Crosstalk between the circatidal and circadian clocks mediates behavioral adaptation to tidal patterns.

The ability to anticipate tides is critical for a wide range of marine organisms, but this task is complicated by the diversity of tidal patterns on Earth. Previous findings suggest that organisms whose geographic range spans multiple types of tidal cycles can produce distinct patterns of rhythmic behavior that correspond to the tidal cycles they experience. How this behavioral plasticity is achieved, however, is unclear. Here, we show that Parhyale hawaiensis adapts its rhythmic behavior to various naturally occurring tidal regimens through the plastic contribution of its circatidal and circadian clocks. After entrainment to a tidal cycle that deviated only mildly from a regular 12.4 h tidal cycle, animals exhibited strong circatidal rhythms. By contrast, following entrainment to more irregularly spaced tides or to tides that occurred every 24.8 h, a significant fraction of animals instead synchronized to the light/dark (LD) cycle and exhibited circadian behavior, while others showed rhythmic behavior with both circatidal and circadian traits. We also show that the circatidal clock, while able to entrain to various naturally occurring tidal patterns, does not entrain to an unnatural one. We propose that Parhyale hawaiensis's ecological success around the world relies in part on the plastic interactions between the circatidal and circadian clocks, which shape its rhythmic behavior appropriately according to tidal patterns.

Timely Questions Emerging in Chronobiology: The Circadian Clock Keeps on Ticking.

Chronobiology investigations have revealed much about cellular and physiological clockworks but we are far from having a complete mechanistic understanding of the physiological and ecological implications. Here we present some unresolved questions in circadian biology research as posed by the editorial staff and guest contributors to the Journal of Circadian Rhythms. This collection of ideas is not meant to be comprehensive but does reveal the breadth of our observations on emerging trends in chronobiology and circadian biology. It is amazing what could be achieved with various expected innovations in technologies, techniques, and mathematical tools that are being developed. We fully expect strengthening mechanistic work will be linked to health care and environmental understandings of circadian function. Now that most clock genes are known, linking these to physiological, metabolic, and developmental traits requires investigations from the single molecule to the terrestrial ecological scales. Real answers are expected for these questions over the next decade. Where are the circadian clocks at a cellular level? How are clocks coupled cellularly to generate organism level outcomes? How do communities of circadian organisms rhythmically interact with each other? In what way does the natural genetic variation in populations sculpt community behaviors? How will methods development for circadian research be used in disparate academic and commercial endeavors? These and other questions make it a very exciting time to be working as a chronobiologist.

Clocks at sea: the genome-editing tide is rising.

The coastline is a particularly challenging environment for its inhabitants. Not only do they have to cope with the solar day and the passing of seasons, but they must also deal with tides. In addition, many marine species track the phase of the moon, especially to coordinate reproduction. Marine animals show remarkable behavioral and physiological adaptability, using biological clocks to anticipate specific environmental cycles. Presently, we lack a basic understanding of the molecular mechanisms underlying circatidal and circalunar clocks. Recent advances in genome engineering and the development of genetically tractable marine model organisms are transforming how we study these timekeeping mechanisms and opening a novel era in marine chronobiology.

Behavioral circatidal rhythms require Bmal1 in Parhyale hawaiensis.

Organisms living in the intertidal zone are exposed to a particularly challenging environment. In addition to daily changes in light intensity and seasonal changes in photoperiod and weather patterns, they experience dramatic oscillations in environmental conditions due to the tides. To anticipate tides, and thus optimize their behavior and physiology, animals occupying intertidal ecological niches have acquired circatidal clocks. Although the existence of these clocks has long been known, their underlying molecular components have proven difficult to identify, in large part because of the lack of an intertidal model organism amenable to genetic manipulation. In particular, the relationship between the circatidal and circadian molecular clocks, and the possibility of shared genetic components, has been a long-standing question. Here, we introduce the genetically tractable crustacean Parhyale hawaiensis as a system for the study of circatidal rhythms. First, we show that P. hawaiensis exhibits robust 12.4-h rhythms of locomotion that can be entrained to an artificial tidal regimen and are temperature compensated. Using CRISPR-Cas9 genome editing, we then demonstrate that the core circadian clock gene Bmal1 is required for circatidal rhythms. Our results thus demonstrate that Bmal1 is a molecular link between circatidal and circadian clocks and establish P. hawaiensis as a powerful system to study the molecular mechanisms underlying circatidal rhythms and their entrainment.

Rhythms and Clocks in Marine Organisms.

