RNAi of the circadian clock gene period disrupts 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.     

Rhythms of locomotion expressed by Limulus polyphemus, the American horseshoe crab: II. Relationship to circadian rhythms of visual sensitivity

In the laboratory, horseshoe crabs express a circadian rhythm of visual sensitivity as well as daily and circatidal rhythms of locomotion. The major goal of this investigation was to determine whether the circadian clock underlying changes in visual sensitivity also modulates locomotion. To address this question, we developed a method for simultaneously recording changes in visual sensitivity and locomotion. Although every animal (24) expressed consistent circadian rhythms of visual sensitivity, rhythms of locomotion were more variable: 44% expressed a tidal rhythm, 28% were most active at night, and the rest lacked statistically significant rhythms. When exposed to artificial tides, 8 of 16 animals expressed circatidal rhythms of locomotion that continued after tidal cycles were stopped. However, rhythms of visual sensitivity remained stable and showed no tendency to be influenced by the imposed tides or locomotor activity. These results indicate that horseshoe crabs possess at least two biological clocks: one circadian clock primarily used for modulating visual sensitivity, and one or more clocks that control patterns of locomotion. This arrangement allows horseshoe crabs to see quite well while mating during both daytime and nighttime high tides.     

Environmental entrainment of tidally rhythmic behaviour in marine animals

Field and laboratory experiments show that endogenous circatidal rhythms in coastal animals are entrained by exposure to real or simulated tidal cycles of hydrostatic pressure, temperature, salinity, wave agitation, immersion and light. Short pulses (2–3 h) of simulated high tide induce slight phase advances or delays in the free-running circatidal rhythm of groups of experimental animals, depending upon the time of application. Phase-response curves derived in this way are less clear-cut than for typical circadian rhythms, but their pattern suggests that tidally rhythmic behaviour is controlled by truly circatidal (and not circadian) oscillators. The underlying circatidal oscillators appear, in general, to be fairly stable, suggesting that populations of coastal animals are relatively unsusceptible to irregularly timed environmental stimuli associated, say, with severe storms.     

Entrainment of bimodal circatidal rhythms in the shore crab Carcinus maenas

Individuals of the shore crab Carcinus maenas were exposed to artificial cycles, applied in tidal antiphase, of pairs of the three major environmental variables that entrain circatidal rhythmicity in this species: salinity, temperature, and hydrostatic pressure. During entrainment, the observed locomotor activity patterns were dominated by exogenous responses to high pressure, low temperature, or low salinity. In subsequent constant conditions, many of the crabs showed bimodal circatidal rhythms, with peaks phased to the times of expected high-tide characteristics of high pressure, low temperature, or high salinity. Similar bimodal rhythms were induced by exposing freshly captured crabs, with free-running circatidal rhythms, to tidal antiphase cycles of each of the three environmental variables applied individually. The hypothesis that circatidal rhythmicity in this species is controlled by at least two separate circatidal oscillators, with differential sensitivities to specific cyclical environmental variables, is discussed.     

Genetic architecture of local adaptation in lunar and diurnal emergence times of the marine midge Clunio marinus (Chironomidae, Diptera)

Circadian rhythms pre-adapt the physiology of most organisms to predictable daily changes in the environment. Some marine organisms also show endogenous circalunar rhythms. The genetic basis of the circalunar clock and its interaction with the circadian clock is unknown. Both clocks can be studied in the marine midge Clunio marinus (Chironomidae, Diptera), as different populations have different local adaptations in their lunar and diurnal rhythms of adult emergence, which can be analyzed by crossing experiments. We investigated the genetic basis of population variation in clock properties by constructing the first genetic linkage map for this species, and performing quantitative trait locus (QTL) analysis on variation in both lunar and diurnal timing. The genome has a genetic length of 167-193 centimorgans based on a linkage map using 344 markers, and a physical size of 95-140 megabases estimated by flow cytometry. Mapping the sex determining locus shows that females are the heterogametic sex, unlike most other Chironomidae. We identified two QTL each for lunar emergence time and diurnal emergence time. The distribution of QTL confirms a previously hypothesized genetic basis to a correlation of lunar and diurnal emergence times in natural populations. Mapping of clock genes and light receptors identified ciliary opsin 2 (cOps2) as a candidate to be involved in both lunar and diurnal timing; cryptochrome 1 (cry1) as a candidate gene for lunar timing; and two timeless (tim2, tim3) genes as candidate genes for diurnal timing. This QTL analysis of lunar rhythmicity, the first in any species, provides a unique entree into the molecular analysis of the lunar clock.     

