic analysis, we found that the antioxidant and immune responses were activated, and glucose metabolism, lipid metabolism, and amino acid metabolism were enhanced, however, vitamin metabolism was suppressed under moderate hypoxia stress. Antioxidant and immune responses were activated, and glucose metabolism, lipid metabolism, and amino acid metabolism were enhanced, and nucleotide metabolism also was activated under severe hypoxia stress. From the analysis of gene expression pattern, we found that the expression of hsp90a, tnf, caspase3, mgst1, fih1, hif1a, ddit4, and egln3 showed a tendency to increase and then decrease and recovered after reoxygenation under moderate and severe hypoxia stress. Except for mgst1, the expression of other genes was significantly higher than the control at 12 h of stress. Under hypoxia stress, antioxidant and metabolic responses were first elevated and then decreased with time, and metabolic and immune pathways were activated, which coordinated with each other in response to hypoxia stress. These results provide basic information for an in-depth investigation of the physiological and molecular mechanisms in gills of rainbow trout under hypoxia stress.
Project description:As an important cold-water economic fish species, rainbow trout (Oncorhynchus mykiss) exhibits several intra-specific variation in skin pigmentation that can give rise to distinctive phenotypes, and wild-type rainbow trout with black skin (WR) and yellow mutant rainbow trout with yellow skin (YR) are the major two types in the farms, whose distinct skin colors make them suitable model for elucidating the skin pigmentation process. Skin color as a key indicator for selection in rainbow trout farming as well as has a strong visual impact on the consumer when rainbow trout are marketed. Previously, extensive studies have been conducted on skin color in rainbow trout, including the observation of skin spots and the expression analysis of some important pigment genes. However, up to date, no studies have systematically examined the molecular regulation mechanism of skin color difference between WR and YR through a high throughput method. Therefore, the aim of this study was to reveal the molecular regulation mechanism of skin color difference between these two strains at the mRNA and miRNA transcriptome level, and candidate genes, miRNAs and miRNA-mRNA pairs that may be responsible for rainbow trout albinism were obtained.
Project description:As an important cold-water economic fish species, rainbow trout (Oncorhynchus mykiss) exhibits several intra-specific variation in skin pigmentation that can give rise to distinctive phenotypes, and wild-type rainbow trout with black skin (WR) and yellow mutant rainbow trout with yellow skin (YR) are the major two types in the farms, whose distinct skin colors make them suitable model for elucidating the skin pigmentation process. Skin color as a key indicator for selection in rainbow trout farming as well as has a strong visual impact on the consumer when rainbow trout are marketed. Previously, extensive studies have been conducted on skin color in rainbow trout, including the observation of skin spots and the expression analysis of some important pigment genes. However, up to date, no studies have systematically examined the molecular regulation mechanism of skin color difference between WR and YR through a high throughput method. Therefore, the aim of this study was to reveal the molecular regulation mechanism of skin color difference between these two strains at the mRNA and miRNA transcriptome level, and candidate genes, miRNAs and miRNA-mRNA pairs that may be responsible for rainbow trout albinism were obtained.
Project description:The frequency of fish encountering hypoxia has risen dramatically as global warming and water pollution intensify, posing a serious threat to survival. Rainbow trout (Oncorhynchus mykiss), which is an economically important fish worldwide, belongs to a typical hypoxia-sensitive fish. However, little is known about the response mechanisms employed by rainbow trout to hypoxia stress. In this study, we systematically analyzed the changes of liver biochemical parameters in rainbow trout exposed to moderate hypoxic conditions (DO: 4.5 ± 0.1 mg/L) for different durations (4, 8, 12, 24 h and 1 month) and 12 h and 24 h reoxygenation, and histologic and transcriptome (lncRNA, miRNA and mRNA) under hypoxia stress for 12 h (Tm12L) and 1 month (TmlL) compared with control, and the interaction between LOC110520201, miR-2188-y and sod1 was verified by dual-luciferase reporter, overexpression and silencing assays. Histological observations revealed that liver suffered severe damage in Tm12L but not in TmlL. Integrating biochemical parameters and mRNA expression profiles, we found that exposure to short-term hypoxia stress resulted in enhanced immune response, aerobic and anaerobic glycolysis, gluconeogenesis and lipid metabolism. In TmlL, increased anaerobic glycolysis and decreased aerobic glycolysis were observed, and antioxidant and immune capacity has returned to normal. Additionally, several key hypoxia-related genes (epo, vegfc, epor, sod1, ppara, foxo1a, gk, dusp1, nlrc3 and nlrp3) and ceRNA regulatory networks were identified from 2711 differentially expressed (DE) mRNAs, 575 DElncRNAs and 395 DEmiRNAs, including LOC110520201-miR-2188-y-sod1, LOC110533332-miR-144-y-vegfc, LOC110526066-miR-465-x-ppara and LOC110535032-miR-743-y-nlrp3. The analysis of expression patterns in liver and rainbow trout liver cells treated with hypoxia suggested that changes in LOC110520201, miR-2188-y and sod1 showed a ceRNA regulatory relationship. Further experiments demonstrated that sod1 is a target of miR-2188-y, and LOC110520201 silencing increased the expression of miR-2188-y and inhibited sod1 expression. These results facilitated our understanding of the molecular mechanisms of hypoxia response in rainbow trout and provided genetic resources for breeding hypoxia-tolerant varieties.
