Bonamiosis and disseminated neoplasia (DN) are the most important diseases affecting cultured flat oysters (Ostrea edulis) in Galicia (NW Spain). Previous research of the response of O. edulis against bonamiosis by suppression subtractive hybridisation yielded a partial expressed sequence tag of tumour necrosis factor (TNF) and allograft inflammatory factor (AIF), as well as the whole open reading frame for dermatopontin and vesicle-associated membrane (VAMP). Herein, the complete open reading frames of TNF and AIF genes were determined by the rapid amplification of cDNA, and the deduced amino acid sequences of the four genes were characterised. Phylogenetic relationships for each gene were studied using maximum likelihood parameters. Quantitative-PCR assays were also performed in order to analyse the modulation of the expression of these genes by bonamiosis and disseminated neoplasia. Gene expression profiles were studied in haemolymph cells and in various organs (gill, gonad, mantle and digestive gland) of oysters affected by bonamiosis, DN, and both diseases with regard to non-affected oysters (control). TNF expression in haemolymph cells was up-regulated at heavy stage of bonamiosis but its expression was not affected by DN. AIF expression was up-regulated at heavy stage of bonamiosis in haemolymph cells and mantle, which is associated with heavy inflammatory response, and in haemolymph cells of oysters affected by DN. AIF expression was, however, down-regulated in other organs as gills and gonads. Dermatopontin expression was down-regulated in haemolymph cells and digestive gland of oysters affected by bonamiosis, but DN had no significant effect on its expression. Gills and gonads showed up-regulation of dermatopontin expression associated with bonamiosis. There were significant differences in the expression of TNF and VAMP depending on the bonamiosis intensity stage whereas no significant differences were detected between light and heavy severity degrees of DN for the studied genes. VAMP expression showed also differences among haemolymph cells and the organs studied. The occurrence of both diseases in oysters involved haemolymph cell gene expression patterns different from those associated to each disease separately: no significant effect was observed in TNF expression, dermatopontin was up-regulated and marked up-regulation of AIF and VAMP was recorded, which suggests a multiplier effect of the combination of both diseases for the latter two genes.
Keywords: AIF; Allograft inflammatory factor; Bonamia; CDD; CDR; Complementary deoxyribonucleic acid; Conserved domain; Cysteine rich domain; DG; DN; Dermatopontin; Digestive glands; Disseminated neoplasia; EST; Expressed sequence tag; Heat shock cognate; Heat shock protein; Hsc; Hsp; Isoelectrical point; ORF; Open reading frame; Ostrea edulis; PI; Q-PCR; Quantitative polymerase chain reaction; RACE; RNA; Rapid amplification of cDNA ends; Ribonucleic acid; SSH; SUMO; Small ubiquitin-like modifier; Suppressive subtractive hybridisation; THD; TIMP; TNF; TNF homology domain; TNF-receptor associated factor 6; TRAF6; Tissue inhibitor of metalloproteinase; Tumour necrosis factor; UTR; Untranslated region; cDNA.
© 2013.