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

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.