RESUMO
In aquaculture, fish species may experience stressful episodes caused by poor farming conditions. The exponential increase of global aquaculture has raised the number of research studies aimed at demonstrating the sensitivity of aquatic animals in confined environments. The development of a real-time PCR and immunohistochemistry methods were investigated to evaluate the presence, localization, and quantity of biomarkers of oxidative stress in European sea bass (Dicentrarchus labrax). In particular, stress tests such as manipulation and temperature changes were conducted through molecular methods to identify the expression level of heat shock protein 70 (HSP70) in stressed animals compared with a control group. The immunohistochemical technique was also applied to locate and study the trends-levels of nitrotyrosine (NT), heat shock protein 70 (HSP70), malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) in different tissues from stressed animals and control group. The presence of the rodlet cell (RCs) was evaluated by histology in both a control and stressed group. Our results show that the real-time PCR method developed is specific for the evaluated target gene and that manipulation and temperature increase are strong stressors for animals. Relative quantification data revealed a gene expression increase of HSP70 in the stressed group of animals compared to the control group. The antibodies used for the immunohistochemical staining were efficient, and it was possible to appreciate the increase of immunoprecipitates in European sea bass either manipulated or stressed by temperature increase. The present study can be a starting point to allow the quantification of HSP70 and the identification of other stress biomarkers in D. labrax.