Protein synthesis is a required process for the circadian clock in the optic lobe of the cricket Gryllus bimaculatus

K. Tomioka

Department of Physics, Biology and Informatics, Faculty of Science, Yamaguchi University, Yamaguchi 753-8512, Japan

Circadian rhythms are now considered to be a common biological property in living systems, including insects. The underlying timing machinery, the so-called circadian clock, drives rhythms of a variety of physiological functions such as eclosion, locomotor activity and hormonal secretion. The optic lobe is the locus of the circadian clock that controls the circadian locomotor rhythm in the cricket, Gryllus bimaculatus. We found that protein synthesis is required for the movement of the clock. To monitor the movement of the clock, we made longterm records of electrical activity of the optic lamina-medulla compound eye complex which was isolated and cultured in vitro in Mitsuhashi and Maramorosh culture medium in a constant darkness at 20 o C. The complex showed a clear circadian electrical activity rhythm peaking during the night with a freerunning period of 22.74+0.31 h. We examined the effect of continuous treatment with various concentrations of cycloheximide (CHX), a translation inhibitor, on the rhythm of optic lobe compound eye complex. When 10 -5 – 10 -4 M CHX was applied, no clear circadian oscillation was observed. Continuous application of CHX also abolished circadian rhythms of the response evoked by light stimulation. As washed with fresh medium after CHX treatment, the rhythm was soon restored and the subsequent phase was clearly correlated to the termination time of the treatment. At 10 -6 M the rhythm was often unstable with the freerunning period being significantly lengthened beyond the circadian range (34.25+1.89 h, n=7). The rhythm again clearly persisted when the concentration was lowered to 10 -7 M, and average freerunning period (22.56+0.56, n=4) was close to that of the control. We then examined the effect of CHX treatment on the phase of the rhythm. When the complex was treated with 10 -5 M CHX for 6 h, the rhythm exhibited a marked phase shift. The magnitude and direction of the phase shift were dependent on the phase at which the complex was treated with CHX; phase advances occurred around the late subjective night, whereas phase delays occurred during the late subjective day to early subjective night. These results suggest that protein synthesis is also involved in the cricket optic lobe circadian clock, and that the clock-related protein synthesis may be activated during the late subjective day to subjective night. Similar time courses of protein synthesis are known for some clock proteins in Drosophila and Anthraea pernyi. The abundance of the proteins increases from the late subjective day to the late subjective night. Taken together, these facts suggest that the requirement of nighttime protein synthesis for the circadian clock is common in insects.

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