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The aim of the present study was to understand the rhythmic changes in innate immune response in freshwater fish Channa punctatus. Furthermore, the putative role of melatonin as the zeitgeber was explored. The phagocytic activity of splenic phagocytes assessed at 6-h intervals showed higher phagocytic activity during light phase than dark phase. The increased phagocytic activity during light phase was diminished by melatonin administration at 09:00 h. Implication of melatonin in control of diurnal variation in phagocytic activity was substantiated by administering irreversible tryptophan hydroxylase inhibitor, para-chlorophenylalanine (pCPA) at 18:00 h. pCPA abrogated the decrease of phagocytosis observed during dark phase, and the same was restored after melatonin administration. The direct involvement of melatonin in modulation of phagocytosis was demonstrated following in vitro experiments. Melatonin suppressed the phagocytic activity in a concentration-dependent manner without affecting the viability of phagocytes. The existence of functional membrane-bound melatonin receptors on fish phagocytes was pharmacologically demonstrated. Luzindole, melatonin membrane receptor antagonist, completely blocked the inhibitory effect of melatonin on phagocytosis. Further receptor-coupled adenylate cyclase-protein kinase A (PKA) pathway was implicated in transducing the melatonin effect as both adenylate cyclase and PKA inhibitor completely nullified the melatonininduced suppression. An increased intracellular cAMP level in response to melatonin ascertained the second messenger status of cAMP for downstream signaling. However, manipulation of phospholipase C/PKC failed to influence the effect of melatonin on phagocytic activity. These observations in C. punctatus evidenced the diurnal rhythmicity in phagocytic activity that is regulated by melatonin following membrane-bound receptorcoupled cAMP-PKA pathway.

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