Disrupting the cockroach circadian clock through RNAi-mediated knockdown of clock proteins

1 Abstract Endogenous circadian clocks control circadian rhythms in physiology and behavior. The predominant hypothesis of biological timing suggests that the responsible master clock for all endogenous circadian rhythms is constituted by an evolutionary conserved transcriptional-translational feedback loop (TTFL) clock consisting of positive feedforward and negative feedback elements. Unexpectedly, in contrast to the evolutionary derived insect Drosophila, RNAi-dependent knockdown of any of the negative feedback elements of the core TTFL clock in the basal Madeira cockroach Rhyparobia maderae does not delete circadian rhythms in locomotor activity. Shown here, neither RNAi-dependent triple knockdowns of all three negative feedback elements Period, Timeless 1, and Cryptochrome 2, nor single and double knockdown of the positive elements Clock and Cycle did directly delete circadian locomotor rhythms as mRNA levels declined. Thus, our experimental data do not support the predominant hierarchical hypothesis of circadian timing. To explore alternative mechanisms, we constructed a computational model of a neuronal circadian pacemaker network using planar switching affine systems (PSAS). The PSAS model comprises plasma membrane-associated posttranslational feedback loop (PTFL) clocks that are coupled to the TTFL nuclear clocks. Modeling results aligned with our experimental results. Therefore, both our experimental and modeling data support a systemic hypothesis of biological timing. Significance statement Based mostly upon genetic studies in derived taxa like Drosophila it is hypothesized that circadian timing of behavior is strictly controlled by specific circadian clock neurons in the brain, realized through a transcriptional-translational feedback loop (TTFL) clock. In contrast to this common hierarchical model that requires transcription, we provide evidence in a basal taxon - the Madeira cockroach - for a systemic explanation of circadian timing of behavior that is based on coupled TTFL and posttranslational feedback loop (PTFL) clocks in adaptive neuronal networks. Competing Interest Statement The authors have declared no competing interest. Footnotes - Changing title - Fixing typos - Fixing yellow shading in Fig. 7 - removing references to the mathematical model of a hierarchical network organization