*3.3. Changes in the Expression Levels of Immune Genes in Mackerel Tuna*

Expression levels of *TRIM35* under sunny conditions were not significantly rhythmic in the liver (Table 7). The expression level of *TRIM35* at 18:00 was significantly higher (*p* < 0.05) than the other three groups at different times in sunny conditions (Figure 3a; Table 7). Expression levels of *NF-kB1*, *MHC-I*, *ALT*, *IFNA3*, *ISY1*, *ARHGEF13* and *GCLC* were significantly rhythmic in the liver under sunny conditions (Table 7).

**Table 7.** Cosinor analysis board for immune genes expression under sunny and cloudy conditions.



**Table 7.** *Cont.*

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.

**Figure 3.** Expression of liver immune genes in different weather conditions in mackerel tuna over a 24 h period. (**a**): *TRIM35*; (**b**): *NF-kB1*; (**c**): *MHC-I*; (**d**): *ALT*; (**e**): *IFNA3*; (**f**): *ISY1*; (**g**): *ARHGEF13*; (**h**): *GCLM*; (**i**): *GCLC*. 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).

7LPH

Under the cloudy condition, the expression levels of *TRIM35*, *MHC-I* and *IFNA3* were not significantly rhythmic in the liver (Table 7). The expression levels of *TRIM35* at 18:00 were significantly higher than the other three time points at different times in the cloudy condition (*p* < 0.05), the expression levels of *MHC-I* at 24:00 were significantly higher than the other three time points in the cloudy condition (*p* < 0.05), and the expression levels of IFNA3 at 12:00 were significantly higher than the other three time points in the cloudy condition (*p* < 0.05; Figure 2a,b,e; Table 7). The expression levels of *NF-kB1*, *ALT*, *ISY1*, *ARHGEF13*, *GCLM* and *GCLC* were significantly rhythmic in the liver under overcast conditions (Table 7).

The expression levels of *TRIM35* at 6:00 and 12:00 were not significantly different between sunny and cloudy days at the same time point; the expression levels of *TRIM35* at 18:00 and 24:00 were significantly higher (*p* < 0.05) under the sunny condition than the under cloudy condition (Figure 3a). The expression levels of *NF-kB1* at 6:00 were not significantly different in the comparison at the same time point; *NF-kB1* expression levels were significantly higher under the cloudy condition at 12:00 than under the sunny conditions (*p* < 0.05); *NF-kB1* expression levels were significantly higher under the sunny condition at 18:00 and 24:00 than under the cloudy condition (*p* < 0.05) (Figure 3b). The expression levels of *MHC-I* at 6:00 and 12:00 were not significantly different in the same time point comparison; the expression levels of *MHC-I* at 18:00 and 24:00 under the sunny condition were significantly higher than those under the cloudy condition (*p* < 0.05) (Figure 3c). The expression levels of *ALT* at 6:00, 18:00 and 24:00 were significantly higher (*p* < 0.05) under the sunny condition than under the cloudy condition (Figure 3d). The expression levels of *ISY1* at 6:00 and 12:00 were not significantly different in the comparison at the same time point; the expression levels of *ISY1* at 18:00 and 24:00 under the cloudy condition were significantly higher than those under the sunny condition (*p* < 0.05; Figure 3f). The expression levels of *ARHGEF1* were significantly higher (*p* < 0.05) under the sunny condition than under the cloudy condition at 6:00, 18:00 and 24:00; the expression levels of *ARHGEF1* at 12:00 were not significantly different at the same time point comparison (Figure 3g). *GCLM* expression levels were significantly higher at 6:00, 18:00 and 12:00 in the same time point comparison between sunny and cloudy conditions (*p* < 0.05); the expression level of *GCLM* at 12:00 was significantly higher under cloudy conditions than under sunny conditions (*p* < 0.05; Figure 3h). The expression level of *GCLC* was significantly higher at 6:00 than in the same time point comparison between sunny and cloudy conditions (*p* < 0.05); The expression level of *GCLC* at 12:00 was significantly higher under cloudy conditions than under sunny conditions (*p* < 0.05; Figure 3i).

The results of the two-way analysis of different weather and time of day on immune genes in mackerel tuna are shown in Table 8. The main effects of time and weather were significant (*p* < 0.05), and there was a significant interaction between time and different weather on immune gene expression levels in mackerel tuna (*p* < 0.05).

**Table 8.** Effects of light intensity and duration on immune genes in mackerel tuna under different weather conditions.


#### **4. Discussion**

#### *4.1. Rhythmic Gene Expression Patterns*

In nature, in both animals and humans, there is a 24 h circadian rhythm called the biological clock [22]. *CREB1* (cyclic adenosine monophosphate response element binding protein 1) is a protein that regulates gene transcription and can participate in cycle regulation by regulating the expression of downstream target genes [23]. In this study, the daily rhythmicity of *CREB1* was only present under the cloudy condition, and the average *CREB1* gene expression was found to be higher under the sunny condition than under the cloudy condition. The light intensity may stimulate the *CREB1* gene expression in mackerel tuna, but the regulatory mechanism remains to be investigated.

