ABSTRACT: Fish respond to stressors by means of an array of changes that involve many biological compartments including molecular, cellular, metabolic, physiological and behavioral acclimation. Among these changes, the immune system also experiences significant alterations that are characterized by an induction of the innate non-specific responses and changes in the acquired specific response. The present investigation was undertaken to check whether the innate response of the complement system is affected after a combination of an immune challenge and a husbandry stressor. In European seabass, two different transcripts of the complement C3 protein, which correspond to two tissue-specific isoforms, have been detected in liver. They may come from post-transcriptional modifications at the pre-mRNA level or from alternative splicing in order to generate a diversified repertoire of protein isoforms in some fish species according to the tissue were they are located. After cloning the hepatic C3, the quantification of seabass isoforms showed specific responses, thus confirming the possible specialized roles of the C3 different isoforms in terms of defence immunity. On one hand, a stronger esbC3_2 expression was observed in front to the viral challenge, suggesting a specific participation of this molecule in front to viruses. This observation is in agreement with previous studies in trout macrophages in our lab, demonstrating that the immune response to viral infections was stronger than to bacterial ones. On the other hand, the esbC3_1 expression appeared stronger after bacterial challenge, especially under high density conditions. Our results confirm the hypothesis of an immune-activation of specific isoforms of the C3 protein of the complement system as a non-specific response in front to either immune or other type of stressors. In order to confirm our hypothesis, we created an oligonucleotide microarray (Agilent Technologies) containing 6275 annotated transcripts, each with x3 specific probes, and 4516 ESTs with 1 probe/target sequence. Gene expression profiles obtained from the livers of fish held under high density culture conditions highlighted a significant contrast in the liver response. In total, 54 transcripts were differentially expressed (GDE; fold change FC >2) in treatment over the control (p<0.01)/201 (p<0.05). Oligonucleotide probes were used to construct a high-density seabass microarray based on the Agilent 4 × 44 K design format (http://www.agilent.com/), which covers 13,199 unique transcripts of Dicentrarchus labrax. Oligonucleotides, 60-mer plus a “linker”, are synthesized directly on glass slides using the Agilent’s SurePrint Tecnology; oligo design was carried out by Agilent and three or more non-overlapping probes were designed for each EST cluster. The number of transcripts represented by three is 6,275, while 6,924 targets EST have only one probe.
