Circatidal Clock Research Articles

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 12.4h 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.

Plasticity of circadian and circatidal rhythms in activity and transcriptomic dynamics in a freshwater snail

Organisms have diverse biological clocks synchronised with environmental cycles depending on their habitats. Anticipation of tidal changes has driven the evolution of circatidal rhythms in some marine species. In the freshwater snail, Semisulcospira reiniana, individuals in nontidal areas exhibit circadian rhythms, whereas those in tidal areas exhibit both circadian and circatidal rhythms. We investigated whether the circatidal rhythms are genetically determined or induced by environmental cycles. The exposure to a simulated tidal cycle did not change the intensity of circatidal rhythm in individuals in the nontidal population. However, snails in the tidal population showed different activity rhythms depending on the presence or absence of the exposure. Transcriptome analysis revealed that genes with circatidal oscillation increased due to entrainment to the tidal cycle in both populations and dominant rhythmicity was consistent with the environmental cycle. These results suggest plasticity in the endogenous rhythm in the gene expression in both populations. Note that circatidal oscillating genes were more abundant in the tidal population than in the nontidal population, suggesting that a greater number of genes are associated with circatidal clocks in the tidal population compared to the nontidal population. This increase of circatidal clock–controlled genes in the tidal population could be caused by genetic changes in the biological clock or the experience of tidal cycle in the early life stage. Our findings suggest that the plasticity of biological rhythms may have contributed to the adaptation to the tidal environment in S. reiniana.

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.

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.

Possible biological processes controlled by the circatidal clock in the mangrove cricket inferred from transcriptome analysis

ABSTRACT Intertidal dwellers exhibit a circatidal rhythm corresponding to the tidal cycles, but its molecular mechanism remains largely unknown. The mangrove cricket Apteronemobius asahinai simultaneously exhibits circatidal and circadian rhythms in its locomotor activity. In A. asahinai, it is considered that the circatidal rhythm is generated by the circatidal clock, and its molecular mechanism appears to differ from that of the circadian clock. For the molecular dissection, it is necessary to investigate the effects of a candidate gene disruption in several circatidal rhythms. However, the circatidal rhythms other than locomotor activity have not been examined in this species. Here, to identify biological processes related to the circatidal rhythm, we performed whole-transcriptome analysis using RNA sequencing with RNA samples extracted from whole crickets. Under a light-dark cycle without tidal stimuli, more contigs showed a diel expression pattern than a tidal expression pattern. Gene ontology (GO) enrichment analysis revealed that most GO terms overrepresented in contigs related to the tidal cycle were also overrepresented in contigs related to the diel cycle. GO terms specific to tide-related contigs were related to cuticle development, melanization, and sclerotization. These results suggest that physiological events in the cuticle may be controlled by the circatidal clock.

12-h clock regulation of genetic information flow by XBP1s.

Our group recently characterized a cell-autonomous mammalian 12-h clock independent from the circadian clock, but its function and mechanism of regulation remain poorly understood. Here, we show that in mouse liver, transcriptional regulation significantly contributes to the establishment of 12-h rhythms of mRNA expression in a manner dependent on Spliced Form of X-box Binding Protein 1 (XBP1s). Mechanistically, the motif stringency of XBP1s promoter binding sites dictates XBP1s’s ability to drive 12-h rhythms of nascent mRNA transcription at dawn and dusk, which are enriched for basal transcription regulation, mRNA processing and export, ribosome biogenesis, translation initiation, and protein processing/sorting in the Endoplasmic Reticulum (ER)-Golgi in a temporal order consistent with the progressive molecular processing sequence described by the central dogma information flow (CEDIF). We further identified GA-binding proteins (GABPs) as putative novel transcriptional regulators driving 12-h rhythms of gene expression with more diverse phases. These 12-h rhythms of gene expression are cell autonomous and evolutionarily conserved in marine animals possessing a circatidal clock. Our results demonstrate an evolutionarily conserved, intricate network of transcriptional control of the mammalian 12-h clock that mediates diverse biological pathways. We speculate that the 12-h clock is coopted to accommodate elevated gene expression and processing in mammals at the two rush hours, with the particular genes processed at each rush hour regulated by the circadian and/or tissue-specific pathways.

