HEAT-SHOCK STRESS ON THE DEVELOPING IMMUNE SYSTEM OF SEA BASS

Temperature controls and limits all physiological and behavioural parameters ofectotherms. Within a range of non-lethal temperatures, fishes are generally able tocope with gradual temperature changes that are common in natural systems. However,rapid increases or decreases in ambient temperature, as potentially occur during theaquaculture starvation, may result in sublethal physiological and, or behaviouralresponses. Responses to environmental stress are broadly grouped into threecategories: primary (e.g. neuroendocrine response and corticosteroid–catecholaminerelease), secondary (e.g. metabolic, cellular, haematological, osmoregulatory andimmunological changes) and tertiary (e.g. whole organism physiological andbehavioural stress responses).Apoptosis is a pathway serving to remove unwanted or damaged cells in thecourse of development and differentiation, or in response to various forms of cellulardamage (due for instance to temperature-stress) that did not surpass the thresholdleading to necrotic, accidental, or other forms of cell death. It is conducible to thesecondary response when the process involved lympho-myeloid organs. The commontarget of the diverse stimuli trigger intrinsic or extrinsic apoptosis pathways, bothmediated by a family of cysteine proteases known as caspases. Particularly, themitochondrial compartment is involved in intrisic patways, which via opening of aproteinaceous pore releases molecules into the cytosol. These released molecules thenact as mediators of apoptosis by causing DNA fragmentation and chromatincondensation (AIF, apoptosis inducing factor; endonuclease G), by de-inhibitingcaspases (Smac/DIABLO, second mitochondria-derived activator of caspases/directinhibitor of apoptosis protein-binding protein with low pI; Omi/HtrA2), or by actingas an essential component of a caspase activating platform (cytochrome c). HeatShock Proteins (HSPs) have important anti-apoptotic properties, blocking cell deathand promoting survival, proliferation or differentiation. HSPs are a wide family ofconserved proteins, present in all organisms including fish, and HSP70 kDa familyhas a strong cytoprotective effect, acting as molecular chaperone and mediating therepair or degradation of denaturated proteins.The purpose of this study is to evaluate the sublethal consequences of heatshockon the developing immune system of sea bass (from 50 to 70 days post-hatch)obtained from a commercial hatchery. To this aim we performed an experiment wherefish were exposed for 40 minutes to thermal stress (24 °C) and then sampled after 2hour (T2h), 24 hour (T24h), 10 days (T10gg) and 20 days (T20gg). Control organisms weremaintained at standard temperature condition (19 °C). Samples were analysed byimmunohistochemistry/ELISA to evidence HSP70 and Caspase-3 expression.TUNEL technique was applied to reveal apoptotic cells. Immunohistochemistry wasperformed to evaluate the presence of phagocytes (granulocytes and macrophages)and lymphocytes. RT-PCR analyses were conducted using specific primers for TcRbeta,TcR-gamma and BcR. In the control organisms, immunohistochemistry revealedno particular differences related to organs (thymus, gill, spleen, cephalic kidney) orage, except for a significant increase in apoptosis, HSP70 and caspase-3 activeexpressions in the thymus at 70 days. These results support the role of apoptosis in thelymphoid organ maturation and the function of HSP70 in controlling the process.Compared to control, the stressed sea bass showed a significant increase inapoptosis/caspase-3 (40') followed by an enhanced HSP70 expression (from T2h).[...]