Monika Stengl

The mating behavior of nocturnal Manduca sexta hawkmoths is under strict temporal control. It is orchestrated via circadian and ultradian oscillations in sex-pheromone stimuli as social Zeitgeber. The extremely sensitive pheromone-detecting olfactory receptor neurons (ORNs) that innervate the long trichoid sensilla on the male’s antennae are peripheral circadian clocks. They express the transcriptional-translational feedback loop (TTFL) circadian clockwork, best characterized in Drosophila melanogaster. In hawkmoths, it is still unknown whether or how the ORN TTFL clockwork regulates the daily rhythms in pheromone sensitivity and in temporal resolution of ultradian pheromone pulses as prerequisites to the temporal regulation of hawkmoth mating behavior. We hypothesize that, rather than the slow TTFL clock, a more rapidly adaptive post-translational feedback loop (PTFL) clockwork, assembled in a signalosome in the ORN plasma membrane, allows for temporal control of pheromone detection via generation of multiscale endogenous membrane potential oscillations. The potential oscillations of the PTFL clock could rapidly synchronize to oscillations of pheromone stimuli at different time scales, thus enabling the prediction of stimulus patterns as a mechanism for active sensing. With in vivo long-term tip recordings of long trichoid sensilla of male hawkmoths, we analyzed the spontaneous spiking activity indicative of the ORNs’ endogenous membrane potential oscillations. Consistent with our hypothesis of a multiscale PTFL clock in hawkmoth ORNs, spontaneous spiking was modulated on ultradian and circadian time scales, with maximum activity at night. When we blocked the evolutionarily conserved olfactory receptor coreceptor (Orco), the circadian modulation was abolished but the ultradian frequency modulation of the spontaneous activity remained. Consistent with PTFL control, Orco was not under the transcriptional control of the TTFL clock, but its modulation of spontaneous spiking activity was dependent on cAMP. We could replicate the experimental data in a conductance-based computational model of an ORN. In this model, Orco conductance changed as a function of fluctuating 2nd messenger levels. This study demonstrates that a PTFL clock is sufficient to impose a circadian pattern on ORN sensitivity. Future experiments are necessary to further challenge our novel hypothesis.

Circadian rhythms, originating in endogenous cellular clockworks, are the evolutionary solution that allows organisms to anticipate and synchronize their internal processes with the predictable changes in their environment on a daily basis. Across evolution, circadian oscillators share a conserved design based on delayed negative feedbacks, yet differ in their molecular implementation in prokaryotes and eukaryotes. Although eukaryotic circadian timing is conventionally assumed to originate from nuclear transcription-translation feedback loop (TTFL) clockworks, mounting evidence indicates that daily rhythms can also persist in cells devoid of such nuclear clocks, including the circadian variation of metabolism and redox state in red blood cells. Here, we demonstrate the generation of endogenous circadian oscillations through a membrane-associated post-translational feedback loop (PTFL) mechanism that operates independently of the nuclear TTFL clock and relies on the dynamic regulation of ion channel gating. This mechanism accounts for the circadian oscillations observed in potassium transport, redox state, and metabolism in anucleate red blood cells. The same membrane-associated mechanism may operate in nucleated cells as well. In circadian clock neurons, it could clarify rhythmic changes in ion channel conductances that cannot be attributed to the nuclear TTFL oscillator. Oscillations in ion fluxes directly modulate neuronal excitability, potentially giving rise to spontaneous circadian firing rhythms.

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.

The mating behavior of nocturnal Manduca sexta hawkmoths is under strict temporal control. It is orchestrated via circadian and ultradian oscillations in sex-pheromone stimuli as social Zeitgeber. The extremely sensitive pheromone-detecting olfactory receptor neurons (ORNs) that innervate the long trichoid sensilla on the male’s antennae are peripheral circadian clocks. They express the transcriptional-translational feedback loop (TTFL) circadian clockwork, best characterized in Drosophila melanogaster. In hawkmoths, it is still unknown whether or how the ORN TTFL clockwork regulates the daily rhythms in pheromone sensitivity and in temporal resolution of ultradian pheromone pulses as prerequisites to the temporal regulation of hawkmoth mating behavior. We hypothesize that, rather than the slow TTFL clock, a more rapidly adaptive post-translational feedback loop (PTFL) clockwork, assembled in a signalosome in the ORN plasma membrane, allows for temporal control of pheromone detection via generation of multiscale endogenous membrane potential oscillations. The potential oscillations of the PTFL clock could rapidly synchronize to oscillations of pheromone stimuli at different time scales, thus enabling the prediction of stimulus patterns as a mechanism for active sensing. With in vivo long-term tip recordings of long trichoid sensilla of male hawkmoths, we analyzed the spontaneous spiking activity indicative of the ORNs’ endogenous membrane potential oscillations. Consistent with our hypothesis of a multiscale PTFL clock in hawkmoth ORNs, spontaneous spiking was modulated on ultradian and circadian time scales, with maximum activity at night. When we blocked the evolutionarily conserved olfactory receptor coreceptor (Orco), the circadian modulation was abolished but the ultradian frequency modulation of the spontaneous activity remained. Consistent with PTFL control, Orco was not under the transcriptional control of the TTFL clock, but its modulation of spontaneous spiking activity was dependent on cAMP. We could replicate the experimental data in a conductance-based computational model of an ORN. In this model, Orco conductance changed as a function of fluctuating 2nd messenger levels. This study demonstrates that a PTFL clock is sufficient to impose a circadian pattern on ORN sensitivity.

