Project description:High-throughput proteomics was used to determine the role of the fish liver in defense responses to bacterial infection, done using a rainbow trout (Oncorhynchus mykiss) model following infection with Aeromonas salmonicida, the causative agent of furunculosis. The vertebrate liver has multifaceted roles in innate immunity, metabolism, and growth; we hypothesize this tissue serves a dual function in supporting host defense in parallel to metabolic adjustments that promote effective immune function. While past studies have reported mRNA responses to A. salmonicida in salmonids, the impact of bacterial infectionon the liver proteome remains uncharacterized in fish.
Project description:The Crown-of-thorns starfish (COTS) Acanthaster planci feeds on hard corals and its outbreaks are a major cause of destruction of coral communities on the Australian Great Barrier Reef. Whilst population booms and the social behaviour of COTS have been well studied, little is known about the neural mechanisms underlying COTS metabolism and behaviour. One of the major classes of chemical messengers that regulate metabolic and behavioural processes in animals are neuropeptides. Here, we have analysed COTS genome and transcriptome sequence data to identify neuropeptide precursor proteins in this species. Mass spectrometry was employed to identify neuropeptides extracted from radial nerve cords. Forty-nine neuropeptide precursors were identified, including homologs of neuropeptide signaling systems that are evolutionarily conserved throughout the Bilateria.
Project description:The transcriptional programs of ectothermic teleosts are directly influenced by water temperature. Although various cold-responsive transcriptional patterns have been determined in fishes, the systematic molecular networks governing the temperature responses are still unknown. We profiled the transcriptional responses in eight tissues of zebrafish exposed to graded cold temperatures, ranging from normal (28°C) to mild (18°C) and severe (10°C) cold, using RNA-seq. The tissues varied in the number of cold-responsive genes, of which the kidney appeared to be most sensitive, whereas the brain was the least. Fuzzy k-means clustering revealed 34 gene clusters of distinct expression patterns, demonstrating diverse tissue-specific responses in conjunction with multiple aspects of ubiquitous cross-tissue responses to cold. Thirty-one GO terms were over-represented upon cold treatment. These terms are involved in basic cellular processes, such as RNA splicing and proton transport, as well tissue-specific processes, such as ‘negative regulation of endopeptidase activity’ in the kidney. To identify the cis-regulatory elements governing the concerted cold responses, the promoters of the genes that demonstrated strong co-regulation were analyzed using an enriched motif discovery program, DREME. Eleven motifs, 6 known and 5 novel, were identified. These motifs belong to the genes corresponding to the 16 over-represented GO terms identified above. Some motifs, such as the AP-1 and STAT1 binding sites, are known to be stress responsive. By integrating comprehensive cold-induced transcriptional changes with a cis-motif identification tool, we identified genome-wide regulatory networks for the cold response in zebrafish. The identified networks provided new insights into molecular mechanisms of thermal responses in teleosts. Examination of gene expression of 24 samples (eight tissues at three temperatures)
Project description:The skin mucus of gilthead sea bream was mapped by 1-DE followed by liquid chromatography coupled to high resolution mass spectrometry using a quadrupole time-of-flight mass analyzer. More than 2000 proteins were identified with a protein score filter of 30. The identified proteins were represented in 418 canonical pathways of the Ingenuity Pathway software. After filtering by canonical pathway overlapping, the retained proteins were clustered in three groups. The mitochondrial cluster contained 59 proteins related to oxidative phosphorylation and mitochondrial dysfunction. The second cluster contained 79 proteins related to antigen presentation and protein ubiquitination pathways. The third cluster contained 257 proteins where proteins related to protein synthesis, cellular assembly, and epithelial integrity were over-represented. The latter group also included acute phase response signaling. In parallel, 2-DE methodology identified six proteins spots of different protein abundance when comparing unstressed fish with chronically stressed fish in an experimental model that mimicked daily farming activities. The major changes were associated with a higher abundance of cytokeratin 8 in the skin mucus proteome of stressed fish, which was confirmed by immunoblotting. Overall, these results indicate that skin mucus is a reliable tissue for alternative or complementary stress phenotyping in fish farming.
Project description:The Squalius alburnoides complex (Steindachner) is one of the most intricate hybrid polyploid systems known in vertebrates. In this complex, the constant switch of the genome composition in consecutive generations, very frequently involving a change on the ploidy level, promotes repetitive situations of potential genomic shock. Previously in this complex, it was shown that in response to the increase in the genome dosage, triploid hybrids could regulate gene expression to a diploid state. In this work, we compared the small RNA profiles in the different genomic compositions interacting in the complex. Using high-throughput arrays and sequencing technologies, we were able to verify that diploid and triploid hybrids were closely related: they shared most of their sequences and their miRNA expression profiles were highly correlated. However, an overall view indicates an up-regulation of a substantial number of miRNAs in triploids. Also, the global miRNA expression in triploids was higher than predicted from an additive model. These results point to a participation of miRNAs in the cellular functional stability needed when the ploidy change. 4 samples were analyzed corresponding to 4 genomic constitutions: PAA, PA, AA and PP. For each sample, a library based on 3 individuals of the same genomic constitution was prepared. From each individual, 3 types of tissues were collected for RNA extraction (brain, liver and muscle).
