Dissociation of Circadian and Circatidal Timekeeping in the Marine Crustacean Eurydice pulchra

BACKGROUND Tidal (12.4 hr) cycles of behavior and physiology adapt intertidal organisms to temporally complex coastal environments, yet their underlying mechanism is unknown. However, the very existence of an independent "circatidal" clock has been disputed, and it has been argued that tidal rhythms arise as a submultiple of a circadian clock, operating in dual oscillators whose outputs are held in antiphase i.e., ~12.4 hr apart. RESULTS We demonstrate that the intertidal crustacean Eurydice pulchra (Leach) exhibits robust tidal cycles of swimming in parallel to circadian (24 hr) rhythms in behavioral, physiological and molecular phenotypes. Importantly, ~12.4 hr cycles of swimming are sustained in constant conditions, they can be entrained by suitable stimuli, and they are temperature compensated, thereby meeting the three criteria that define a biological clock. Unexpectedly, tidal rhythms (like circadian rhythms) are sensitive to pharmacological inhibition of Casein kinase 1, suggesting the possibility of shared clock substrates. However, cloning the canonical circadian genes of E. pulchra to provide molecular markers of circadian timing and also reagents to disrupt it by RNAi revealed that environmental and molecular manipulations that confound circadian timing do not affect tidal timing. Thus, competent circadian timing is neither an inevitable nor necessary element of tidal timekeeping. CONCLUSIONS We demonstrate that tidal rhythms are driven by a dedicated circatidal pacemaker that is distinct from the circadian system of E. pulchra, thereby resolving a long-standing debate regarding the nature of the circatidal mechanism.