1 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. 3 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.

The circadian clock of Rhyparobia maderae is the accessory medulla (AME) in the optic lobes of the brain. Controlled by compound ocular photoreceptors, the cockroach clock orchestrates circadian rhythms in physiology and behavior synchronized with environmental light-dark cycles. Approximately 240 neurons, rich in partially colocalized neuropeptides, innervate the AME. Since the identity and function of most of these neuropeptides are unknown in the cockroach, the transcriptome of the Madeira cockroach was prepared, revealing 68 neuropeptides, neuropeptide-like and protein hormone genes. To identify the neuropeptidome, a combination of analyses of brain/retrocerebral complex extracts by Q-Exactive Orbitrap mass spectrometry (MS) and direct tissue profiling of different parts of the nervous system by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS was used. The resulting data set revealed 192 mature neuropeptides, with 155 identified for the first time and 57 precursor peptides. The AME neuropeptidomic profile revealed 166 mature neuropeptides with potential bioactive circadian clock functions. The precision of our MS data was validated by immunostaining, as illustrated by an example of immunoreactive patterns for proctolin in AME neurons. This peptidergic data set serves as the foundation for future research on neuropeptide-based functional studies that regulate the timing of disparate neuronal circuits.

1 The mating behavior of nocturnal Manduca sexta hawkmoths is under strict temporal control. It is orchestrated via circadian and ultradian oscillations in sex-pheromone stimuli as social Zeitgeber. The extremely sensitive pheromone-detecting olfactory receptor neurons (ORNs) that innervate the long trichoid sensilla on the male’s antennae are peripheral circadian clocks. They express the transcriptional-translational feedback loop (TTFL) circadian clockwork, best characterized in Drosophila melanogaster . In hawkmoths, it is still unknown whether or how the ORN TTFL clockwork regulates the daily rhythms in pheromone sensitivity and in temporal resolution of ultradian pheromone pulses as prerequisites to the temporal regulation of hawkmoth mating behavior. We hypothesize that, rather than the slow TTFL clock, a more rapidly adaptive post-translational feedback loop (PTFL) clockwork, assembled in a signalosome in the ORN plasma membrane, allows for temporal control of pheromone detection via generation of multiscale endogenous membrane potential oscillations. The potential oscillations of the PTFL clock could rapidly synchronize to oscillations of pheromone stimuli at different time scales, thus enabling the prediction of stimulus patterns as a mechanism for active sensing. With in vivo long-term tip recordings of long trichoid sensilla of male hawkmoths, we analyzed the spontaneous spiking activity indicative of the ORNs’ endogenous membrane potential oscillations. Consistent with our hypothesis of a multiscale PTFL clock in hawkmoth ORNs, spontaneous spiking was modulated on ultradian and circadian time scales, with maximum activity at night. When we blocked the evolutionarily conserved olfactory receptor coreceptor (Orco), the circadian modulation was abolished but the ultradian frequency modulation of the spontaneous activity remained. Consistent with PTFL control, Orco was not under the transcriptional control of the TTFL clock, but its modulation of spontaneous spiking activity was dependent on cAMP. We could replicate the experimental data in a conductance-based computational model of an ORN. In this model, Orco conductance changed as a function of fluctuating 2 ^nd messenger levels. This study demonstrates that a PTFL clock is sufficient to impose a circadian pattern on ORN sensitivity. 2 Significance statement: It is generally assumed that all circadian rhythms in an organism are driven by a transcriptional-translational feedback loop (TTFL) clock. In this study, we demonstrate with in vivo recordings of hawkmoth pheromone-sensitive olfactory receptor neurons (ORNs) that the olfactory receptor coreceptor (Orco) is the key pacemaker channel for controlling circadian, but not ultradian, rhythms in spontaneous spiking activity. Since Orco expression is not driven by the TTFL clock, its conductance appears to be controlled by a post-translational feedback loop (PTFL) membrane clock assembled in a signalosome controlling 2 ^nd messengers. Accordingly, our computational model suggests that ORN sensitivity is tuned by periodic changes in the conductance of an Orco ion channel, which is mediated by cycling levels of cyclic nucleotides. This highlights the role of the contribution of posttranslational modifications to the generation of circadian rhythmicity.

