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RNA Interference of the Clock Gene period Disrupts Circadian Rhythms in the Cricket Gryllus bimaculatus

Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan

Tomoaki Sakamoto

Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan

Svetlana G. Karpova

Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan

Akira Matsumoto

Center for Research and Advancement in Higher Education, Kyushu University, Fukuoka, Japan

Sumihare Noji

Department of Life Systems, Institute of Technology and Science, University of Tokushima, Tokushima City, Japan

Kenji Tomioka

Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan, tomioka{at}cc.okayama-u.ac.jp

Periodic expression of so-called clock genes is an essential part of the circadian clock. In Drosophila melanogaster the cyclic expression of per and tim through an autoregulatory feedback loop is believed to play a central role in circadian rhythm generation. However, it is still elusive whether this hypothesis is applicable to other insect species. Here it is shown that per gene plays a key role in the rhythm generation in the cricket Gryllus bimaculatus. Measurement of per mRNA levels in the optic lobe revealed the rhythmic expression of per in light cycles with a peak in the late day to early night, persisting in constant darkness. A single injection of per double-stranded RNA (dsRNA) into the abdomen of the final instar nymphs effectively knocked down the mRNA levels as adult to about 50% of control animals. Most of the per dsRNA-injected crickets completely lost the circadian locomotor activity rhythm in constant darkness up to 50 days after the injection, whereas those injected with DsRed2 dsRNA as a negative control clearly maintained it. The electrical activity of optic lobe efferents also became arrhythmic in the per dsRNA-injected crickets. These results not only suggest that per plays an important role in the circadian rhythm generation also in the cricket but also show that RNA interference is a powerful tool to dissect the molecular machinery of the cricket circadian clock.

Key Words: circadian rhythm • clock genes • cricket • locomotor activity • period • RNAi

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