Effects of constant temperature on liver phospholipid fatty acid composition (PLFA) during seawater acclimation
In trial 1, after rearing in freshwater for four weeks (FW-28), the liver PLFA composition of rainbow trout was significantly altered by different constant temperatures (Table 1). In treatments of 9 (T9) and 12.5 °C (T12.5), the proportions of 16:0 and 18:0 significantly lowered ratio of saturated fatty acid (SFA) in liver phospholipids. The proportion of monounsaturated fatty acid (MUFA) in liver phospholipids was significantly lower in treatment of 16 °C (T16) than in T12.5, but significantly higher than in treatment T9, mainly caused by 18:1n9. The proportion of polyunsaturated fatty acids (PUFAs) in the liver phospholipids of rainbow trout was negatively correlated with temperature; the proportion of PUFAs in T9 was the highest, followed by T12.5 and T16. The ratio of unsaturated to saturated fatty acid (U/S) in T9 and T12.5 was significantly higher than in T16, while the unsaturation index (UI) in T9 was significantly higher than in T16. The ratio of n-3 PUFA to n-6 PUFA (n3/n6) in T9 and T12.5 was significantly higher than in T16 (Fig. 1A–D). In addition, the average chain length (ACL) of fatty acid in T9 was significantly higher than in the other groups.
At the first day after seawater acclimation (SW-1), the liver PLFA composition in rainbow trout was significantly altered (Table 1). The proportion of SFA in liver phospholipids significantly decreased in T16, whereas those in T9 and T12.5 increased, significantly higher than in T16. The total MUFA in T12.5 significantly decreased, while those in other groups remained unchanged. The proportion of PUFAs in T9 increased to 55.75%, close to the level in T12.5. Fig. 1 shows a significantly low U/S in T9 and a significantly high ACL in T12.5, but no significant difference in UI among groups.
Two weeks after seawater acclimation, the total SFA in T9 significantly decreased and was significantly lower than in T12.5 (Table 1). The total MUFA in T16 considerably increased and was significantly higher than in other groups. Consequently, the UI in T9 and T16 increased and was significantly higher than in T12.5. These changes resulted in significantly higher UI, U/S, and ACL in T9 (Fig. 1A–D).
Effects of constant temperature on intestinal PLFA composition during seawater acclimation
In trial 1, after rearing in freshwater for four weeks, the intestinal PLFA composition in rainbow trout was altered significantly (Table 2). The total SFA in T9 and T12.5 decreased significantly, while the total PUFAs in T9 and T12.5 were significantly higher in T16. The total MUFA in T12.5 significantly decreased and was significantly lower than in T16. These changes resulted in significantly higher U/S, UI, and ACL in T9 and T12.5 than in T16 (Fig. 1E–H). The n3/n6 ratio was positively associated with temperature.
On the first day after seawater acclimation, the total SFA in T16 significantly decreased. The total MUFA in T9 and T16 significantly decreased whereas that in T12.5 increased and was significantly higher than in other groups. The total PUFA in T9 and T16 significantly increased, while that in T12.5 was relatively steady. Consequently, significant increases in n3/n6, U/S, UI, and ACL were recorded in T16.
Two weeks after seawater acclimation (SW-14), the total SFA in T16 decreased while the other groups showed no significant difference (Table 2). The total MUFA in all groups decreased significantly. Accordingly, the total PUFA in all groups increased but showed no difference. Thus, the UI in T9 was significantly higher than in other groups, whereas no difference was found in UI and ACL in liver phospholipids in rainbow trout (Fig. 1E–H).
Effects of diel cyclic temperature on liver PLFA composition during seawater acclimation
In trial 2, at the final day of growth trial (FW-42), the proportion of 16:0 in the treatment of 13 °C (CT) was significantly decreased while that of 20:4n6 in the treatment of 13 ± 1 °C (VT2) and 13 ± 2 °C (VT4) remained steady (Table 3). However, no significant change was found in total SFA, MUFA, and PUFA among the groups. Therefore, there was no difference among groups in terms of U/S, UI, and n3/n6, except for a significantly higher ACL in VT2 (Fig. 2A–D).
At SW-1, the total SFA of liver phospholipids in CT and VT2 increased significantly, while VT4 showed no significant change (Table 3). An overall decrease in total MUFA was detected in all groups, and the CT group showed significantly lower total MUFA than the VT2 and VT4 groups. Furthermore, the proportion of 22:6n3 was significantly lower in VT2 and VT4 than in CT, while total PUFA showed no difference among groups. These changes resulted in significantly higher UI in CT than in VT2. ACL and n3/n6 were significantly higher in CT than in VT2 and VT4 (Fig. 2A–D).
In trial 2, two weeks after seawater acclimation, the total SFA in all groups increased significantly and showed no difference in liver phospholipids of rainbow trout (Table 3). The total MUFA in CT increased to 7.88%, while in VT2 and VT4 it was comparatively steady. The total PUFA in VT2 and VT4 decreased significantly and showed no difference among groups. The PLFA-related parameters showed no significant difference among groups.