Pigment-dispersing factors (PDFs) are neuropeptides that play key roles in controlling the circadian rhythms in various insects, whereas their function remains elusive in other protostomes including tardigrades (water bears). Here we show that the three PDFs of the tardigrade Hypsibius exemplaris are co-localized in two pairs of inner lobe cells in the brain, whereas only one PDF occurs in four additional cerebral and two extracerebral cells. The axons of the inner lobe cells pass through the contralateral brain hemisphere, descend to the ventral nerve cord and terminate in two pairs of potential release sites in the posteriormost trunk ganglion. Using in vitro assays, we demonstrate that all three PDFs and their deorphanized receptor (PDFR) are functional. Widespread localization of PDFR suggests that tardigrade PDFs may act as multifunctional hormones and neuromodulators that control major functions including light detection, neural processing, locomotion, feeding, digestion, osmoregulation, growth, embryonic development and oogenesis/reproduction.

Evolutionary pressures adapted insect chemosensation to their respective physiological needs and tasks in their ecological niches. Solitary nocturnal moths rely on their acute olfactory sense to find mates at night. Pheromones are detected with maximized sensitivity and high temporal resolution through mechanisms that are mostly unknown. While the inverse topology of insect olfactory receptors and heteromerization with the olfactory receptor coreceptor suggest ionotropic transduction via odorant-gated receptor–ion channel complexes, contradictory data propose amplifying G-protein–coupled transduction. Here, we used in vivo tip-recordings of pheromone-sensitive sensilla of maleManduca sextahawkmoths at specific times of day (rest vs activity). Since the olfactory receptor neurons distinguish signal parameters in three consecutive temporal windows of their pheromone response (phasic; tonic; late, long-lasting), respective response parameters were analyzed separately. Disruption of G-protein–coupled transduction and block of phospholipase C decreased and slowed the phasic response component during the activity phase of hawkmoths without affecting any other component of the response during activity and rest. A more targeted disruption of G_αsubunits by blocking G_αoor sustained activation of G_αsusing bacterial toxins affected the phasic pheromone response, while toxins targeting G_αqand G_α12/13were ineffective. Consistent with these data, the expression of phospholipase Cβ4 depended on zeitgeber time, which indicates circadian clock-modulated metabotropic pheromone transduction cascades that maximize sensitivity and temporal resolution of pheromone transduction during the hawkmoth's activity phase. Thus, discrepancies in the literature on insect olfaction may be resolved by considering circadian timing and the distinct odor response components.

1 Evolutionary pressures adapted insect chemosensation to the respective insect’s physiological needs and tasks in their ecological niches. Solitary nocturnal moths rely on their acute olfactory sense to find mates at night. Pheromones are detected with maximized sensitivity and high temporal resolution through mechanisms that are mostly unknown. While the inverse topology of insect olfactory receptors and heteromerization with the coreceptor Orco suggest ionotropic transduction via odorant-gated receptor-ion channel complexes, contradictory data propose amplifying G protein-coupled transduction. Here, we used in vivo tip-recordings of pheromone-sensitive sensilla of male Manduca sexta hawkmoths at specific times of day (rest vs. activity). Since the olfactory receptor neurons distinguish signal parameters in three consecutive temporal windows of their pheromone response (phasic; tonic; late, long-lasting), respective response parameters were analyzed separately. Disruption of G protein-coupled transduction and block of phospholipase C decreased and slowed the phasic response component during the activity phase of hawkmoths without affecting any other component of the response during activity and rest. A more targeted disruption of G _α subunits by blocking G _αo or sustained activation of G _αs using bacterial toxins affected the phasic pheromone response, while toxins targeting G _αq and G _α12/13 were ineffective. Consistent with these data, the expression of phospholipase Cβ4 depended on zeitgeber time, which indicates circadian clock-modulated metabotropic pheromone transduction cascades that maximize sensitivity and temporal resolution of pheromone transduction during the hawkmoth’s activity phase. Thus, discrepancies in the literature on insect olfaction may be resolved by considering circadian timing and the distinct odor response components. 2 Significance statement: Insect chemosensory transduction is typically thought to be ionotropic, but data from different insect species suggests that metabotropic olfactory signaling may occur, either alongside or instead of ionotropic mechanisms. Nocturnal moths, known for their extraordinarily sensitive pheromone-detecting olfactory receptor neurons, likely use metabotropic signal amplification. To overcome limitations of previous in vitro studies, we conducted tip-recordings of pheromone-sensitive sensilla in healthy hawkmoths at specific zeitgeber times. Disrupting G protein signaling and phospholipase Cβ reduced sensitivity and altered response kinetics, revealing strict temporal control of transduction. Thus, contradictory findings in insect olfaction may be reconciled by considering diverse evolutionary pressures for distinct chemosensory signals in different species, zeitgeber time, and disparate odor response parameters.