Project description:Abstract. The molecular pathways in embryonic vertebrates leading to gonad formation in each sex are incompletely understood. The purpose of this study was to identify novel genes that could be associated with sex-specific gonadal differentiation in a fish, the rainbow trout (Oncorhynchus mykiss). This study was facilitated by a custom microarray based on 7,681 genes derived from embryonic rainbow trout gonad cDNA libraries and public databases. Gonad samples for total RNA isolation were obtained from pvasa-green fluorescent protein (pvasa-GFP) transgenic rainbow between 300 and 700 degree days of development post-fertilization. The transgenic fish permitted the collection of gonads from embryonic rainbow trout during the period of molecular sex differentiation in advance of any morphologically distinguishable characteristics of sex. A bioinformatic method was used with the microarray data that looked for strong associations in gene expression patterns between known sex differentiation genes (the target genes) and novel genes (the target-associated genes) previously not allied with sex differentiation in fishes. The expression patterns of representative targets genes from both sexes and their target-associated genes were independently confirmed by real-time reverse transcription-polymerase chain reaction to support the validity of the bioinformatics method employed. Numerous, novel genes were identified in the gonads of embryonic female and male rainbow trout that could be involved in sex-specific differentiation pathways in this fish. Embryonic gonads were removed by dissection from known genetic female (XX) and male (XY) rainbow trout (Oncorhynchus mykiss) from a transgenic population where expression of GFP is controlled by vasa-gene regulatory elements (Yoshizaki et al. 2000; Takeuchi et al. 2002). These fish were maintained in incubators containing flowing freshwater (10°C) at the Ooizumi Research Station, Yamanashi, Japan. Ten female or male transgenic rainbow trout were randomly selected every 5 days, beginning at 30 days (i.e., 300 degree days = incubation temperature in °C x number of days) post fertilization through until 70 days (i.e., 700 degree days) post fertilization. The gonads from each sex, at each sampling time, were pooled and immediately frozen for subsequent total RNA isolation.
Project description:The aim of present study is to identify and quantify proteins involved in the events of fertilization and early embryo development using a label-free protein quantification method in rainbow trout (Oncorhynchus mykiss) as an economically important fish species in aquaculture.
Project description:A rapid decline in temperature poses a major challenge for poikilothermic fish. The gene expression of rainbow trout Oncorhynchus mykiss having undergone such a cold shock (0 °C) and a control (5 °C) were compared in a microarray-based study.
Project description:Abstract. The molecular pathways in embryonic vertebrates leading to gonad formation in each sex are incompletely understood. The purpose of this study was to identify novel genes that could be associated with sex-specific gonadal differentiation in a fish, the rainbow trout (Oncorhynchus mykiss). This study was facilitated by a custom microarray based on 7,681 genes derived from embryonic rainbow trout gonad cDNA libraries and public databases. Gonad samples for total RNA isolation were obtained from pvasa-green fluorescent protein (pvasa-GFP) transgenic rainbow between 300 and 700 degree days of development post-fertilization. The transgenic fish permitted the collection of gonads from embryonic rainbow trout during the period of molecular sex differentiation in advance of any morphologically distinguishable characteristics of sex. A bioinformatic method was used with the microarray data that looked for strong associations in gene expression patterns between known sex differentiation genes (the target genes) and novel genes (the target-associated genes) previously not allied with sex differentiation in fishes. The expression patterns of representative targets genes from both sexes and their target-associated genes were independently confirmed by real-time reverse transcription-polymerase chain reaction to support the validity of the bioinformatics method employed. Numerous, novel genes were identified in the gonads of embryonic female and male rainbow trout that could be involved in sex-specific differentiation pathways in this fish.
Project description: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:The objective of this study was to identify and quantify proteomic profiles of intestine of rainbow trout (Oncorhynchus mykiss). Specific pathogen free rainbow trout (mean length 15 ± 1 cm) were maintained in recirculating de-chlorinated water at 19±1 °C. Prior to the experiment, fish were distributed between aquaria. The test groups were infected by immersion of Yersinia ruckeri CSF007-82 (biotype 1) and 7959-11 (biotype 2) strains. The control group was immersed similar with sterile broth medium. Fish were anaesthetized and sampled aseptically at different time points. Each intestine was washed three times with sterile phosphate-buffered saline containing a cocktail of mammalian protease inhibitors. Intestinal mucosa was scraped with a sterile large scalpel blade. Intestinal samples were snap-frozen in liquid nitrogen and stored at –80 °C.