Circatidal gene expression in the mangrove cricket Apteronemobius asahinai

The mangrove cricket Apteronemobius asahinai is endemic to mangrove forest floors. It shows circatidal rhythmicity, with a 12.6-h period of locomotor activity under constant conditions. Its free-running activity also has a circadian component; i.e. it is more active during the subjective night than during the day. In this study, we investigated rhythmic gene expression under constant darkness by RNA sequencing to identify genes controlled by the biological clock. Samples collected every 3 h for 48 h were analysed (one cricket per time-point). We identified 284 significant circatidal cycling transcripts (period length 12–15 h). Almost half of them were annotated with known genes in the NCBI nr database, including enzymes related to metabolic processes and molecular chaperones. There were less transcripts with circadian rhythmicity than with circatidal rhythmicity, and the expression of core circadian clock genes did not show significant rhythmicity. This may reflect the nature of the mangrove cricket or may be due to the paucity of the sampling repeats: only two periods for circadian cycle with no replications. We evaluated for the first time the rhythmic transcriptome of an insect that shows circatidal rhythmic activity; our findings will contribute to future studies of circatidal clock genes.

Unveiling "Musica Universalis" of the Cell: A Brief History of Biological 12-Hour Rhythms.

“Musica universalis” is an ancient philosophical concept claiming the movements of celestial bodies follow mathematical equations and resonate to produce an inaudible harmony of music, and the harmonious sounds that humans make were an approximation of this larger harmony of the universe. Besides music, electromagnetic waves such as light and electric signals also are presented as harmonic resonances. Despite the seemingly universal theme of harmonic resonance in various disciplines, it was not until recently that the same harmonic resonance was discovered also to exist in biological systems. Contrary to traditional belief that a biological system is either at stead-state or cycles with a single frequency, it is now appreciated that most biological systems have no homeostatic “set point,” but rather oscillate as composite rhythms consisting of superimposed oscillations. These oscillations often cycle at different harmonics of the circadian rhythm, and among these, the ~12-hour oscillation is most prevalent. In this review, we focus on these 12-hour oscillations, with special attention to their evolutionary origin, regulation, and functions in mammals, as well as their relationship to the circadian rhythm. We further discuss the potential roles of the 12-hour clock in regulating hepatic steatosis, aging, and the possibility of 12-hour clock–based chronotherapy. Finally, we posit that biological rhythms are also musica universalis: whereas the circadian rhythm is synchronized to the 24-hour light/dark cycle coinciding with the Earth’s rotation, the mammalian 12-hour clock may have evolved from the circatidal clock, which is entrained by the 12-hour tidal cues orchestrated by the moon.

Circadian and circatidal clocks control the mechanism of semilunar foraging behaviour

How animals precisely time behaviour over the lunar cycle is a decades-old mystery. Experiments on diverse species show this behaviour to be endogenous and under clock control but the mechanism has remained elusive. We present new experimental and analytical techniques to test the hypotheses for the semilunar clock and show that the rhythm of foraging behaviour in the intertidal isopod, Scyphax ornatus, can be precisely shifted by manipulating the lengths of the light/dark and tidal cycles. Using light T-cycles (Tcd) the resultant semilunar beat period undergoes shifts from 14.79 days to 6.47 days under T = 23 hours (h), or to 23.29 days under T = 24.3 h. In tidal T-cycles (Tt) of natural length Tt = 12.42 h, the semilunar rhythm is shifted to 24.5 days under Tt = 12.25 h and to 9.7 days under Tt = 12.65 h. The implications of this finding go beyond our model species and illustrate that longer period rhythms can be generated by shorter period clocks. Our novel analysis, in which periodic spline models are embedded within randomization tests, creates a new methodology for assessing long-period rhythms in chronobiology. Applications are far-reaching and extend to other species and rhythms, potentially including the human-ovarian cycle.

Glass eels (Anguilla anguilla) have a magnetic compass linked to the tidal cycle.

The European eel (Anguilla anguilla) has one of the longest migrations in the animal kingdom. It crosses the Atlantic Ocean twice during its life history, migrating between the spawning area in the Sargasso Sea and Europe, where it is widely distributed. The leptocephalus larvae drift with the Gulf Stream and other currents for more than a year and metamorphose into glass eels when they arrive on the continental shelf and move toward coastal areas. The mechanisms underlying glass eel orientation toward the coast and into freshwater systems are poorly known. However, anguillid eels, including the glass eel life stage, have a geomagnetic sense, suggesting the possibility that they use Earth's magnetic field to orient toward the coast. To test this hypothesis, we used a unique combination of laboratory tests and in situ behavioral observations conducted in a drifting circular arena. Most (98%) of the glass eels tested in the sea exhibited a preferred orientation that was related to the tidal cycle. Seventy-one percent of the same eels showed the same orientation during ebb tide when tested in the laboratory under a manipulated simulated magnetic field in the absence of any other cue. These results demonstrate that glass eels use a magnetic compass for orientation and suggest that this magnetic orientation system is linked to a circatidal rhythm.

