## 4 Discussion
### Expression of Stress-Related Proteins  
Deviations from optimum ambient salinity may disrupt the equilibrium between generation and neutralization of reactive oxygen species (ROS), thus leading to induction of oxidative stress in aquatic organisms (see review in [65]). The capacity of animals to cope with severe environmental stress, including extreme salinity fluctuations largely relies on the cellular stress response (CSR), an evolutionarily conserved physiological mechanism [66]. Exposure to severe salinities induces several CSR components including Hsps, which, among other functions, protect and repair protein structure due to alterations in the cellular ion regulation [66; 67]. Herein, the gilthead sea breams subjected to different salinities showcased a tissue- and time-specific induction of Hsp60. Specifically, regardless of the salinity, hepatic levels of Hsp60 were initially elevated during the first 15 days and subsequently decreased following prolonged exposure. In accordance with the latter results, _Liza haematochelia_ subjected to salinity of 2, 28, and 42 ppt displayed an initial increase in the Hsp90 mRNA expression during the first days, while decreased levels were prominent following further exposure [68]. On the contrary, Hsp60 induction was evident herein in the gills as a response to prolonged exposure to 14 and 20 ppt, as well as during the first 30 days of exposure to the hypotonic environment (8 ppt). Gills, the major respiratory and ionoregulatory organ of fish, are directly exposed to the surrounding water, and thus have to continuously contend with fluctuations in environmental factors [69]. Salinity alterations are known to result in unchanged Hsps levels in the gills of several fish species [70; 71]. For instance, shifting from fresh water to seawater may exert no influence on the levels of Hsp90 mRNA in the gills of Chinook salmon (_Oncorhynchus sthawyrtscha_) [71]. However, increased Hsps expression as observed herein was also reported in the gills of several fish species including black porgy (_Acanthopagrus schlegeli_) [72] and sea bream (_Sparrus sarbu_) [73] in response to osmotic stress. The contrast in the Hsp60 expression pattern observed herein among the two examined tissues indicates a more prominent and constant protective role in the gills and an immense recruitment of molecular chaperones to facilitate the adaptive response to salinity stress. On the other hand, liver seemed to be subjected to an initial salinity-induced stress state, thus requiring the functional involvement of Hsp60 facilitating the subsequent recovery period. Previous studies suggested that metabolically active organs are able to efficiently cope with environmental perturbations and restore the prestress state [67]. In addition, cellular response to environmental stressors may include the activation of the MAPK signaling pathway, which was demonstrated in a plethora of fish species (e.g., [40; 74; 75]). During changes in salinity, MAPKs are involved in osmosensory signaling pathways, such as the regulation of inorganic ions and organic osmolytes intracellular levels, thus mediating the physiological acclimation [76; 77]. Several fish species subjected to hypotonic and/or hypertonic environments were reported to display up-regulation of MAPKs members [78; 79]. Herein, the phosphorylation of p38 MAPK in gilthead sea bream gills during exposure to 8 and 14 ppt showcased an elevation at the first 15 days, where simultaneously phosphorylated p44/42 MAPK levels were also increased under the hypotonic regime. Similarly, previous studies [78] demonstrated that MAPKs activity in the gill epithelium of the euryhaline teleost _Fundulus heteroclitus_ is more prominent during hyposmotic stress. Thereby, due to the direct interaction with the surrounding water, gills may be more prone to a hypotonic environment, and MAPKs phosphorylation is probably required in gilthead sea bream physiological acclimation and adaptation.