The regular movements of waves and tides are obvious representations of the oceans' rhythmicity. But the rhythms of marine life span across ecological niches and timescales, including short (in the range of hours) and long (in the range of days and months) periods. These rhythms regulate the physiology and behavior of individuals, as well as their interactions with each other and with the environment. This review highlights examples of rhythmicity in marine animals and algae that represent important groups of marine life across different habitats. The examples cover ecologically highly relevant species and a growing number of laboratory model systems that are used to disentangle key mechanistic principles. The review introduces fundamental concepts of chronobiology, such as the distinction between rhythmic and endogenous oscillator-driven processes. It also addresses the relevance of studying diverse rhythms and oscillators, as well as their interconnection, for making better predictions of how species will respond to environmental perturbations, including climate change. As the review aims to address scientists from the diverse fields of marine biology, ecology, and molecular chronobiology, all of which have their own scientific terms, we provide definitions of key terms throughout the article.

Towards an Understanding of Circatidal Clocks.

Circadian clocks are an intrinsic element of life that orchestrate appropriately timed daily physiological and behavioural rhythms entrained to the solar cycle, thereby conferring increased fitness. However, it is thought that the first archaic 'proto-clocks' evolved in ancient cyanobacteria in a marine environment, where the dominant time cues (zeitgebers) probably would have been lunar-driven and included tidal cycles. To date, non-circadian 'marine clocks' have been described with circatidal (~12.4 h), circasemilunar (~14.8 days), and circalunar (~29.5 days) periodicity, mostly studied in accessible but temporally complex intertidal habitats. In contrast to the well-described circadian clock, their molecular machinery is poorly understood, and fundamental mechanisms remain unclear. We propose that a multi-species approach is the most apposite strategy to resolve the divergence that arose from non-circadian clockwork forged in an evolutionary environment with multiple zeitgebers. We review circatidal clock models with a focus on intertidal organisms, for which robust behavioural, physiological, or genetic underpinnings have been explicated, and discuss their relative experimental merits. Developing a comprehensive mechanistic understanding of circatidal clocks should be a priority because it will ultimately contribute to a more holistic understanding of the origins and evolution of chronobiology itself.

De novo assembly and annotation of the mangrove cricket genome.

OBJECTIVES: The mangrove cricket, Apteronemobius asahinai, shows endogenous activity rhythms that synchronize with the tidal cycle (i.e., a free-running rhythm with a period of ~ 12.4 h [the circatidal rhythm]). Little is known about the molecular mechanisms underlying the circatidal rhythm. We present the draft genome of the mangrove cricket to facilitate future molecular studies of the molecular mechanisms behind this rhythm. DATA DESCRIPTION: The draft genome contains 151,060 scaffolds with a total length of 1.68 Gb (N50: 27 kb) and 92% BUSCO completeness. We obtained 28,831 predicted genes, of which 19,896 (69%) were successfully annotated using at least one of two databases (UniProtKB/SwissProt database and Pfam database).

Circadian and circatidal rhythms of protein abundance in the California mussel (Mytilus californianus).

Coastal habitats fluctuate with the 12.4 h tidal and 24 h light/dark cycle to predictably alter conditions such as air exposure, temperature, and food availability. Intertidal sessile bivalves exhibit behavioural and physiological adjustments to minimize the challenges of this environment. We investigated a high-resolution time course of the changes in protein abundance in the gill tissue of the intertidal mussel Mytilus californianus in a simulated tidal environment of 12:12 h light:dark cycles and a matching 6:6 h high:low tide cycle within each 12 h period. Approximately 38% of detected proteins showed significant rhythms in their abundances, with diversity in the phases of rhythmic isoforms. The circadian rhythm was dominant in protein abundance changes, particularly with oxidative metabolism. A tidal cycle elicited changes within functional groups, including in cytoskeletal proteins, chaperones, and oxidative stress proteins. In addition to protein abundance changes, we found the possibility for post-translational modifications driving rhythms, including methylation, mitochondrial peptide processing (proteolysis), and acylation. Dynamic changes in the proteome across functional categories demonstrate the importance of the tidal environment in entraining cellular processes, confirming that differential expression studies should not assume a static baseline of cellular conditions in intertidal organisms.

Intensive longitudinal characterization of multidimensional biobehavioral dynamics in laboratory rats.