Circadian components of semilunar and lunar timing mechanisms

The timing of semilunar as well as lunar reproductive rhythms has been analyzed in different geographic populations of the intertidal chironomid Clunio. In stocks of three populations differing in period and phase relationship with the lunar month, these long-term rhythms were synchronized in the laboratory by using artificial moonlight cycles of 30 days in otherwise 24-hr light-dark (LD) cycles (0.4 lux during 4 successive nights every 30 days in LD 12:12). In LD cycles of various periods, a strong synchronization was only possible in LD 12:12 and LD 11:11, whereas in LD 10:10 and LD 15:15 the synchronization by the 30-"day" moonlight cycle was weak or even absent. The study demonstrates a limited range of circadian periods for entrainment of the long-term rhythms. It is concluded that an LD cycle with a period near 24 hr is an essential zeitgeber condition for semilunar and lunar timing in this marine insect. Further, it is suggested that the underlying physiological timing mechanism of Clunio consists of a circadian function for the perception of the monthly moonlight zeitgeber cycles that entrain the endogenous, temperature-compensated oscillator of the circasemilunar (or circalunar) period. The long-term oscillator triggers the metamorphosis of the insect, and thereby determines the time of its eclosion and reproduction on the shorelines, in correlation with days of spring tides recurring about every 14-15 days.     

Endogenous rhythms of locomotion in the American horseshoe crab, Limulus polyphemus

American horseshoe crabs (Limulus polyphemus) exhibit clear circadian rhythms of visual sensitivity in the laboratory and in the field they exhibit seasonal patterns of mating behavior that are closely associated with the tides. Recent reports suggest that Limulus locomotor activity may be controlled by endogenous circadian and/or circatidal clocks and that light:dark (LD) cycles may affect the rhythmic output of both of these clocks. In this study, we examined locomotor behavior in the laboratory to determine the extent of this endogenous activity and to examine the influence of LD cycles on these rhythms. Thirty-three L. polyphemus were captured during the breeding season and their activity was monitored with activity boxes and “running wheels” in seawater kept at constant temperature and salinity. Activity patterns were analyzed using visual inspection of actograms and Chi-square and Lomb-Scargle periodograms. Overall, 36% of the animals was significantly more active during L, while only 12% was more active during D (52% showed no preference). Circatidal rhythms were observed in LD in 67% of the horseshoe crabs. Surprisingly, LD cycles appeared to synchronize these rhythms at times. In DD, the majority of animals tested (63%) exhibited circatidal rhythms that persisted for at least seven days. Overall, the results demonstrate that an endogenously controlled tidal rhythm of locomotion operates during, and significantly after, the breeding season in this species. In addition, the present results are consistent with the presence of circalunidian oscillators controlling these rhythms.     

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.     

Crab Clockwork: The Case for Interactive Circatidal and Circadian Oscillators Controlling Rhythmic Locomotor Activity of Carcinus Maenas

The circalunidian hypothesis that tidal rhythms in coastal animals are controlled by two lunar-day (c. 24.8 h) oscillators coupled in antiphase is challenged. Rhythmic locomotor activity patterns of the shore crab Carcinus maenas, and probably of some other species too, are more economically explained by interacting circadian (c. 24 h) and true circatidal (c. 12.4 h) physiological oscillators. A testable hypothesis is proposed that combines a circadian promotor and a circatidal inhibitor of locomotor activity.     

An antibody to the Drosophila period protein recognizes circadian pacemaker neurons in Aplysia and Bulla

The molecular mechanisms of the pacemakers underlying circadian rhythms are not well understood. One molecule that presumably functions in the circadian clock of Drosophila is the product of the period (per) gene, which dramatically affects biological rhythms when mutated. An antibody specific for the per protein labels putative circadian pacemaker neurons and fibers in eyes of two marine gastropods, Aplysia and Bulla. As was found for the Drosophila per protein, there is a daily rhythm in the levels of the per-like antigen in Aplysia eyes. Thus, certain molecular features of the per protein, as well as aspects of the temporal regulation of its expression, may be conserved in circadian pacemakers of widely divergent species.     

