Project description:The aim of this sequencing experiment was to make available tissue expression panels for selected fish species for comparative expression studies between the species. Tissue samples were collected for zebrafish (Danio rerio), medaka (Oryzias latipes), and rainbow trout (Oncorhynchus mykiss). Tissue types included liver, skin, muscle, heart, gut, gill, eye, brain for all three species, with additionally pyloric caeca, kidney, head kidney, and spleen for rainbow trout. Only liver samples were taken in replicate of four or three for rainbow trout. All fish were raised under standard rearing conditions for the species. Total RNA was extracted from the tissue samples and paired‐end sequencing of sample libraries was completed on an Illumina HiSeq 2500 with 125‐bp reads. Processed count tables per species as raw counts, FPKM, or TPM, were generated from read alignment to the Ensembl genomes of the respective species using STAR and gene level counting using RSEM and Ensembl gene annotation.
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:Purpose:Our data significantly advance understanding of heat stress regulatory mechanism of miRNA in the head kidney of rainbow trout Methods:miRNAs of rainbow trout were involved in heat stress were identified by high-throughput sequencing of six small RNA libraries of the kidney tissues under control (18℃) and heat-treated (24℃) conditions Results:high-throughput sequencing was performed to identify miRNAs responsive to heat stress. We obtained 41,991,119 and 43,882,123 raw reads and 39,756,736 and 42,538,331 clean reads from under control (18℃) and heat-treated (24℃) .A total of 392 conserved miRNAs and 989 novel miRNAs were identified, of which 78 miRNAs were expressed in different response to heat stress. In addition to, including 393 negative correlation miRNA-target gene pairs Conclusions:through high-throughput sequencing of the six libraries from head kidney tissue of rainbow trout, the expression level of miRNA has significant changes after heat stress.
Project description:Purpose: a transcriptomic analysis was performed to extend our understanding on the immune response picture of rainbow trout exposed to I. multifiliis. Methods: Gill samples were collected from fish in each tank (control and infected group) at day 8 after infection. Total RNA was extracted using RTN350 (Sigma-Aldrich), according to the manufacturer’s instruction and subsequently, DNase treated with DNase I (Thermo Scientific, USA). Quality and integrity of the total RNA were checked on an Agilent Bioanalyzer 2100 total RNA Nano series II chip (Agilent, Amstelveen, Netherlands). Illumina RNAseq libraries were prepared from 500 ng total RNA using the Illumina TruSeqTM Stranded mRNA LT Sample Prep Kit according to the manufacturer’s instructions (Illumina Inc. San Diego, CA, USA). All RNAseq libraries (150-750 bp inserts) were sequenced on an Illumina HiSeq2500 sequencer as 1 x 50 nucleotides single-end reads according to the manufacturer’s protocol. Image analysis and base calling were done using the Illumina pipeline. Reads were aligned to the Rainbow trout reference genome (http://www.genoscope.cns.fr/trout/data/) using TopHat (version 2.0.5) and on average 53.4% of the RNAseq reads could be mapped. The resulting files were filtered using SAMtools (version 0.1.18) to exclude secondary alignment of reads. For statistical comparison of gene expression levels between groups, aligned fragments per predicted gene were counted from SAM alignment files using the Python package HTSeq (version 0.5.3p9). To make comparisons across samples possible, these fragment counts were corrected for the total amount of sequencing performed for each sample. As a correction scalling factor, we employed library size estimates determined using the R/Bioconductor (release 2.11) package DESeq. Read counts were normalized by dividing the raw counts obtained from HTSeq by its scale factor. Correction for false positives is included in the statistical analysis of gene expression through DESeq. The cut-off for significance was set to adjusted p<0.05 and at least 2-fold change. Results: a transcriptomic analysis was performed on infected rainbow trout gills and it showed that a total of 3,352 (7.2%) out of 46,585 identified genes were revealed significantly expressed after parasite infection. Of differentially expressed genes, 1.796 genes were up-regulated and 1.556 genes down-regulated. These were classified into 61 Gene Ontology (GO) terms and mapped to 282 reference canonical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Infection of I. multifiliis induced a clear differential expression of immune genes, related to both innate and adaptive immunity. A total of 268 (6.86%) regulated genes was known to take part in 16 immune-related pathways. These involved pathways related to the innate immune system such as Chemokine signaling pathway, Platelet activation, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, and Leukocyte transendothelial migration. Conclusion: a transcriptomic profile of rainbow trout gills exposed to the parasitic I. multifiliis was reported for the first time. A total of 3,355 differentially expressed unigenes were identified. Of these were 1,184 unigenes (mapped to 952 genes) annotated 282 KEGG pathways and 268 unigenes were associated with 16 immune pathways. Most unigenes were related to innate immune system pathways (Chemokine signaling pathway, Platelet activation, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, and Leukocyte transendothelial migration) although a number of unigenes was related to adaptive responses (antigen processing and presentation, T and B cell receptor signaling pathway). The present study gave a far better resolution of the immune response of rainbow trout exposed to a parasitic protozoan than has ever been presented previously. The identification of a series of immune genes involved in several but important was useful for understanding of immune mechanism of the rainbow trout responding to the parasite I. multifiliis. Our results provide tools to link innate and adaptive immune elements in the process and present basic information which will be useful in the future studies related to immunoprophylaxis.
Project description:Rainbow trout (1000 fish) was exposed to the bacterial pathogen F. psychrophilum by simple bath challenge without any pre-treatment with hydrogen peroxide. Samples (fin clip for Affymetrix QTL analysis) were taken from 167 moribund fish during the course of infection. When mortality/morbidity ended (day 40) we euthanized a total of 197 specimens of the remaining fish and took samples for DNA (QTL analysis) and assigned the status: Survivor. For gene expression analysis we took samples from gill, spleen and liver between day 11 and 15 from fish with clinical signs (CS) and no clinical signs (NCS), whereas samples from survivors were taken at day 40.
Project description:Stress represents a major factor negatively affecting fish welfare in aquaculture. The objective of the present study was to identify and evaluate informative indicators for the welfare of rainbow trout Oncorhynchus mykiss exposed to (A) a critical water temperature of 27 °C and (B) acute crowding of 100 kg/m3 combined with water temperature of 27 °C. In order to make an approximate assessment of the overall condition, we determined health index, spleen-somatic index and haematocrit and recorded the blood concentrations of haemoglobin, cortisol and glucose of rainbow trout under challenging versus control conditions. Moreover, we analysed the transcriptomic profiles of the spleen of the two stress-treatment and the reference groups to identify gene sets, which are specific for temperature stress alone or combined temperature and crowding 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 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: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: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.