Zolmon, H. | Search bioRxiv

bioRxiv2026

Characterizing rhythmic wheel-turning behavioral patterns in cockroaches Rhyparobia maderae using machine learning

Organisms must adapt to environmental changes occurring across multiple time scales, with endogenous multiscale clocks coordinating physiology and behavior with recurring environmental rhythms, including the dominant 24-hour cycle and faster ultradian rhythms. The Madeira cockroach (Rhyparobia maderae) provides a suitable model for investigating such multiscale temporal organization. Here, locomotor activity was recorded in running-wheel experiments under constant darkness. While the endogenous circadian clock produces a clearly visible 24-hour rhythm, it remains unknown whether locomotor behavior also exhibits temporal patterns at additional time scales. These temporal patterns cannot be found by classical frequency analysis, as they are veiled by higher harmonics of the circadian rhythm which are in the same frequency range. Unsupervised machine learning methods such as K-Means clustering, self-organizing maps and Gaussian mixture are used in search for fast ultradian rhythms possibly linked to circadian cycles in locomotor activity. Prior to applying these methods, data metrics are defined which characterize bouts of activity (called activity impulses) compared to periods of reduced activity. A stochastic pattern was found in these activity metrics which characterizes the time distance between activity impulses. Across all approaches, a consistent ultradian rhythm of approximately one hour was identified in the timing of the activity maxima. This rhythm was mainly detected during the subjective night, suggesting circadian control, and appears to consist of two components with periods of approximately 40 minutes and 1.5 hours. The method proposed in this paper is applied to two cockroach groups with different levels of activity, and is generalizable to diverse datasets occurring in the form of a time series with a dominant rhythm.

animal behavior and cognition↗

bioRxiv2026

Disrupting the clock of the Madeira cockroach through RNAi-mediated knockdown of CLOCK and CYCLE

1Endogenous 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, TIMELESS1, and CRYPTOCHROME2, 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 indicates the presence of compensatory elements, likely through posttranslational feedback loop (PTFL) controlled modifications. To explore alternative mechanisms, we constructed a computational model of a neuronal circadian pacemaker network using a network of coupled limit cycle oscillators, specifically planar switching affine systems (PSAS). The PSAS model comprises plasma membrane-associated 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. 3 Significance statementBased 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 first 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.

physiology↗