**3. Results**

#### *3.1. Changes in Gene Expression Levels in the Mackerel Tuna Rhythm*

Under the sunny condition, the expression levels of *CREB1*, *CLOCK*, *RORA*, *PER1*, *PER3* and *CRY1* were not significantly rhythmic in the liver (Table 3). The expression levels of *CREB1* and *CLOCK* were significantly higher at 18:00 than at the other time points (*p* < 0.05). The expression levels of *RORA*, *PER1* and *PER3* were significantly higher at 24:00 than at the other time points (*p* < 0.05; Figure 1a–f). The expression level of *CRY1* was significantly lower at 6:00 than at the remaining time points (*p* < 0.05, Figure 1g). The expression levels of *PER2*, *CRY2*, *REVERBA* and *BMAL1* were significantly rhythmic in the liver under the sunny condition (Table 3). The expression levels of *PER2* and *CRY2* were significantly higher at 24:00 than at the other time points (*p* < 0.05; Figure 1e,h; Table 3). The expression levels of *REVERBA* were significantly higher at 18:00 than at the other time points < 0.05; Figure 1i; Table 3). The expression levels of *BMAL1* were significantly higher at 24:00 than at the other time points (*p* < 0.05; Figure 1h; Table 3).


**Table 3.** Cosinor analysis board for rhythm gene expression under sunny and cloudy conditions.

n.s. denotes statistical differences between the sampling points. Acrophases (circadian peak times) were calculated by a non-linear regression fit of a cosine function. Data are expressed as acrophase ± 95% confidence intervals.

Under the cloudy condition, the expression levels of *RORA*, *PER2*, *CRY1* and *CRY2* were not significantly rhythmic in the liver (Table 3). The expression levels of *CREB1*, *PER1*, *PER3* and *REVERBA* were significantly higher at 12:00 than the other three time points under the cloudy condition (*p* < 0.05). The expression levels of *CLOCK* and *BMAL1* were significantly higher at 6:00 than at the other time points (*p* < 0.05; Figure 1a,b,d,f,i,j; Table 3). The expression levels of *RORA*, *PER2*, *CRY1* and *CRY2* were significantly rhythmic in the liver under cloudy conditions (Table 3). The expression levels of *RORA*, *PER2* and *CRY2* were significantly higher at 12:00 than at the other three time points (*p* < 0.05). The expression level of *CRY1* was significantly higher at 6:00 than at the other three time points (*p* < 0.05; Figure 1c,e,g,h; Table 3).

A comparison of rhythm genes' expression levels at the same time point under different weather (sunny versus cloudy days) is shown in Figure 1. Expression levels of *CREB1*, *RORA*, *PER1*, *PER3*, *CRY1*, *REVERBA* and *BMAL1* at all the time points were significantly different between sunny and cloudy days at the same time point (*p* < 0.05; Figure 1a,c,d,f,g,i,j). Expression levels of *CLOCK* at 6:00, 18:00 and 24:00 were significantly different between sunny and cloudy days at the same time point (*p* < 0.05; Figure 1b). The expression levels of *PER2* at 6:00, 12:00 and *PER2* at 6:00, 12:00 and 24:00 were significantly different between sunny and cloudy days at the same time point (*p* < 0.05; Figure 1e). The expression levels of *CRY2* at 12:00, 18:00 and 24:00 were significantly different between sunny and cloudy days at the same time point (*p* < 0.05; Figure 1h).

**Figure 1.** Expression of liver rhythm genes during 24 h in different weather in mackerel tuna. (**a**): *CREB1*; (**b**): *CLOCK*; (**c**): *RORA*; (**d**): *PER1*; (**e**): *PER2*; (**f**): *PER3*; (**g**): *CRY1*; (**h**): *CRY2*; (**i**): *REVERBA*; (**j**): *BMAL1*. Red in each graph represents sunny days, and blue represents cloudy days. The presence of different letters indicates significance by ANOVA and Tukey's tests (*p* < 0.05). \* represents significant differences at the same time point (*p* < 0.05). Differences between those with different lowercase letters indicate significance (*p* < 0.05), while the opposite difference is not significant (*p* > 0.05); the same for the latter figure.

The results of the two-way analysis of different weather and time of day on mackerel tuna rhythm genes are shown in Table 4. The main effect of time and weather was significant (*p* < 0.05); there was a significant interaction between time and different weather on the level of rhythm gene expression in mackerel tuna (*p* < 0.05).


**Table 4.** Effects of light intensity and duration on the rhythm genes of mackerel tuna under different weather conditions.

Results of the two-way ANOVA with SPSS for the measured factors. When interactions in the analysis are significant (*p* < 0.001), a between-group comparison and an independent samples *t*-test at the same time point are used (the same applies below).

#### *3.2. Changes in the Expression Levels of Lipid Metabolism in Mackerel Tuna*

Under the sunny condition, the expression levels of *SIRT1*, *GST* and *LPL* were not significantly rhythmic in the liver (Table 5). The expression levels of *SIRT1* and *GST* were significantly higher at 18:00 than the other three groups at different times in sunny conditions (*p* < 0.05).



n.s. denotes statistical differences between the different sampling points. Acrophases (circadian peak times) were calculated by a non-linear regression fit of a cosine function. Data are expressed as acrophase ± 95% confidence intervals.

Under the cloudy condition, the expression levels of *GST* and *LPL* were not significantly rhythmic in the liver (Table 5). The expression levels of *GST* and *LPL* were significantly higher (*p* < 0.05) than the other three groups at 18:00 under different times in the cloudy condition (Figure 2b,c; Table 5). The expression levels of *SIRT1* and *SREBP1* were significantly rhythmic in the liver under overcast conditions (Table 5).

The expression levels of metabolic genes at the same time point under different weather on sunny and cloudy days were significantly different in all four groups (*p* < 0.05). No significant differences could be seen between *GST* at 6:00 and 18:00 in the same time point comparison; *GST* expression levels were significantly higher under the cloudy condition than under the sunny condition at 12:00 (*p* < 0.05), and *GST* expression levels were significantly higher under the sunny condition than under the cloudy condition at 24:00 (*p* < 0.05; Figure 2b). The expression levels of *LPL* were significantly higher under sunny light conditions at 6:00 and 24:00 than under the cloudy condition (*p* < 0.05); the expression levels of *LPL* were significantly higher under cloudy light conditions at 12:00 and 18:00 than under cloudy light conditions (*p* < 0.05) (Figure 2c). The expression level of SREBP1 at 12:00 under the cloudy condition was significantly higher than that of the sunny condition (*p* < 0.05); the expression level of *SREBP1* at 18:00 was not significantly different in the comparison at the same time point (Figure 2d).

**Figure 2.** Expression of hepatic metabolic genes during 24 h in different weather in mackerel tuna. (**a**): *SIRT1*; (**b**): *GST*; (**c**): *LPL*; (**d**): *SREBP1*. Red in each graph represents sunny days, and blue represents cloudy days. The presence of different letters indicates significance by ANOVA and Tukey's tests (*p* < 0.05). \* represents significant differences at the same time point (*p* < 0.05).

The results of the two-way analysis of different weather and time of day on metabolic genes in mackerel tuna are shown in Table 6. The main effect of time and weather was significant (*p* < 0.05); time and different weather had a significant interaction effect (*p* < 0.05) on metabolic gene expression levels in mackerel tuna.

**Table 6.** Effects of light intensity and duration on metabolic genes in mackerel tuna under different weather conditions.