Rats have been used as animal models for human diseases for more than a century, yet a systematic understanding of basal biobehavioral phenotypes of laboratory rats is still missing. In this study, we utilize wireless tracking technology and videography, collect and analyze more than 130 billion data points to fill this gap, and characterize the evolution of behavior and physiology of group-housed male and female rats (n = 114) of the most commonly used strains (Lister Hooded, Long-Evans, Sprague-Dawley, and Wistar) throughout their development. The resulting intensive longitudinal data suggest the existence of strain and sex differences and bi-stable developmental states. Under standard laboratory 12-h light/12-h dark conditions, our study found the presence of multiple oscillations such as circatidal-like rhythms in locomotor activity. The overall findings further suggest that frequent movement along cage walls or thigmotaxic activity may be a physical feature of motion in constrained spaces, critically affecting the interpretation of basal behavior of rats in cages.

Bivalve mollusc circadian clock genes can run at tidal frequency.

Marine coastal habitats are complex cyclic environments as a result of sun and moon interactions. In contrast with the well-known circadian orchestration of the terrestrial animal rhythmicity (approx. 24 h), the mechanism responsible for the circatidal rhythm (approx. 12.4 h) remains largely elusive in marine organisms. We revealed in subtidal field conditions that the oyster Crassostrea gigas exhibits tidal rhythmicity of circadian clock genes and clock-associated genes. A free-running (FR) experiment showed an endogenous circatidal rhythm. In parallel, we showed in the field that oysters' valve behaviour exhibited a strong tidal rhythm combined with a daily rhythm. In the FR experiment, all behavioural rhythms were circatidal, and half of them were also circadian. Our results fuel the debate on endogenous circatidal mechanisms. In contrast with the current hypothesis on the existence of an independent tidal clock, we suggest that a single 'circadian/circatidal' clock in bivalves is sufficient to entrain behavioural patterns at tidal and daily frequencies.

RhythmicAlly: Your R and Shiny-Based Open-Source Ally for the Analysis of Biological Rhythms.

Research on circadian rhythms often requires researchers to estimate period, robustness/power, and phase of the rhythm. These are important to estimate, owing to the fact that they act as readouts of different features of the underlying clock. The commonly used tools, to this end, suffer from being very expensive, having very limited interactivity, being very cumbersome to use, or a combination of these. As a step toward remedying the inaccessibility to users who may not be able to afford them and to ease the analysis of biological time-series data, we have written RhythmicAlly, an open-source program using R and Shiny that has the following advantages: (1) it is free, (2) it allows subjective marking of phases on actograms, (3) it provides high interactivity with graphs, (4) it allows visualization and storing of data for a batch of individuals simultaneously, and (5) it does what other free programs do but with fewer mouse clicks, thereby being more efficient and user-friendly. Moreover, our program can be used for a wide range of ultradian, circadian, and infradian rhythms from a variety of organisms, some examples of which are described here. The first version of RhythmicAlly is available on Github, and we aim to maintain the program with subsequent versions having updated methods of visualizing and analyzing time-series data.

Adaptation of the Hierarchical Factor Segmentation method to noisy activity data.

The Hierarchical Factor Segmentation (HFS) method is a non-parametric statistical method for detection of the phase of a biological rhythm shown in an actogram. The detection accuracy of this method was measured on actograms showing only circadian rhythms with a constant ratio of signal to noise (S/N). In the present study, we generated 84 types of artificial actograms including circadian or circatidal rhythms by using three parameters: α/ρ, S/N and period length τ, and evaluated the effectiveness of our devised adaptation of the HFS method, the cycle-by-cycle adaptation. The results showed the effectiveness of the cycle-by-cycle adaptation was high even though S/N or τ was fluctuating through a whole actogram. These suggested that the cycle-by-cycle adaptation could be effectively applied to various kinds of rhythmic activity data. The C++ source code of the cycle-by-cycle adaptation is available on the website at https://github.com/KazukiSakura/cHFS.git .

Effects of pars intercerebralis removal on circatidal rhythm in the mangrove cricket, Apteronemobius asahinai.

The circatidal rhythm is an endogenous rhythm corresponding to the tidal cycles, and its neural mechanism remains unknown. The mangrove cricket, Apteronemobius asahinai, possesses both circatidal and circadian clocks, and simultaneously exhibits circatidal and circadian rhythms in its locomotor activity. In a previous study, we showed that surgical removal of the optic lobes, the principal circadian clock locus in crickets, disrupted their circadian rhythm, but not their circatidal rhythm. In this study, we focused on the pars intercerebralis (PI) because surgical removal of the PI disrupts the circadian rhythm and causes arrhythmic activity in some cricket species. After surgical removal of the PI, the proportion of crickets displaying circatidal rhythm decreased, and more than half of the crickets exhibited arrhythmic activity. Surgical removal of the regions around the PI also caused a similar effect on locomotor activity. Our results indicate that the PI and/or its surrounding regions are important not only for circadian but also for circatidal rhythm. This suggests the presence of a neural or hormonal pathway in the PI and/or its surrounding regions that is common to the circatidal and circadian rhythms.