Project description:To study the effects of bacterial infection upon gene expression changes in flounder liver fish were artificially "infected" by injection with a commercial water-based vaccine containing killed Aeromonas salmonicida, the bacterium responsible for furunculosis, which, as its name describes, presents as external lesions (furuncles, "lumps"). This disease occurs in flounder as well as in salmonids. Flounders were treated by intraperitoneal injection with 1ml/kg Furunculosis vaccine (Schering-Plough, Aquavac Furovac 5, batch number FNM/C/007). 30 control fish were injected with 1% saline. Animals were then maintained unfed in static aerated sea water tanks in a constant temperature containment aquarium. Water was renewed every 2 days. After 1, 2, 4, 8, and 16 days, 6 vaccine treated and 6 saline control fish were removed, killed by a blow to the head and samples of liver tissue were immediately homogenized in TriReagent prior to gene expression profiling. Microarray experiments consisted an individual array for each of 4 or 5 fish from each timepoint and each treatment compared with an artificial reference sample.
Project description:The present work was designed to assess the effects of high plasma cortisol levels induced by slow-release cortisol implants in the mRNA transcription of the GR in the different organs of the Sparus aurata, including liver. For that purpose fish were intraperitoneally injected with the implants containing two different concentrations of cortisol (50 or 200 µg/g body weight) and blood and organs were sampled after 7 and 14 days of implantation. Only fish with 200 µg/g implants exhibited a significant rise in the plasma cortisol. For microarray analysis we used livers of gilthead sea bream (N=36 fish). Fish were injected with 200 µg/g body weight of cortisol and sampled after 7 and 14 days (n=6 for each condition). RNA samples were grouped into pools of 2 animals for each time point. The analysis were carried out considering the transcripts differentially expressed in the group of fish implanted during 7 days with cortisol comparing to control group. Additionally, the transcripts differentially expressed at day 14 were compared with day 7.
Project description:Effect of ethanol or nicotine exposure on gene expression compared to control. Duplicate arrays from ethanol or nicotine treated animals compared with triplicate arrays from paired control animals. In total 4 treatment arrays (2 ethanol, 2 nicotine) and 3 control arrays (from control animals treated in parallel with ethanol-treated fish and nicotine-treated fish.)
Project description:Perfluorooctanoic acid (PFOA) is a potent hepatocarcinogen and peroxisome proliferator (PP) in rodents. Humans are not susceptible to peroxisome proliferation and are thought to be refractory to carcinogenesis by PFOA and other PPs. However, previous studies with rainbow trout have shown that they are also insensitive to peroxisome proliferation by the PP, dehydroepiandrosterone (DHEA), but are still susceptible to enhanced hepatocarcinogenesis after chronic exposure. In this study, we determined whether PFOA is also a tumor promotor in trout and then examined hepatic gene expression profiles to further investigate possible mechanisms of action. Trout were initiated as fry to the hepatocarcinogen, aflatoxin B1, and then fed 200-1800 ppm PFOA in the diet for 30 weeks. Two structurally diverse PPs, clofibrate (CLOF) and DHEA, were included for comparison. Hepatic gene expression profiles were subsequently examined in animals exposed to similar doses of PFOA, DHEA and CLOF along with 5 ppm 17β-estradiol (E2; a known tumor promotor) in the diet. PFOA (1800 ppm) and DHEA treatments resulted in enhanced liver tumor incidence and multiplicity while CLOF showed no effect. Carcinogenesis seemed independent of peroxisome proliferation as no induction of peroxisomal β-oxidation and catalase activity were observed. Alternately, plasma VTG was elevated in fish fed PFOA and DHEA suggesting that estrogenic mechanisms may play a role. Both tumor promotors, PFOA and DHEA, resulted in strong correlation of transcriptional profiles with E2 by Pearson correlation (R=0.81 and 0.78, respectively). In comparison, CLOF regulated no genes in common with E2. Overall, these data suggest that the tumor promoting activities of DHEA and PFOA in trout are independent of peroxisome proliferation and may involve estrogenic mechanisms. Juvenile trout, 12-18 months old, were fed experimental diets containing 500 or 1800 ppm PFOA, 1800 ppm CLOF, 750 ppm DHEA, 5 ppm E2 or 0.15 % dimethyl sulfoxide vehicle control for 14 days. Liver samples were collected for microarray analysis. Hybridizations were performed using standard reference design with dye-swapping. For each sample, equal amounts of RNA (µg) were pooled from five fish per tank for every treatment (n=3 biological replicates per treatment). cDNA from two of the three biological replicates was dye-swapped and hybridized to two slides as technical replicates (5 arrays per treatment).
Project description:The skin mucus of gilthead sea bream was mapped by 1-DE followed by liquid chromatography coupled to high resolution mass spectrometry using a quadrupole time-of-flight mass analyzer. More than 2000 proteins were identified with a protein score filter of 30. The identified proteins were represented in 418 canonical pathways of the Ingenuity Pathway software. After filtering by canonical pathway overlapping, the retained proteins were clustered in three groups. The mitochondrial cluster contained 59 proteins related to oxidative phosphorylation and mitochondrial dysfunction. The second cluster contained 79 proteins related to antigen presentation and protein ubiquitination pathways. The third cluster contained 257 proteins where proteins related to protein synthesis, cellular assembly, and epithelial integrity were over-represented. The latter group also included acute phase response signaling. In parallel, 2-DE methodology identified six proteins spots of different protein abundance when comparing unstressed fish with chronically stressed fish in an experimental model that mimicked