Abstract
Circadian clocks control rhythms in physiology and behavior entrained to 24 h light–dark cycles. Despite of conserved general schemes, molecular circadian clockworks differ between insect species. With RNA interference (RNAi) we examined an ancient circadian clockwork in a basic insect, the hemimetabolous Madeira cockroach Rhyparobia maderae. With injections of double-stranded RNA (dsRNA) of cockroach period (Rm´per), timeless 1 (Rm´tim1), or cryptochrome 2 (Rm´cry2) we searched for essential components of the clock´s core negative feedback loop. Single injections of dsRNA of each clock gene into adult cockroaches successfully and permanently knocked down respective mRNA levels within ~two weeks deleting daytime-dependent mRNA rhythms for Rm´per and Rm´cry2. Rm´perRNAi or Rm´cry2RNAi affected total mRNA levels of both genes, while Rm´tim1 transcription was independent of both, also keeping rhythmic expression. Unexpectedly, circadian locomotor activity of most cockroaches remained rhythmic for each clock gene knockdown employed. It expressed weakened rhythms and unchanged periods for Rm´perRNAi and shorter periods for Rm´tim1RNAi and Rm´cry2RNAi.As a hypothesis of the cockroach´s molecular clockwork, a basic network of switched differential equations was developed to model the oscillatory behavior of clock cells expressing respective clock genes. Data were consistent with two synchronized main groups of coupled oscillator cells, a leading (morning) oscillator, or a lagging (evening) oscillator that couple via mutual inhibition. The morning oscillators express shorter, the evening oscillators longer endogenous periods based on core feedback loops with either PER, TIM1, or CRY2/PER complexes as dominant negative feedback of the clockwork. We hypothesize that dominant morning oscillator cells with shorter periods express PER, but not CRY2, or TIM1 as suppressor of clock gene expression, while two groups of evening oscillator cells with longer periods either comprise TIM1 or CRY2/PER suppressing complexes. Modelling suggests that there is an additional negative feedback next to Rm´PER in cockroach morning oscillator cells.
Highlights
The molecular circadian clockwork [1] that controls rest-activity rhythms in insects is studied best in the fruitfly Drosophila melanogaster [2]
Comparably to crickets we expected that knockdown of the mRNA of Rmper, and Rmcry2, but not of Rmtim1 alone hits essential negative feedback loops of the core clock deleting circadian locomotor activity rhythms [28, 7, 29]
With RNA interference (RNAi)-dependent knockdown of mRNA levels of the circadian clock genes Rmper, Rmtim1,and Rmcry2 of the Madeira cockroach we examined which of these negative feedback loops of the core feedback loop of the circadian clockwork are indispensable for circadian locomotor activity rhythms
Summary
The molecular circadian clockwork [1] that controls rest-activity rhythms in insects is studied best in the fruitfly Drosophila melanogaster [2]. It is built of several interlocked transcriptional/ posttranscriptional feedback loops resulting in circadian oscillations of mRNA- and protein levels. During the middle of the day to the early night, they activate the transcription of the E-box containing clock genes period (Dm’per) and timeless (Dm’tim). They can accumulate in the cytoplasm and move to the nucleus to act as transcriptional inhibitors until Dm’PER protein is degraded phosphorylation- and Dm’CRY1/TIM-dependently. A new cycle of transcription starts during the day [2]
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