Opportunistic suspension feeding in the intertidal gastropod Olivella columellaris and its implications for the regulation of tidal migrations

The gastropod Olivella columellaris inhabits sandy beaches of the Tropical East Pacific. These snails perform tidal migrations forcontinuous suspension feeding in the moving backwash zone, possibly controlled by an endogenous circatidal clock. However, O. columellaris readily modifies its circatidal behavior, for example when flow channels develop below natural and artificial tide pools at ebb tide. Suspension feeding continues in such channels while water is running, delaying tidal migration. Such behavioral plasticity questions the significance ofendogenous rhythms for the regulation of tidal migrations in O. columellaris.

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.

Changes to Physiological Entomology: New Co‐Editor and members of the Editorial Board

With the first issue of 2015 (vol. 40:1) we celebrate the 40th consecutive year of publication of Physiological Entomology, from its inauguration by the first editor, John Brady as one of the Royal Entomological Society's new triad of journals that evolved after 50 years of prior publication of the antecedent journals Journal of Entomology (A) and earlier Proceedings of the (Royal) Entomological Society of London (1926–1976). As one of several current editorial changes, we welcome a new co-editor, Shin Goto from Osaka City University, Japan, who has recently agreed to be our first editor representing the wider interests of the journal in east and south-east Asia, as well as his own research interests into the physiological and molecular mechanisms underlying environmental adaptation in insects related to diapause, circadian clock, circatidal clock, photoperiodism, and stress tolerance. Readers will recall that Shin served previously as guest editor for a dedicated issue entitled ‘Adaptations to Changing Environments’ (vol. 38:2), along with former editor Jim Hardie (now a member of the editorial board ) and Kyeong-Yeoll Lee of Kyungpook National University, Daegu, Korea. As a major international journal, Physiological Entomology receives submissions from more than 30 different countries each year, and it is our aim that this broader editorial team (representing U.K., U.S.A. & Japan) should reflect those interests more fittingly. With the appointment of a new co-editor, it is timely also to review the composition of the editorial board. After deliberation by the editors, and consultation with the Society's Honorary Editorial Officer, Professor Lin Field, we are pleased to announce that Maya Evenden, University of Alberta, Canada; Vladimír Košt'ál, Institute of Entomology, Czech Republic; and Kenji Tomioka, Okayama University, Japan, have all agreed to add their expertise by joining us as members of the editorial board. To both the long-standing and newest members of the board, we look forward to your continuing judgement and opinions, and to the encouragement of relevant submissions from your laboratories, students, co-workers and associates. It is also a useful time to re-state the aims and broad interests of Physiological Entomology, which are reflected in the strapline ‘: from biochemistry to behaviour’, which was added in 2003. Physiological Entomology is designed primarily to serve the interests of experimentalists who work on the physiology and behaviour of insects and other arthropods, but also to retain the Royal Entomological Society's interests in the general physiology of arthropods. The subjects that it covers include, but are not limited to: chemical ecology; behavioural physiology and biochemistry, experimental analysis of behaviour; neurobiology and sensory physiology, general physiology; endocrinology, photoperiodism and circadian rhythms, and these days, of course, elements of molecular biology. We welcome both original manuscripts within the journal's remit, as well as relevant review papers. All submissions to the journal should be submitted online at http://mc.manuscriptcentral.com/phen.

Silencing the circadian clock gene Clock using RNAi reveals dissociation of the circatidal clock from the circadian clock in the mangrove cricket

Whether a clock that generates a circatidal rhythm shares the same elements as the circadian clock is not fully understood. The mangrove cricket, Apteronemobius asahinai, shows simultaneously two endogenous rhythms in its locomotor activity; the circatidal rhythm generates active and inactive phases, and the circadian rhythm modifies activity levels by suppressing the activity during subjective day. In the present study, we silenced Clock (Clk), a master gene of the circadian clock, in A. asahinai using RNAi to investigate the link between the circatidal and circadian clocks. The abundance of Clk mRNA in the crickets injected with double-stranded RNA of Clk (dsClk) was reduced to a half of that in control crickets. dsClk injection also reduced mRNA abundance of another circadian clock gene period (per) and weakened diel oscillation in per mRNA expression. Examination of the locomotor rhythms under constant conditions revealed that the circadian modification was disrupted after silencing Clk expression, but the circatidal rhythm remained unaffected. There were no significant changes in the free-running period of the circatidal rhythm between the controls and the crickets injected with dsClk. Our results reveal that Clk is essential for the circadian clock, but is not required for the circatidal clock. From these results we propose that the circatidal rhythm of A. asahinai is driven by a clock, the molecular components of which are distinct from that of the circadian clock.

