Gb'Clock Is Expressed in the Optic Lobe and Is Required for the Circadian Clock in the Cricket Gryllus bimaculatus

Reverse genetic studies have revealed that common clock genes, such as period (per), timeless (tim), cycle (cyc), and Clock (Clk), are involved in the circadian clock mechanism among a wide variety of insects. However, to what degree the molecular oscillatory mechanism is conserved is still to be elucidated. In this study, cDNA of the clock gene Clk was cloned in the cricket Gryllus bimaculatus, and its function was analyzed using RNA interference (RNAi). In adult optic lobes, the Clk mRNA level showed no significant rhythmic changes both under light-dark cycle (LD) and constant darkness (DD). A single injection of Clk double-stranded RNA (dsRNA) resulted in a knockdown of the mRNA level to about 25% of the peak level of control animals. The injected crickets lost their locomotor rhythms in DD. The arrhythmicity in locomotor activity persisted for up to 50 days after the Clk dsRNA injection. Control animals injected with DsRed2 dsRNA showed a clear locomotor rhythm like intact animals. Injection of Clk dsRNA not only suppressed the mRNA levels of both per and tim but also abolished their rhythmic expression. per RNAi down-regulates the Clk mRNA levels, suggesting that per is required for sufficient expression of Clk. These results suggest that Clk is an essential component and plays an important role in the cricket's circadian clock machinery like in Drosophila, but regulation of its expression is probably different from regulation in Drosophila.