Setting the pace: host rhythmic behaviour and gene expression patterns in the facultatively symbiotic cnidarian Aiptasia are determined largely by Symbiodinium.

BACKGROUND: All organisms employ biological clocks to anticipate physical changes in the environment; however, the integration of biological clocks in symbiotic systems has received limited attention. In corals, the interpretation of rhythmic behaviours is complicated by the daily oscillations in tissue oxygen tension resulting from the photosynthetic and respiratory activities of the associated algal endosymbiont Symbiodinium. In order to better understand the integration of biological clocks in cnidarian hosts of Symbiodinium, daily rhythms of behaviour and gene expression were studied in symbiotic and aposymbiotic morphs of the sea-anemone Aiptasia diaphana. RESULTS: The results showed that whereas circatidal (approx. 12-h) cycles of activity and gene expression predominated in aposymbiotic morphs, circadian (approx. 24-h) patterns were the more common in symbiotic morphs, where the expression of a significant number of genes shifted from a 12- to 24-h rhythm. The behavioural experiments on symbiotic A. diaphana displayed diel (24-h) rhythmicity in body and tentacle contraction under the light/dark cycles, whereas aposymbiotic morphs showed approximately 12-h (circatidal) rhythmicity. Reinfection experiments represent an important step in understanding the hierarchy of endogenous clocks in symbiotic associations, where the aposymbiotic Aiptasia morphs returned to a 24-h behavioural rhythm after repopulation with algae. CONCLUSION: Whilst some modification of host metabolism is to be expected, the extent to which the presence of the algae modified host endogenous behavioural and transcriptional rhythms implies that it is the symbionts that influence the pace. Our results clearly demonstrate the importance of the endosymbiotic algae in determining the timing and the duration of the extension and contraction of the body and tentacles and temporal gene expression.

Local tidal regime dictates plasticity of expression of locomotor activity rhythms of American horseshoe crabs, Limulus polyphemus.

While horseshoe crabs Limulus polyphemus from regions with two daily tides express endogenous circatidal (~ 12.4 h) activity rhythms, much less is known about locomotor rhythm expression in horseshoe crabs from other tidal regimes. This study investigated whether horseshoe crabs (1) always express activity rhythms consistent with their natural tides, and (2) can alter activity rhythm expression in response to novel tide cycles. Activity rhythms of animals from environments with two daily tides (Gulf of Maine, 43°6' N/70°52' W, and Massachusetts, 41°32' N/70°40'W), one dominant daily tide (Apalachee Bay, Florida, 29°58' N/84°20' W), and microtides (Indian River Lagoon, Florida, 28°5' N/80°35' W) were recorded in 2011-2013 during three artificial tide conditions: no tides, a 12.4 h tidal cycle, and a 24.8 h tidal cycle. Interestingly, L. polyphemus from the microtidal site (n = 7) appeared "plastic" in their responses; they were able to express both bimodal and unimodal rhythms in response to different tide cycles. In contrast, the other two populations exhibited more fixed responses: regardless of the tides they were exposed to, animals from areas with one dominant daily tide (n = 18) consistently expressed unimodal rhythms, while those from areas with two daily tides (n = 28) generally expressed bimodal rhythms. Rhythms expressed by L. polyphemus thus appear to be a function of endogenous clocks, the tidal cues to which animals are exposed, and tidal cues that animals experience throughout ontogeny.

A Cell-Autonomous Mammalian 12 hr Clock Coordinates Metabolic and Stress Rhythms.

Besides circadian rhythms, oscillations cycling with a 12 hr period exist. However, the prevalence, origin, regulation, and function of mammalian 12 hr rhythms remain elusive. Utilizing an unbiased mathematical approach identifying all superimposed oscillations, we uncovered prevalent 12 hr gene expression and metabolic rhythms in mouse liver, coupled with a physiological 12 hr unfolded protein response oscillation. The mammalian 12 hr rhythm is cell autonomous, driven by a dedicated 12 hr pacemaker distinct from the circadian clock, and can be entrained in vitro by metabolic and ER stress cues. Mechanistically, we identified XBP1s as a transcriptional regulator of the mammalian 12 hr clock. Downregulation of the 12 hr gene expression strongly correlates with human hepatic steatosis and steatohepatitis, implying its importance in maintaining metabolic homeostasis. The mammalian 12 hr rhythm of gene expression also is conserved in nematodes and crustaceans, indicating an ancient origin of the 12 hr clock. Our work sheds new light on how perturbed biological rhythms contribute to human disease.