Project description:The objective of this study was to identify and quantify proteomic profiles of spleen of rainbow trout Oncorhynchus mykiss. Specific pathogen free rainbow trout (mean length 15 ± 1 cm) were maintained in recirculating de-chlorinated water at 19±1 °C. Prior to the experiment, fish were distributed between 9 aquaria, 18 fish per aquarium. The test groups were infected by immersion of Yersinia ruckeri strains: CSF007-82 (biotype 1) and 7959-11 (biotype 2). The control group was immersed similar with sterile broth medium. There were 3 aquaria per each group (CSF007-82-infected, 7959-11-infected and control). Nine fish from infected and control fish groups were anaesthetized with MS-222 at 3, 9 and 28 days post exposure and sampled aseptically. Each spleen was washed three times with sterile phosphate-buffered saline containing a cocktail of mammalian protease inhibitors. Spleen samples were snap-frozen in liquid nitrogen and stored at –80 °C.
Project description:The objective of this study was to identify and quantify proteomic profiles of head kidney of rainbow trout Oncorhynchus mykiss. Specific pathogen free rainbow trout (mean length 15 ± 1 cm) were maintained in recirculating de-chlorinated water at 19±1 °C. Prior to the experiment, fish were distributed between 9 aquaria, 18 fish per aquarium. The test groups were infected by immersion of Yersinia ruckeri strains: CSF007-82 (biotype 1) and 7959-11 (biotype 2). The control group was immersed similar with sterile broth medium. There were 3 aquaria per each group (CSF007-82-infected, 7959-11-infected and control). Nine fish from infected and control fish groups were anaesthetized with MS-222 at 3, 9 and 28 days post exposure and sampled aseptically. Each head kidney was washed three times with sterile phosphate-buffered saline containing a cocktail of mammalian protease inhibitors. Head kidney samples were snap-frozen in liquid nitrogen and stored at –80 °C.
Project description:Stocking density is considered as a key factor determining the productivity of fish aquaculture systems. The transcriptomic response to crowding stress is, however, still poorly investigated. We aimed at the identification of potential biomarker genes via microarray analyses to get insight into molecular pathways modulated through density-induced stress in farmed rainbow trout Oncorhynchus mykiss. Transcriptome profiling in liver, kidney, and gills was complemented with behaviarol observation and analysis of classical plasma parameters. Individuals of two trout strains were exposed for eight days to definite stocking densities, 1 kg/m³ (low density); 10 kg/m³ (moderate); 18 kg/m³ (elevated); and 35 kg/m³ (high). Whereas stocking density had no significant effect on cortisol levels, plasma glucose levels were elevated in trout kept at high density. Pathway enrichment analyses confirmed the upregulation of HIF1a signaling in liver contributing to glucose homeostasis during stress conditions, while mTOR and PI3K/AKT signaling pathways were downregulated. Further perturbed hepatic pathways were involved in protein ubiquitination and the biosynthesis of cholesterol, retinol and glutathione. Three stocking density conditions were investigated: an uncrowded âmoderateâ density (MD: 10 kg trout/m³) , an elevated density (ED: 18 kg/m³ ), and high density (HD: 35 kg/m³). The experiment was performed twice with two strains of Steelhead rainbow trout (Troutlodge and Born trout), randomly assigned to identical glass tanks with MD (30 and 34 individuals), ED (60 and 64 individuals), and HD (120 and 140 individuals). Trout were sampled 8 d after experimental onset.
Project description:Infectious hematopoietic necrosis virus (IHNV) can cause widespread death of rainbow trout (Oncorhynchus mykiss), understanding the molecular mechanisms that occur in the rainbow trout in response to IHNV infection will be useful to decrease IHN-related morbidity and mortality in trout aquaculture. However, the molecular mechanisms of rainbow trout in response to IHNV are very limited. This study performed analysis of mRNAs and miRNAs based on RNA-seq technology on the intestine of rainbow trout infected with IHNV and control. There were 80 differentially expressed miRNAs that regulated 3355 target mRNAs, which overlapped with differentially expressed mRNAs obtained from RNA-seq. The expression patterns of DEGs and miRNAs differentially expressed were validated by qRT-PCR. GO enrichment and KEGG pathway analyses of the potential target genes of the DE miRNAs, revealed DEGs were mainly enriched in immune-related pathways such as Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway and IL-17 signaling pathway. These findings improve our understanding of the molecular mechanisms of IHNV infection. The study analyzed the immune regulatory target gene pairs and signal pathways of rainbow trout intestine against IHNV infection at the transcriptional level, and provided basic data for the study of rainbow trout against IHNV immune regulatory.