Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs.
This study describes the development and validation of an enriched oligonucleotide-microarray platform forSparus aurata(SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response inS. auratamacrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene expression inS. auratawith an emphasis upon immunity and the immune response.
Institute of Biotechnology and Biomedicine, University Autónoma de Barcelona, Barcelona 08193, Spain. sboltana@udec.cl.|University of Stirling, School of Natural Sciences, Stirling FK9 4LA, Scotland, UK. sboltana@udec.cl.|Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Aquaculture Research (INCAR), Biotechnology Center, University of Concepción, Concepción 4030000, Chile. sboltana@udec.cl.
Sebastian
S
Boltaña
The Child & Family Research Institute, Vancouver, BC V5Z 4H4, Canada. bcastellana@cfri.ca.|Department of Obstetrics & Gynecology, The University of British Columbia, Vancouver, BC V6T 1Z4, Canada. bcastellana@cfri.ca.
Barbara
B
Castellana
Northwest Fisheries Science Centre, Seattle, WA 98112, USA. giles.goetz@noaa.gov.
Giles
G
Goetz
Department Biologia Cellular, Immunologia I Fisiologia, Universitat Autónoma de Barcelona, Barcelona 08193, Spain. lluis.tort@uab.cat.
Lluis
L
Tort
Department Biologia Cellular, Immunologia I Fisiologia, Universitat Autónoma de Barcelona, Barcelona 08193, Spain. mteles0@gmail.com.
Mariana
M
Teles
Department de Biología Celular e Histología, Facultad de Biología, Universidad de Murcia, Murcia 30100, Spain. vmulero@um.es.
Victor
V
Mulero
Institute of Marine Research, Spanish National Research Council (CSIC), Eduardo Cabello 6, Vigo 36208, Spain. virus@iim.csic.es.
Beatriz
B
Novoa
Institute of Marine Research, Spanish National Research Council (CSIC), Eduardo Cabello 6, Vigo 36208, Spain. antoniofigueras@iim.csic.es.
Antonio
A
Figueras
Northwest Fisheries Science Centre, Seattle, WA 98112, USA. Rick.Goetz@noaa.gov.
Frederick W
FW
Goetz
Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Aquaculture Research (INCAR), Biotechnology Center, University of Concepción, Concepción 4030000, Chile. crisgallardo@udec.cl.
Cristian
C
Gallardo-Escarate
Department of Physiology and Immunology, University of Barcelona, Barcelona 08028, Spain. jplanas@ub.edu.
Josep V
JV
Planas
Institute of Biotechnology and Biomedicine, University Autónoma de Barcelona, Barcelona 08193, Spain. simon.mackenzie@stir.ac.uk.|University of Stirling, School of Natural Sciences, Stirling FK9 4LA, Scotland, UK. simon.mackenzie@stir.ac.uk.
Simon
S
Mackenzie
D000818:Animals; D016000:Cluster Analysis; D019295:Computational Biology / Q000379:methods; D020224:Expressed Sequence Tags; D005393:Fish Diseases / Q000235:genetics* / Q000276:immunology*; D020869:Gene Expression Profiling; D015723:Gene Library; D007249:Inflammation / Q000662:veterinary*; D008070:Lipopolysaccharides / Q000009:adverse effects; D000069452:Pathogen-Associated Molecular Pattern Molecules; D021541:Sea Bream / Q000235:genetics* / Q000276:immunology*; D059467:Transcriptome
D008070:Lipopolysaccharides; D000069452:Pathogen-Associated Molecular Pattern Molecules
expressed sequence tags (EST); lipopolysaccharide (LPS); macrophages; oligo-nucleotide microarray; pathogen-associated molecular patterns (PAMPs); peptidoglycan (PGN)