Circalunidian clocks control tidal rhythms of locomotion in the American horseshoe crab, Limulus polyphemus.

While many intertidal animals exhibit circatidal rhythms, the nature of the underlying endogenous clocks that control these rhythms has been controversial. In this study American horseshoe crabs, Limulus polyphemus, were used to test the circalunidian hypothesis by exposing them to four different tidal regimes. Overall, the results obtained support the circalunidian hypothesis: each of the twice-daily rhythms of activity appears to be controlled by a separate clock, each with an endogenous period of approximately 24.8h. First, spontaneous "skipping" of one of the daily bouts was observed under several different conditions. Second, the presence of two bouts of activity/day, with different periods, was observed. Lastly, we were able to separately synchronize bouts of activity to two artificial tidal regimes with different periods. These results, taken together, argue in favor of two separate circalunidian clocks in Limulus, each of which controls one of the two bouts of their daily tidal activity rhythms.

Identification of putative circadian clock genes in the American horseshoe crab, Limulus polyphemus.

While the American horseshoe crab, Limulus polyphemus, has robust circadian and circatidal rhythms, virtually nothing is known about the molecular basis of these rhythms in this species or any other chelicerate. In this study, next generation sequencing was used to assemble transcriptomic reads and then putative homologs of known core and accessory circadian genes were identified in these databases. Homologous transcripts were discovered for one circadian clock input gene, five core genes, 22 accessory genes, and two possible output pathways. Alignments and functional domain analyses showed generally high conservation between the putative L. polyphemus clock genes and homologs from Drosophila melanogaster and Daphnia pulex. The presence of both cry1 and cry2 in the L. polyphemus transcriptome would classify its system as an "ancestral", type 2 clock system. In addition, a novel duplication of CYCLE, and a novel triplication of PERIOD were found. Investigations are currently underway to determine if any of these "circadian" genes also participate in the molecular processes that drive the Limulus circatidal clock.

Common features in diverse insect clocks.

This review describes common features among diverse biological clocks in insects, including circadian, circatidal, circalunar/circasemilunar, and circannual clocks. These clocks control various behaviors, physiological functions, and developmental events, enabling adaptation to periodic environmental changes. Circadian clocks also function in time-compensation for celestial navigation and in the measurement of day or night length for photoperiodism. Phase response curves for such clocks reported thus far exhibit close similarities; specifically, the circannual clock in Anthrenus verbasci shows striking similarity to circadian clocks in its phase response. It is suggested that diverse biological clocks share physiological properties in their phase responses irrespective of period length. Molecular and physiological mechanisms are best understood for the optic-lobe and mid-brain circadian clocks, although there is no direct evidence that these clocks are involved in rhythmic phenomena other than circadian rhythms in daily events. Circadian clocks have also been localized in peripheral tissues, and research on their role in various rhythmic phenomena has been started. Although clock genes have been identified as controllers of circadian rhythms in daily events, some of these genes have also been shown to be involved in photoperiodism and possibly in time-compensated celestial navigation. In contrast, there is no experimental evidence indicating that any known clock gene is involved in biological clocks other than circadian clocks.

Lunisolar tidal force and its relationship to chlorophyll fluorescence in Arabidopsis thaliana.

The yield of chlorophyll fluorescence Ft was measured in leaves of Arabidopsis thaliana over periods of several days under conditions of continuous illumination (LL) without the application of saturating light pulses. After linearization of the time series of the chlorophyll fluorescence yield (ΔFt), oscillations became apparent with periodicities in the circatidal range. Alignments of these linearized time series ΔFt with the lunisolar tidal acceleration revealed high degrees of synchrony and phase congruence. Similar congruence with the lunisolar tide was obtained with the linearized quantum yield of PSII (ΔФII), recorded after application of saturating light pulses. These findings strongly suggest that there is an exogenous timekeeper which is a stimulus for the oscillations detected in both the linearized yield of chlorophyll fluorescence (ΔFt) and the linearized quantum yield of PSII (ΔФII).