The role of circadian clock genes in the photoperiodic timer of the linden bug Pyrrhocoris apterus during the nymphal stage

Many insects survive seasonal adversities during diapause, a form of programmed developmental and metabolic arrest. Photoperiodically regulated entry into diapause allows multivoltine insect species to optimize the number of generations. The molecular mechanism of the photoperiodic timer is unknown in insects. In the present study, we take advantage of the robust reproductive diapause response in the linden bug Pyrrhocoris apterus and explore the fifth-instar nymphal stage, which is the most photoperiod-sensitive stage. The nymphs display daily changes in locomotor activity during short days; this differs from the activity observed during long days. We find evidence of cyclical expression of the circadian clock genes, per and cyc, in nymphal heads; in addition, per expression is also photoperiod-dependent. The RNA interference-mediated knockdown of the two circadian clock genes, Clk and cyc, during the nymphal stage results in reproductive arrest in adult females. Furthermore, Clk and cyc knockdown induces the expression of the storage protein hexamerin in the fat body, whereas the expression of vitellogenin diminishes. Taken together, these data support the involvement of circadian clock genes in photoperiodic timer and/or diapause induction.