Project description:Background: Rainbow trout (Oncorhynchus mykiss) is a salmonid species with a complex life-history. Wild populations are naturally divided into freshwater residents and sea-run migrants. Migrants undergo an energy-demanding adaptation for life in seawater, known as smoltification, while freshwater residents display these changes in an attenuated magnitude and rate. Despite this, in seawater rainbow trout farming all animals are transferred to seawater. Under these circumstances, weeks after seawater transfer, a significant portion of the fish die (around 10%) or experience growth stunting (GS; around 10%), which represents an important profitability and welfare issue. The underlying causes leading to GS in seawater-transferred rainbow trout remain unknown. In this study, we aimed at characterising the GS phenotype in seawater-transferred rainbow trout using untargeted and targeted approaches. To this end, the liver proteome (LC-MS/MS) and lipidome (LC-MS) of GS and fast-growing phenotypes were profiled to identify molecules and processes that are characteristic of the GS phenotype. Moreover, the transcription, abundance or activity of key proteins and hormones related to osmoregulation (Gill Na+, K+–ATPase activity), growth (plasma IGF-I, and liver igf1, igfbp1b, ghr1 and ctsl) and stress (plasma cortisol) were measured using targeted approaches. Results: No differences in Gill Na+, K+–ATPase activity and plasma cortisol were detected between the two groups. However, a significant downregulation in plasma IGF-I and liver igf1 transcription pointed at this growth factor as an important pathomechanism for GS. Changes in the liver proteome revealed reactive-oxygen-species-mediated endoplasmic reticulum stress as a key mechanism underlying the GS phenotype. From the lipidomic analysis, key observations include a reduction in triacylglycerols and elevated amounts of cardiolipins, a characteristic lipid class associated with oxidative stress, in GS phenotype. Conclusion: While the triggers to the activation of endoplasmic reticulum stress are still unknown, data from this study point towards either an unresolved infection or a nutritional deficiency as underlying drivers of this phenotype.
Project description:Dissolved oxygen (DO) has an important impact on fish survival and reproduction, and is also one of the keys limiting conditions for healthy fish life. Rainbow trout (Oncorhynchus mykiss), an economically significant cold-water fish globally, are highly susceptible to hypoxia stress. However, the physiological changes and molecular mechanisms of rainbow trout under different hypoxic conditions are still unknown. In the present study, we used RNA-seq analysis and measurement of biochemical parameters at different time points (0-, 4-, 8-, 12-, 24 h) under acute (3.0 ± 0.1 mg/L), chronic (4.5 ± 0.1 mg/L) hypoxic stress and reoxygenation (R12-, R24 h) to reveals the physiological changes and molecular mechanism of the hypoxic stress response in rainbow trout. The transcriptome results showed that a total of 424 differentially expressed genes (DEGs) were identified in the CM-vs-Tm12M, CM-vs-Ts12M and Tm12M-vs-Ts12M groups and the genes for FOXO signaling pathway, p53 signaling pathway, Adipocytokine signaling pathway, Autophagy, Glycine, serine and threonine metabolism, etc. were significantly enriched under hypoxic stress, as shown by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes enrichment analyses (KEGG) and Gene Set Enrichment Analysis (GSEA). Based on DEGS expression trend analysis, the most significant 2 clusters were selected to construct protein-protein interaction (PPI) networks, which resulted in the prediction of ddit4, txnip, slc3a2 and p4ha1 as the hypoxia-induced hub genes in cluster 7. fgg, f7, serpina1 and serpinf2 are the hypoxia-induced hub genes of cluster 3. This study preliminarily elucidated the adaptive mechanism of rainbow trout in response to hypoxic stress and provide a basis for the study of the molecular mechanism of fish adaptation to different hypoxic stresses.
Project description:Proteomics represents a powerful tool for the analysis of fish spermatozoa, since these cells are transcriptionally inactive. The aim of the present study was to generate an inventory of the most prominent rainbow trout sperm proteins with the use of one-dimensional electrophoresis prefractionation combined with performance liquid chromatography electrospray ionization tandem mass spectrometry. This study provides the first in-depth analysis of the rainbow trout sperm proteome, with a total of 204 identified proteins. We found that rainbow trout spermatozoa are equipped with functionally diverse proteins related to energetic metabolism, signal transduction, protein turnover, transport, cytoskeleton, oxidative injures and stress and reproduction. The availability of a catalogue of rainbow trout sperm proteins provides a crucial tool for the understanding of fundamental molecular processes in fish spermatozoa for ongoing research in the development of novel markers of sperm quality and for the optimization of short- and long-term sperm preservation procedures.