*4.3. Immune Gene Expression Patterns*

The TRIM family of proteins belongs to the RING family of E3 ubiquitin ligases. *NF-kB* is an important nuclear transcription factor involved in regulating the body's immune response, and abnormal regulation can lead to immune diseases, metabolic diseases, etc. The TRIM family of proteins is involved in the regulation of the NF-kB signaling pathway as E3 ubiquitin ligases [35–37]. Interferon (IFN) receptor proteins are a class of cytokines secreted by host cells that regulate the immune response. When a pathogen is present, interferon is usually released by the host cell, which is sensed by surrounding undisturbed cells and activates appropriate cellular defense mechanisms to eliminate the pathogen [38]. The composition, signaling pathways, function and evolutionary relationships of the *IFN* were extensively studied in fish many years ago [39–41]. It has been shown that *TRIM35* is a positive regulatory molecule in the natural immune signaling pathway [42]. In this study, *TRIM35* did not show daily rhythmicity under sunny conditions, but there were significant differences between groups. The expression level of *TRIM35* was up-regulated by sunny weather conditions. *NF-kB1* showed significant daily rhythmicity in both sunny and cloudy conditions. However, under sunny conditions, the expression level of NF-kB1 showed an increasing trend and started to decrease by 24:00. *IFNA3* showed daily rhythmicity only in sunny conditions. It has been shown that the expression of EcTRIM21 significantly increased the IFN promoter activity and simultaneously increased the transcriptional levels of interferon-related molecules, with a positive regulatory effect [43]. This is consistent with the results of the present experimental study.

*MHC-I* (major histocompatibility complex) plays an important role in adaptive immunity in vertebrates, primarily recognizing intracellular antigens and triggering adaptive immunity. This gene is currently expressed in different fish species, such as the Japanese flounder (*Paralichthys olivaceus*) [44], turbot (*Scophthalmus maximus*) [45] and rainbow trout (*Oncorhynchus mykiss*) [46]. Interferons increase the expression levels of *MHC-I* class molecules on the cell surface, and an increase in *MHC-I* molecules on the surface of virus-infected cells contributes to the delivery of antigens to T cells, causing lysis of target cells. It was found that the MHC-I protein is extremely important in immune recognition in zebrafish by positively regulating IFN immunity and inflammatory responses [47]. In the present study, both *MHC-I* and *IFNA3* were expressed in daily rhythms under sunny conditions and were positively regulated. This is consistent with the present study results.

GCL (glutamate-cysteine ligase) is composed of different gene-edited *GCLC* (glutamate cysteine ligase catalytic subunit) and *GCLM* (glutamate cysteine ligase), with *GCLC* playing all catalytic roles and being subject to feedback inhibition by *GSH* and *GCLM* having regulatory functions [48]. It has been shown that altered single nucleotide polymorphisms in the *GCLC* and *GCLM* genes can regulate gene expression processes and thus participate in various disease processes [49]. In the present study, both *GCLM* and *GCLC* were rhythmic under overcast conditions, and expression levels were simultaneously downregulated at 12:00. It has been shown that the expression of *NF-kB1* and *GCLC* are mutually regulated [50]. In the present study, *NF-kB1* expression levels were down-regulated

when *GCLC* expression levels were up-regulated, suggesting that the down-regulation of *GCLC* expression may be related to the inhibition of the signaling pathway.