Effects of diel cyclic temperature on intestinal PLFA composition during seawater acclimation
In trial 2, after six weeks in freshwater, the intestinal PLFA composition of rainbow trout was significantly influenced by diel cyclic temperature (Table 4). The total SFA in VT4 was significantly lower than in CT and VT2. A significantly high proportion of 22:6n3 was recorded in VT4, resulting in a significantly high total PUFA in VT4. Correspondingly, the U/S, UI, n3/n6, and ACL in VT4 were significantly higher than in CT and VT2 (Fig. 2E–H).
At SW-1, significant decreases in the proportions of 16:0 and 18:0 were detected in CT in the intestinal phospholipids of rainbow trout (Table 4). The total MUFA in VT4 significantly increased and was significantly higher than that in CT and VT2. The proportion of 22:6n3 in CT and VT2 increased significantly, whereas that in VT4 decreased. Consequently, the total PUFA in CT was significantly higher than in VT2 and VT4 (Fig. 2E-H). These changes caused significantly higher U/S in CT than VT2 and VT4, whereas the UI and ACL in VT4 were significantly lower than in the other groups.
At SW-21, the intestinal phospholipids contained significantly lower proportions of 18:1n9 and 18:2n6 in CT than in VT2 and VT4, whereas the proportion of 22:6n3 in CT was significantly higher than in VT2 and VT4 (Table 4). However, no significant difference was observed in the total SFA, MUFA, and PUFA among the groups. The UI, n3/n6, and ACL in CT were significantly higher than in VT2 and VT4 (Fig. 2E–H).
A two-way analysis of variance
In trial 1, the two-way analysis of variance (ANOVA) indicated that both the liver and intestine in rainbow trout were significantly influenced by constant temperature, seawater acclimation, and their interaction (see Additional file 3). In trial 2, the diel cyclic temperature had no significant effect on liver PLFA composition, whereas seawater acclimation significantly influenced the total SFA, n3/n6, U/S, UI, and ACL in the liver PLFA composition. Therefore, the total SFA, n3/n6, U/S, UI, and ACL were significantly influenced by the interaction of diel cyclic temperature and seawater acclimation. In the intestinal phospholipids, all selected parameters were significantly affected by diel cyclic temperature, seawater acclimation, and their combination, except total MUFA, which was free of the effect of temperature.
Principal component analysis
In the present study, principle component analysis (PCA) was performed to reveal the tissue differences in rainbow trout at different temperature regimes and seawater acclimation stages. Based on the results of the two-way ANOVA, 16 variables (14:0, 16:0, 18:0, 16:1, 18:1n9, 24:1, 18:2n6, 20:2n6, 20:3n3, 20:4n6, 20:5n3, and 22:6n3, U/S, UI, n3/n6, and ACL) were selected for the liver of rainbow trout, while nine variables (14:0, 18:0, 14:1, 16:1, 18:1n9, 18:2n6, 18:3n3, 20:5n3, 22:6n3, U/S, UI, n3/n6, and ACL) were selected for the intestine.
In trial 1, the first two principal components (PCs) with eigenvalues of 6.68 and 3.72 were found to explain 69.34% of the overall variability. The eigenvectors of 16:1, 18:0, 18:1n9, 18:2n6, 22:6n3, U/S, UI, n3/n6, and ACL were more than 0.4, indicating a strong effect on the PCs. The score plots distinguished the liver PLFA composition of rainbow trout at different constant temperatures and seawater acclimation stages (Fig. 3A). PCA analysis of the intestinal PLFA composition yielded two PCs with eigenvalues of 6.70 and 2.44, explaining 76.06% of the dataset variance. The eigenvectors for 14:0, 14:1, 18:1n9, 18:3n3, 20:5n3, 22:6n3, U/S, UI, n3/n6, and ACL were greater than 0.4, resulting in the difference in intestinal PLFA composition of rainbow trout at different temperatures and seawater acclimation stages (Fig. 3B).
In trial 2, two of three PCs were selected for PCA on liver PLFA composition, and their eigenvalues were 5.69 and 3.54 and explained 65.88% of the total variability. The eigenvectors for 16:1, 16:0, 18:2n6, 18:1n9, 18:0, 20:5n3, 20:3n3, n3/n6 and U/S exceeded 0.4. However, individuals of liver PLFAs only clustered at FW-42 and SW-21 instead of temperature regimes (Fig. 3C). Analysis of the intestinal PLFA composition produced two PCs with eigenvalues of 6.68 and 2.13, explaining 73.45% of the accumulated variability. Eigenvectors of more than 0.4 included 16:0, 18:2n6, 18:1n9, 18:0, 20:5n3, 22:6n3, 24:1, n3/n6, U/S, UI, and ACL, contributing to the comparatively high differentiation in intestinal phospholipids of rainbow trout influenced by diel cyclic temperature and seawater acclimation (Fig. 3D).