Project description:Gynogenetic development in fish is induced by activation of eggs with irradiated spermatozoa followed by exposure of the activated eggs to the temperature or high hydrostatic pressure (HHP) shock that prevents 1st cell cleavage. Produced specimens are fully homozygous fish also known as Doubled Haploids. Gynogenetic DH individuals might be used aquaculture and developmental biology unfortunately; the potential application of DHs is limited by a rather low survival rate of such specimens. However, observed variation in the survival rates of the gynogenetic embryos originated from different clutches suggests that eggs from some females have increased ability for gynogenetic development than others. Taking into account that first 10 cell cleavages in the fish embryos rely on the maternal RNA, it is tempting to assume that the ova showing such a vast difference in potential for gynogenesis may have also had different biological characteristics including alterations in maternal gene expression profiles. If so, then genes that up- or down –regulated expression in eggs increases competence for gynogenetic development in trout might be considered as candidate genes for gynogenesis in rainbow trout. Thus, the main goal of the project is identification of genes that increase ability of rainbow trout eggs for gynogenetic development. Within the project, we tried to verify following hypotheses: 1. Eggs from different females have different potential for gynogenesis in rainbow trout. 2. Eggs with different ability for gynogenetic development with all maternal inheritance have different biological characteristics including morphology and anti-ROS enzyme activities. 3. Eggs with increased competence for gynogenesis have altered transcriptomic profiles. 4. There are some particular genes that altered expression in trout eggs enable development of gynogenetic embryos. Gynogenetic rainbow trout specimens were produced in the course of activation of eggs with UV-irradiated spermatozoa and High Hydrostatic Pressure shock (HHP) applied around 1st cell cleavage. Eggs from several females were used in the experiment. Survival rates of gynogenetic rainbow trout was monitored since fertilization. Quality of eggs was examined by assessment of their morphology and activity of anti-ROS (reactive oxygene species) enzymes. Transcriptome of eggs showing increased and decreased developmental competence for gynogenesis was analyzed using RNA-seq approach and results compared to find out any alterations related to survival of gynogenetic trout.
Project description:Transcriptional profiling of rainbow trout muscle cells comparing muscle cells from small fish with muscle cells from large fish at two time periods.
Project description:Transcriptional profiling of rainbow trout liver cells comparing liver cells from small fish with liver cells from large fish at two time periods.
Project description:Migration is essential for the reproduction and survival of many animals, yet little is understood about its underlying molecular mechanisms. We used the salmonid Oncorhynchus mykiss to gain mechanistic insight into smoltification, which is a morphological, physiological, and behavioral transition undertaken by some juveniles that culminates in a seaward migration. This species is experimentally tractable and, unlike common model species, displays intra- and inter-population variation in migration propensity. Migratory individuals can produce non-migratory progeny and vice versa, indicating a high degree of phenotypic plasticity. One potential way that phenotypic plasticity might be linked to variation in migration-related life history tactics is through epigenetic regulation of gene expression. To explore this, we quantitatively measured genome-scale DNA methylation in fin tissue using reduced representation bisulfite sequencing of F2 siblings produced from a cross between steelhead (migratory) and rainbow trout (non-migratory) lines. We identified 57 differentially methylated regions (DMRs) between smolt and resident O. mykiss juveniles. DMRs were of high magnitude, ranging from 20-62% differential methylation between life history types, and over half of the gene-associated DMRs were in transcriptional regulatory regions. Many of the DMRs encode proteins with activity relevant to migration-related transitions (e.g. circadian rhythm pathway, nervous system development, protein kinase activity). This study provides the first evidence of a relationship between epigenetic variation and life history divergence associated with a migration-related transition in any species. Comparing global DNA methyldation profiles (via RRBS) of resident and smolt O. mykiss siblings using caudal fin tissue.
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.
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.
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:Transcriptional profiling of rainbow trout liver and muscle cells comparing small fish with large fish within a population of neomale offspring.
Project description:The sea-run phenotype of rainbow trout (Oncorhynchus mykiss), like other anadromous salmonids, present a juvenile stage fully adapted to life in freshwater known as parr. Development in freshwater is followed by the smolt stage, where preadaptations needed for seawater life are developed making fish ready to migrate to the ocean, after which event they become post-smolts. While these three life stages have been studied using a variety of approaches, proteomics has never been used for such purpose. The present study characterised the blood plasma proteome of parr, smolt and post-smolt rainbow trout using a gel electrophoresis liquid chromatography tandem mass spectrometry approach alone or in combination with low-abundant protein enrichment technology (combinatorial peptide ligand library). In total, 1,822 proteins were quantified, 17.95% of them being detected only in plasma post enrichment. Across all life stages, the most abundant proteins were ankyrin-2, DNA primase large subunit, actin, serum albumin, apolipoproteins, hemoglobin subunits, hemopexin-like proteins and complement C3. When comparing the different life stages, 17 proteins involved in mechanisms to cope with hyperosmotic stress and retinal changes, as well as the downregulation of nonessential processes in smolts, were significantly different between parr and smolt samples. On the other hand, 11 proteins related to increased growth in post-smolts, and also related to coping with hyperosmotic stress and to retinal changes, were significantly different between smolt and post-smolt samples. Overall, this study presents a series of proteins with the potential to complement current seawater-readiness assessment tests in rainbow trout, which can be measured non-lethally in an easily accessible biofluid. Furthermore, this study represents a first in-depth characterisation of the rainbow trout blood plasma proteome, having considered three life stages of the fish and used both fractionation alone or in combination with enrichment methods to increase protein detection.
Project description:Rainbow trout (Oncorhynchus mykiss) is one of the economically important cold-water fish cultivated in the world. The outbreak of infectious hematopoietic necrosis (IHN) seriously restricted the development of rainbow trout farming industry and caused huge economic losses. Fish skin is the largest mucosal immune organ, providing the first line of defense against pathogen invasion. However, the immune mechanisms associated with fish skin remain unclear. To systematically identify skin mucosal immune genes induced by infectious hematopoietic necrosis virus (IHNV) infection, trout transcriptome profiles following IHNV challenge were examined. Transcriptome analysis identified 6905 differentially expressed genes (DEGs) and revealed numerous immune-related DEGs involved in cytokine-cytokine receptor interactions, NOD-like receptor signaling, RIG-I-like receptor signaling, Toll-like receptor signaling, JAK-STAT signaling, Chemokine signaling pathway, and TNF signaling pathway, and the expression of these DEGs was significantly up-regulated in T48Skm group, including NOD1, NLRC3, NLRC5, TLR3, TLR7/8, TRIM25, DHX58, IFIH1, IRF3/7, STAT1, TRAF3, MX1, and HSP90A1. Additionally, highly interactive DEGs network involving immune-related terms and pathways was shown using protein-protein interaction network. The expression patterns of 12 DEGs were further verified by quantitative real-time PCR, which confirmed the reproducibility and reliability of transcriptome sequencing data. These findings expand our understanding of the innate immune system of rainbow trout skin infected with IHNV, and lay a foundation for further studies of the immune molecular mechanism and disease resistance breeding.