Bidirectional Interaction Between Clock Genes and Reactive Oxygen Species Production in Carp and Zebrafish...

Our study investigated the bidirectional interaction between circadian clock genes and reactive oxygen species (ROS) production in fish monocytes/macrophages (Mo/Mf). Macrophages are key leukocytes whose antimicrobial activity relies on ROS generation. As ROS levels fluctuate across the day and clock genes regulate numerous cellular processes, we examined whether macrophage clock gene activity and ROS production are functionally interconnected. We found that carp Mo/Mf displayed constitutive expression of core clock genes and genes involved in melatonin synthesis and redox balance: Clock genes (per2, clock, cry2, and bmal2) showed rhythmic expression under both light:dark and constant dark conditions and ROS production varied across the day. Ex vivo cell stimulation revealed that immune signals, cortisol, and ROS‑modulating agents altered clock gene expression. Direct ROS donors upregulated multiple clock genes, while inhibition of NADPH oxidase reduced ROS production and changed expression of clock genes. Finally, zebrafish mutants lacking cry1 exhibited changes in ROS production, demonstrating that disruption of the circadian clock affects redox activity. Together, these findings show that carp and zebrafish Mo/Mf possess a functional circadian system and that the ROS and clock pathways influence each other. This interplay may shape daily patterns of immune responsiveness and contribute to time‑dependent variation in host defense.