Expression dynamics of immune-related genes in circadian regulator Bmal1-deficient medaka (Oryzias latipes)

The circadian clock regulates diverse physiological and immune processes through transcriptional feedback loops, with Bmal1 (Brain and Muscle Arnt-like protein 1) as a core component. Although well studied in mammals, its immunological role in teleosts remains unclear. In this study, we examined the effects of Bmal1 deficiency on the fish immune system, by generating a Bmal1 knockout (Bmal1 KO) Japanese medaka (Oryzias latipes) using CRISPR-Cas9, introducing a 16-bp insertion in exon 4, which disrupted the bHLH and PAS domains. Unlike wild-type (WT) fish, Bmal1 KO individuals remained active during ZT21, suggesting altered time-of-day-dependent activity patterns. Exploratory RNA-seq analysis of the head kidneys under phosphate buffered saline and lipopolysaccharide stimulation revealed broad transcriptional changes, with 481 and 767 genes showing altered expression patterns in Bmal1 KO fish under control and LPS-stimulated conditions, respectively. Gene Ontology analysis suggested that among the top-ranked functional groups in Bmal1 KO fish, immune-related categories were less prominent whereas reproductive and vitellogenin-associated processes were more prominent, suggesting alteration of functional profiles rather than direct suppression of immune pathways. Infection assays at ZT2 demonstrated induction of ifng, tnfa, tlr1, tlr5m, tlr9, and tlr21 after bacterial challenge in WT medaka. In contrast, Bmal1 KO fish exhibited an altered response pattern -elevated expression in controls but reduced inducibility after infection. Bmal1 may thus contribute to time-of-day-dependent coordination of circadian and immune signaling, including potential involvement of NF-κB-related pathways. Loss of Bmal1 alters immune gene expression patterns and pathogen responsiveness in a time-of-day-dependent manner. Overall, Bmal1 deficiency was associated with altered clock-gene expression patterns and immune responses at specific Zeitgeber times. Therefore, Bmal1 may contribute to the time-of-day-dependent regulation of innate immune gene expression in medaka; however, circadian rhythmicity of immune responses remains to be directly demonstrated.