Project description:The physiological consequences of an activation of the immune system in fish are not well understood. In particular, skeletal muscle, due to its essential role in locomotion and whole-animal energy homeostasis, is a potentially important target of inflammation. In this study, we have evaluated the in vivo effects of lipopolysaccharide (LPS) on the white and red skeletal muscle transcriptome of the gilthead seabream (Sparus aurata) by microarray analysis at 24 and 72 hours after injection. In white muscle, the transcriptomic response was characterized by an up-regulation of genes involved in carbohydrate catabolism and protein synthesis at 24 hours and a complete reversal of this pattern at 72 hours. In red muscle, an up-regulation of genes involved in carbohydrate catabolism and protein synthesis was observed only at 72 hours after LPS administration. Interestingly, both white and red muscles showed a similar consistent down-regulation of immune genes at 72 hours post-injection. However, genes involved in muscle contraction showed a general up-regulation in response to LPS in both types of muscle. In summary, LPS administration causes muscle type-specific responses regarding the expression of genes involved in carbohydrate and protein metabolism and a common decreased expression of immune genes in skeletal muscle, concomitant with increased expression of genes for contractile elements. Our results evidence a robust and tissue-specific transcriptomic response of the skeletal muscle to an acute inflammatory challenge. Total RNA from pooled control (n = 5) and LPS-treated (n = 5) sea bream white and red muscle tissues was labeled with Cy3-dUTP and Cy5-dUTP (GE Healthcare, Barcelona, Spain). We used a dye swap experimental design and each cDNA from a pooled RNA sample was hybridized to two microarrays. For the first slide, test and control cDNA were labeled with Cy5 and Cy3 respectively, and for the second array dye assignment was reversed. Therefore, samples from individual fish within each group were pooled and expression values shown represent the means of 6 M-CM-^W 2 = 12 technical replicates. A total of eight slides were used in this study.

Project description:This study describes the development and validation of the Aquagenomic Sparus aurata oligonucleotide-microarray (SAQ) based on the Agilent Technology system (eArray) to provide a platform for studies on the gene expression of gilthead seabream. The platform developed used all available public ESTs stored and annotated in the Aquagenomic Consortium seabream library (10K). In fish, lipopolysaccharide (LPS) gives a robust cytokine response that is stimulated by crude LPS preparations, some component of the LPS complex are responsible for this stimulation. Peptidoglycan (PGN) is a component of G-negative bacteria (found as a contaminant of crude LPS preparations) able to be recognized by macrophages inducing depth transcriptional modulations and a strong inflammatory response. For microarray analysis, head kidney macrophage cultures were used (N = 36 fish). Each cell culture was stimulated with equal concentrations of PGN and LPS from E. coli O111:B4 strain (10 ug/mL): non-stimulated cell cultures (control n = 9 fish), stimulated during 6 h with LPS (n = 9), and stimulated during 1 h (n = 9), and 6 h (n = 9) with PGN. A loop microarray design approach was used for the study. All experimental RNA samples were labelled with a single colour dye (Cy3) and each stimulated sample was compared to the control sample (pool without stimulation) labelled with the same dye (Cy3). Our microarray analyses identified differential transcriptional modulations in macrophages stimulated with both LPS and PGN at the level of differentially activated RNA transcripts related with the regulation of transcriptional program, prostaglandin synthesis or highlighting the expression of responsive gene-cassettes tightly related to LPS-PGN host recognition. A total of 43,398 oligonucleotide probes were used to construct a high-density seabream microarray based on the Agilent 4 × 44 K design format. Microarray hybridization validation was made by analyzing the gene expression profiles in primary cultures of seabream macrophages (MC). 7,285 transcripts with annotated sequences were spotted in triplicate onto the slide (total probes 21,855), as well as 8,377 ESTs without annotation, 183 enriched sequences (gene bank) with 15 replicated probes (total probes 2,745), and finally 1,417 internal control probes of Agilent (N = 43,398).

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Sparus aurata

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