The Circatidal Rhythm Persists without the Optic Lobe in the Mangrove Cricket Apteronemobius asahinai

Whether the circatidal rhythm is generated by a machinery common to the circadian clock is one of the important and interesting questions in chronobiology. The mangrove cricket Apteronemobius asahinai shows a circatidal rhythm generating active and inactive phases and a circadian rhythm modifying the circatidal rhythm by inhibiting activity during the subjective day simultaneously. In the previous study, RNA interference of the circadian clock gene period disrupted the circadian rhythm but not the circatidal rhythm, suggesting a difference in molecular mechanisms between the circatidal and circadian rhythms. In the present study, to compare the neural mechanisms of these 2 rhythms, we observed locomotor activity in the mangrove cricket after surgical removal of the optic lobe, which has been shown to be the locus of the circadian clock in other crickets. We also noted the pigment-dispersing factor immunoreactive neurons (PDF-IRNs) in the optic lobe, because PDF is a key output molecule in the circadian clock system in some insects. The results showed that the circadian modulation was disrupted after the removal of the optic lobes but that the circatidal rhythm was maintained with no remarkable changes in its free-running period. Even in crickets in which some PDF-immunoreactive somata remained after removal of the optic lobe, the circadian rhythm was completely disrupted. The remnants of PDF-IRNs were not correlated to the occurrence and free-running period of the circatidal rhythm. These results indicate that the principal circatidal clock is located in a region(s) different from the optic lobe, whereas the circadian clock is located in the optic lobe, as in other crickets, and PDF-IRNs are not important for circatidal rhythm. Therefore, it is suggested that the circatidal rhythm of A. asahinai is driven by a neural basis different from that driving the circadian rhythm.

Looking for the clock mechanism responsible for circatidal behavior in the oyster Crassostrea gigas

Valve activity rhythm of the oyster Crassostrea gigas is mainly driven by tides in the field, but in the laboratory, only a circadian clock mechanism has been demonstrated. In an attempt to reconcile these results, the mechanisms underlying the circatidal rhythm were studied in the laboratory under different entrainment or free-running regimes and in the field at Arcachon (44°39′N/1°09′W) in February–April 2011). Results confirm the existence of a circadian clock in C. gigas. Under entrainment regimes (12-h dark/12-h light photoperiod and tidal cycles simulated by a reversing current flow), oysters exhibited both circadian and circatidal cycles. Under free-running conditions (e.g., continuous darkness), the endogenous rhythm appeared to be circadian. There was no experimental evidence for an endogenous circatidal rhythm, even in oysters just transferred from the field, where a clear tidal cycle was expressed. There are two possible mechanisms to explain tidal behavior in C. gigas: an exogenous tidal cue that drives tidal activity and masks the circadian rhythm and an endogenous circatidal clock that is sensitive to tidal zeitgebers and runs at tidal frequency.

Patterns of activity expressed by juvenile horseshoe crabs.

Adult American horseshoe crabs, Limulus polyphemus, possess endogenous circadian and circatidal clocks controlling visual sensitivity and locomotion, respectively. The goal of this study was to determine the types of activity rhythms expressed by juvenile horseshoe crabs (n = 24) when exposed to a 14:10 light/dark cycle (LD) for 10 days, followed by 10 days of constant darkness (DD). Horseshoe crab activity was recorded with a digital time-lapse video system that used an infrared-sensitive camera so animals could be monitored at night. In LD, 15 animals expressed daily patterns of activity, 6 displayed a circatidal pattern, and the remaining 3 were arrhythmic. Of the 15 animals with daily patterns of locomotion, 7 had a significant preference (P < 0.05) for diurnal activity and 3 for nocturnal activity; the remainder did not express a significant preference for day or night activity. In DD, 13 horseshoe crabs expressed circatidal rhythms and 8 maintained a pattern of about 24 h. Although these results suggest the presence of a circadian clock influencing circatidal patterns of locomotion, these apparent circadian rhythms may actually represent the expression of just one of the two bouts of activity driven by the putative circalunidian clocks that control their tidal rhythms. Overall, these results indicate that, like adults, juvenile horseshoe crabs express both daily and tidal patterns of activity and that at least one, and maybe both, of these patterns is driven by endogenous clocks.

