Cloning and expression analysis of specific and non-specific immune genes in adult and larval turbot (Scophthalmus maximus)
Increased knowledge of the components of the turbot immune system, and the effects of environmental conditions such as water temperature, which influence the appearance of defense mechanisms would allow for the introduction of new strategies to cope with the problem of a high, variable mortality rate observed during the early rearing stages of turbot larvae. Partial sequences for a number of turbot non-specific and specific immune genes have been identified. These include partial sequences for transforming growth factor-beta 1 (TGFb1) (186bp), transferrin (492bp), interleukin-1 beta (IL-1b) (1025bp), recombinase activating gene 1 (RGA-1) (548bp) and immunoglobulin M (IgM) (606bp). Expression studies looking at the dose dependency of lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C) induced gene expression in a turbot cell line (TFC) and turbot leucocytes was studied. The studies using turbot leucocytes show that a number of genes of the turbot immune system are induced in response to stimulation with LPS, these include IgM, lysozyme, transferrin IL-1b and TGFb1. The response to poly I:C was more selective with only the Mx gene and TGFb1 induced in response to stimulation. To determine the effects of the inclusion of exogenous nucleotides in aquaculture diets on the turbot immune system, a feeding study was carried out. When aided to normal fish feed formulations at a combined inclusion level of 0.03% these additional nucleotides were shown to increase the humoral immune response in terms of increased IgM, RAG-1 and TcRa gene expression. RT-PCR expression studies looking at immune gene development in turbot eggs and newly hatched larvae, and the effect of rearing temperature and stimulants was also carried out using the turbot specific primers. The results show that immune gene expression is detectable from early in development. Low rearing temperatures (10-12oC) had a detrimental effect on the development of immune gene expression.