## 3 Results
### Expression of Stress-Related Proteins
Different letters depict significant changes in Hsp60 expression between different exposure times in each salinity, while different symbols (\(\tau\), \(\#\), and \(\$\)) are used to denote significant differences between the three experimental salinities at the same exposure time.  
observed at T30 compared to both T15 and T45. Concerning the exposure time, significant induction of Hsp60 due to the increased salinity (14 and 20 ppt) was evident only at T45.  
Regarding p44/42 MAPK, the results of two-way ANOVA show a significant interaction between the effects of time and salinity on phosphorylation in both examined tissues. Compared to the initial p44/42 MAPK phosphorylation levels (T0), a significant decrease was observed in the liver at 8 and 14 ppt regardless of the exposure time (Figure 3a). Concerning the hepatic p44/42 MAPK at 20 ppt, significantly decreased levels were observed at T15 and T30 compared to T0, while prolonged exposure for 45 days (T45) led to phosphorylation increase at the initial levels. Furthermore, the hepatic phosphorylation levels decreased with increasing salinity at both T0 and T30, while at T45, increased salinity resulted in elevated levels. However, at T15, a shift from 8 ppt to 14 ppt led to a significant increase in the p44/42 MAPK phosphorylation, while decreased levels compared to both 8 and 14 ppt were observed following exposure to 20 ppt.  
In contrast to the liver, two-way ANOVA revealed that salinity did not have a statistically significant effect on the phosphorylation of p44/42 MAPK in the gills (\(p>0.05\)). However, there was a statistically significant interaction between the effects of time and salinity (F(6, 36) = 41.14, \(p<0.001\)). The exposure of gilthead sea bream to 8 ppt resulted in elevated phosphorylation at T15, while further exposure (T30, T45) led to a significant decrease compared to T0 (Figure 3b). During the first 30 days of exposure to 14 ppt, no significant changes were observed in p44/42 MAPK phosphorylation, while at T45 a significant decrease was apparent compared to T0. Compared to the initial p44/42 MAPK  
Figure 3: Phosphorylation levels of p44/42 MAPK in the liver (**a**) and gills (**b**) of gilthead sea bream following exposure to a salinity of 8, 14, or 20 ppt for 0, 15, 30, and 45 days. Different bar colors represent the exposure time to the experimental salinities (T0: 0 days, T15: 15 days, T30: 30 days, and T45: 45 days). Data are expressed as mean values of three replicates \(\pm\) S.D. Different letters depict significant changes in phosphorylated levels of p44/42 MAPK between different exposure times in each salinity, while different symbols (\(\lx@math@degree\), \(\bar{\#}\), and \(\lx@math@degree\)) are used to denote significant differences between the three experimental salinities at the same exposure time.  
phosphorylation levels (T0), a significant reduction was observed during further exposure to 20 ppt. Moreover, initial exposure (T0) showcased increased phosphorylation at 20 ppt compared to 8 and 14 ppt. On the contrary, at T15 exposure at higher salinities significantly decreased the phosphorylation levels compared to 8 ppt. Concerning T30, salinity increase from 8 to 14 ppt elevated p44/42 MAPK phosphorylation in the gills, while decreased levels compared to both 8 and 14 ppt were evident following exposure to 20 ppt. Moreover, phosphorylation levels significantly increased with increasing salinity at T45.  
The phosphorylation of p38 MAPK in response to increasing salinity at different exposure times is presented in Figure 4. According to the two-way ANOVA analysis, salinity, time, and the cumulative effect of the two variables significantly influenced the phosphorylation of p38 MAPK in the liver and the gills (\(p<0.01\)). Following exposure to 8 ppt, significantly decreased phosphorylated p38 MAPK levels were observed in the liver at T15, T30, and T45 compared to T0 (Figure 4a). Despite the increased hepatic phosphorylation of p38 MAPK at T15, prolonged exposure to 14 ppt for 45 days (T45) resulted in a significant decrease compared to T0. In contrast, exposure of gilthead sea bream to 20 ppt for 15, 30, and 45 days significantly elevated the phosphorylated p38 MAPK levels compared to T0. However, a significant reduction in the phosphorylation was apparent at T45 compared to T30. Concerning the exposure time, phosphorylated levels significantly decreased with increasing salinity at T0, while the opposite pattern was observed at T30. Elevation of phosphorylated p38 MAPK levels was observed following shift from 8 to 14 ppt at T15, while further increase in the salinity (20 ppt) led to a significant decrease compared to 14 ppt.  
Figure 4: Phosphorylation levels of p38 MAPK in the liver (**a**) and gills (**b**) of gilthead sea bream following exposure to a salinity of 8, 14, or 20 ppt for 0, 15, 30, and 45 days. Different bar colors represent the exposure time to the experimental salinities (T0: 0 days, T15: 15 days, T30: 30 days, and T45: 45 days). Data are expressed as mean values of three replicates \(\pm\) S.D. Different letters depict significant changes in phosphorylated levels of p38 MAPK between different exposure times in each salinity, while different symbols (*, #, and $) are used to denote significant differences between the three experimental salinities at the same exposure time.