RNAi of the circadian clock geneperioddisrupts the circadian rhythm but not the circatidal rhythm in the mangrove cricket

The clock mechanism for circatidal rhythm has long been controversial, and its molecular basis is completely unknown. The mangrove cricket, Apteronemobius asahinai, shows two rhythms simultaneously in its locomotor activity: a circatidal rhythm producing active and inactive phases as well as a circadian rhythm modifying the activity intensity of circatidal active phases. The role of the clock gene period (per), one of the key components of the circadian clock in insects, was investigated in the circadian and circatidal rhythms of A. asahinai using RNAi. After injection of double-stranded RNA of per, most crickets did not show the circadian modulation of activity but the circatidal rhythm persisted without a significant difference in the period from controls. Thus, per is functionally involved in the circadian rhythm but plays no role, or a less important role, in the circatidal rhythm. We conclude that the circatidal rhythm in A. asahinai is controlled by a circatidal clock whose molecular mechanism is different from that of the circadian clock.

Functional Conservation of Clock Output Signaling between Flies and Intertidal Crabs

Intertidal species have both circadian and circatidal clocks. Although the behavioral evidence for these oscillators is more than 5 decades old, virtually nothing is known about their molecular clockwork. Pigment-dispersing hormones (PDHs) were originally described in crustaceans. Their insect homologs, pigment-dispersing factors (PDFs), have a prominent role as clock output and synchronizing signals released from clock neurons. We show that gene duplication in crabs has led to two PDH genes (β-pdh-I and β-pdh-II). Phylogenetically, β-pdh-I is more closely related to insect pdf than to β-pdh-II, and we hypothesized that β-PDH-I may represent a canonical clock output signal. Accordingly, β-PDH-I expression in the brain of the intertidal crab Cancer productus is similar to that of PDF in Drosophila melanogaster, and neurons that express PDH-I also show CYCLE-like immunoreactivity. Using D. melanogaster pdf-null mutants (pdf(01)) as a heterologous system, we show that β-pdh-I is indistinguishable from pdf in its ability to rescue the mutant arrhythmic phenotype, but β-pdh-II fails to restore the wild-type phenotype. Application of the three peptides to explanted brains shows that PDF and β-PDH-I are equally effective in inducing the signal transduction cascade of the PDF receptor, but β-PDH-II fails to induce a normal cascade. Our results represent the first functional characterization of a putative molecular clock output in an intertidal species and may provide a critical step towards the characterization of molecular components of biological clocks in intertidal organisms.

Circatidal rhythms of locomotion in the American horseshoe crab Limulus polyphemus: Underlying mechanisms and cues that influence them

Abstract While eye sensitivity in the American horseshoe crab Limulus polyphemus has long been known to be under the control of an endogenous circadian clock, only recently has horseshoe crab locomotion been shown to be controlled by a separate clock system. In the laboratory, this system drives clear activity rhythms throughout much of the year, not just during the mating season when horseshoe crabs express clear tidal rhythms in the field. Water temperature is a key factor influencing the expression of these rhythms: at 17̊C tidal rhythms are expressed by most animals, while at 11̊C expression of circatidal rhythms is rarely seen, and at 4̊C rhythms are suppressed. Neither long (16:8 Light:Dark) nor short (8:16) photoperiods modify this behavior at any of these temperatures. Synchronization of these circatidal rhythms can be most readily effected by water pressure cycles both in situ and in the lab, while temperature and current cycles play lesser, but possibly contributory, roles. Interestingly, Light:Dark cycles appear to have synchronizing as well as “masking” effects in some individuals. Evidence that each of two daily bouts of activity are independent suggests that the Limulus circatidal rhythm of locomotion is driven by two (circalunidian) clocks, each with a period of 24.8h. While the anatomical locations of either the circadian clock, that drives fluctuations in visual sensitivity, or the circatidal clock, that controls tidal rhythms of locomotion, are currently unknown, preliminary molecular analyses have shown that a 71 kD protein that reacts with antibodies directed against the Drosophila PERIOD (PER) protein is found in both the protocerebrum and the subesophageal ganglion.