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 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: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 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 13,824 cDNA microarray was constructed by printing 6912 PCR-amplified cDNA clones in duplicate. These were derived from a previous EST study (Clark et al, 2007) and comprised 3840 clones from the Library D2 (fully dehydrated animals, cooled to -14°C) and 3072 clones from the Library D1 (dehydrated animals cooled to -2°C). The Stratagene SpotReport Alien Array Validation System (Stratagene, La Jolla, CA, USA) was included on the microarray. Construction and hybridization of the arrays were performed as described in Purac et al (2008) with the exception of the amino-modified primers used for the initial cDNA amplification for array printing, which in this study were: (pAL32FOR: TTCTCGGGAAGCGCG and M13 forward: GTAAAACGACGGCCAG). Hybridizations were performed using control animals in combination with the groups listed below.<br><br><br><br>Six groups of animals were used for the hybridizations. Treatments were as follows:<br><br>C = control = live animals from +5°C<br><br>M2 = cold dehydrated animals, cooled to -2°C at a rate of 2°C/week<br><br>M7 = cold dehydrated animals, cooled to -7°C at a rate of 2°C/week<br><br>H18 = animals taken from -7°C, and left to recover for 18 hours at 4°C with moisture.<br><br>Salt 0.9 = animals slowly dehydrated over a saturated solution of sodium nitrate (which gives a constant humidity of 98% RH) to produce animals with a similar water content as the M2 animals<br><br>Salt 0.2 = as for Salt 0.9, but the animals were dehydrated to a similar water content to the M7 animals.<br><br>6 biological replicates were performed for each condition, as well as some dye swaps.
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: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
Project description:We investigated ecotoxicological effects and toxicogenomic responses in fathead minnows (Pimephales promelas) exposed to an environmentally-relevant concentration (0.83 mg/L) of the munitions compound cyclotrimethylenetrinitramine (RDX) in one year and multi-generational assays. In the one year assay, RDX effects were discerned by comparing breeding groups reared in control or RDX-exposure conditions for one year. RDX had no detectable effect on gonad-somatic index, or condition factor in females assayed at 1 day and at 1, 3, 6, 9, and 12 months, however the liver-somatic index was significantly increased versus controls only at the 12 month time point. RDX had no effect on live-prey capture rates at all time points assayed and no significant impacts on egg production, fertilization or hatch success in the 1-year exposure trial. Genomic analyses indicated that RDX exposure caused limited differential expression of transcripts within time points and no functional conservation of effects indicative of RDX exposure among time points for either brain or liver tissues in the one year exposure. In the multi-generational assay, the effects of acute (96h) exposure to RDX were compared in fish reared to the F2 generation in either control or RDX-exposure conditions. The RDX-reared fish were not observed to have appreciably enhanced RDX tolerance versus the control-reared fish. However, significant differences in gene expression were observed among the control and RDX-reared fish related to neuro-excitatory glutamate metabolism, sensory signaling and processes in neurological development. In total, our results indicate that exposure to an RDX concentration approximating maximum levels observed in the field (0.83 mg/L) caused limited impacts in fathead minnows in a one year exposure, however caused altered expression in genes involved in neural function in a multi-generational exposure. Adult fathead minnows were exposed to 0.83 mg/L (0.15 mg/L standard deviation) in experimental units including 2 male and 4 fish. Five replicate males were randomly sampled with replacement from each of 8 control and 8 RDX-exposed experimental units at 1d, 1mo, 3mo, 6mo, 9mo and 12mo sampling periods. Liver tissue was investigated for differential expression in response to RDX exposure.
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:This experiment contains the transcriptomic dataset that constitutes part of an integrated transcriptomic and proteomic study monitoring the response of exponential phase E. coli O157:H7 Sakai cultures upon an abrupt downshift in temperature and water activity (from 35°C aw 0.993 to 14°C aw 0.967).