The molecular mechanism of the biological clock is primarily the existence of a cellautonomous transcriptional–translational feedback loop in which a pair of positive regulators (*CLOCK* and *BMAL1*) form a heterodimer that activates its transcription by binding to the negative regulators (*PER* and *CRY* promoters). Subsequently, the *PER* and *CRY* proteins bind to form a complex that enters the nucleus and acts on the *CLOCK* and *BMAL1* heterodimers, thereby feeding back to repress its own transcription [24]. *BMAL1* is thought to regulate the expression of rhythmic genes throughout the liver [25]. In this study, *PER2*, *CRY2* and *BMAL1* showed daily rhythmicity under the sunny condition and *CLOCK*, *PER1*, *PER3* and *BMAL1* showed daily rhythmicity under the cloudy condition. It has been shown that the expression levels of the rhythm genes *PER1*, *PER2*, *CRY1*, *CRY2* and *BMAL1* in the liver of the Atlantic bluefin tuna (*Thunnus thynnus*, L.) exhibit daily rhythmicity [26]. Similar to the results of the present study. However, in this study, PER1 showed daily rhythmicity only under cloudy conditions, while *PER2* and *CRY2* showed daily rhythmicity only under sunny conditions, which may be due to the different changes in light intensity under the cloudy and the sunny conditions, leading to an effect of light stimulation on the circadian system of fish. It has been shown that photoperiod has an effect on core biological clock gene expression in fish [27]. In this study, the expression levels of rhythm genes were at their highest under sunny conditions at 12:00, when light intensity was the strongest of the day, reflecting that light intensity at 12:00 under sunny conditions can cause up-regulation of the expression levels of rhythm genes in mackerel tuna to some extent.

#### *4.2. Metabolic Gene Expression Patterns*

The liver is the main organ of lipid metabolism in fish and plays a huge role in the body as a hub for fat transport, influencing the breakdown and absorption of nutrients and hormonal signaling [28]. Some hepatic metabolic pathways are driven by the circadian biological clock, resulting in a circadian rhythm, and disturbances in the biological clock can cause metabolic disorders in fish. *SIRT1* is involved in a wide range of glucose and lipid metabolism pathways, as well as in the regulation of gene transcription and cellular senescence through the deacetylation of several metabolism-controlling transcription factors [29]. Current research on the *SIRT1* gene focuses on humans, mice, livestock and poultry (pigs, sheep, chickens, etc.) [30]. Little research has been reported on the *SIRT1* gene in fish. It has been found that there is a close correlation between the expression level of the *SIRT1* gene and lipid metabolism in pigs [31]. In this study, there was no daily rhythm in the expression levels of the *SIRT1* gene under sunny conditions, while there was a daily rhythm in the expression levels of *SIRT1* under cloudy conditions. This may result from the involvement of *CLOCK* and *BMAL1* in the regulation. It has been shown that *SIRT1* interacts with *BMAL1*, *PER2* and *CRY1* in the liver of mice [32]. In rainbow trout [33], *SIRT1* interacts with *BMAL1*, *PER2*, *PER3*, *CLOCK* and *BMAL1*, with *CLOCK* and *BMAL1* controlling the rhythm of *SIRT1*, which is in agreement with the results of this study.

*SREBP1* plays a key role in regulating lipid homeostasis, and *REVERBA* is a powerful transcriptional repressor that plays an important role in rhythms. In this study, *SREBP1* showed a clear daily rhythm under sunny conditions, and both *REVERBA* and *SREBP1* had a daily rhythm under cloudy conditions with similar trends, but *REVERBA* peaked at 12:00 and *SREBP1* peaked at 18:00 when the rhythm genes regulate *SREBP1* expression. This is probably due to the delayed expression levels of the *SREBP1* metabolic gene, resulting in a peak at 18:00, but *REVERBA* plays a regulatory role in *SREBP1* gene expression. It has been shown that *REVERBA* can regulate the expression of *SREBP1* in the salmon liver, that *SREBP1* exhibits daily rhythmicity, and that *REVERBA* has a regulatory effect on *SREBP1* [15], which is consistent with the results of this experimental study. In the present study, *SIRT1* and *SREBP1* showed an up-regulation trend both under sunny and cloudy conditions, and the expression level of *SIRT1* continued to increase with time. It has been shown that *LPL* is expressed in the liver of fish and can exhibit changes in lipid metabolism and deposition [34]. In this study, because of the high activity of mackerel tuna during the day under sunny or cloudy conditions, the mackerel tuna was active in the water at this time, prompting an increase in its metabolic levels and making its expression levels up-regulated during the day.