Interaction of endogenous and exogenous factors controlling locomotor activity rhythms in Carcinus exposed to tidal salinity cycles

The shore crab Carcinus maenas (L.) reported hitherto not to express endogenous circatidal rhythmicity in winter, is shown not to lose the ability to express such rhythmicity. Crabs maintained in constant reduced salinity in winter exhibit circatidal and circadian rhythms similar to the normal endogenous rhythms of summer caught crabs.In sinusoidal changes of salinity of tidal periodicity, reductions of salinity and increases to ambient sea water induced increased locomotor activity. The former were purely exogenous responses but the latter were also observed to entrain the underlying endogenously controlled circatidal pattern of behaviour.The occurrence of separate exogenous and endogenous responses to different phases of imposed salinity cycles has implications when seeking to understand rhythmic locomotor activity of crabs on the shore and in the search for components of the underlying physiological clock mechanism.     

Diversity of biological rhythm and food web stability

How ecosystem biodiversity is maintained remains a persistent question in the field of ecology. Here, I present a new coexistence theory, i.e. diversity of biological rhythm. Circadian, circalunar and circannual rhythms, which control short- and long-term activities, are identified as universal phenomena in organisms. Analysis of a theoretical food web with diel, monthly and annual cycles in foraging activity for each organism shows that diverse biological cycles play key roles in maintaining complex communities. Each biological rhythm does not have a strong stabilizing effect independently but enhances community persistence when combined with other rhythms. Biological rhythms also mitigate inherent destabilization tendencies caused by food web complexity. Temporal weak interactions due to hybridity of multiple activity cycles play a key role toward coexistence. Polyrhythmic changes in biological activities in response to the Earth''s rotation may be a key factor in maintaining biological communities.     

Biological timing and the clock metaphor: oscillatory and hourglass mechanisms

Living organisms have developed a multitude of timing mechanisms--"biological clocks." Their mechanisms are based on either oscillations (oscillatory clocks) or unidirectional processes (hourglass clocks). Oscillatory clocks comprise circatidal, circalunidian, circadian, circalunar, and circannual oscillations--which keep time with environmental periodicities--as well as ultradian oscillations, ovarian cycles, and oscillations in development and in the brain, which keep time with biological timescales. These clocks mainly determine time points at specific phases of their oscillations. Hourglass clocks are predominantly found in development and aging and also in the brain. They determine time intervals (duration). More complex timing systems combine oscillatory and hourglass mechanisms, such as the case for cell cycle, sleep initiation, or brain clocks, whereas others combine external and internal periodicities (photoperiodism, seasonal reproduction). A definition of a biological clock may be derived from its control of functions external to its own processes and its use in determining temporal order (sequences of events) or durations. Biological and chemical oscillators are characterized by positive and negative feedback (or feedforward) mechanisms. During evolution, living organisms made use of the many existing oscillations for signal transmission, movement, and pump mechanisms, as well as for clocks. Some clocks, such as the circadian clock, that time with environmental periodicities are usually compensated (stabilized) against temperature, whereas other clocks, such as the cell cycle, that keep time with an organismic timescale are not compensated. This difference may be related to the predominance of negative feedback in the first class of clocks and a predominance of positive feedback (autocatalytic amplification) in the second class. The present knowledge of a compensated clock (the circadian oscillator) and an uncompensated clock (the cell cycle), as well as relevant models, are briefly re viewed. Hourglass clocks are based on linear or exponential unidirectional processes that trigger events mainly in the course of development and aging. An important hourglass mechanism within the aging process is the limitation of cell division capacity by the length of telomeres. The mechanism of this clock is briefly reviewed. In all clock mechanisms, thresholds at which "dependent variables" are triggered play an important role.     

Rockin' in rhythm,growth dynamics of the peat moss Sphagnum

The genus Sphagnum is an essential component in the formation and maintenance of high latitude peatlands, bogs and mires. The species grows in dense, extended mats of agglomerated shoots that allow it to retain water necessary for its growth. These mats are partly responsible for maintaining the right conditions for other species in these wetlands to thrive. In this issue of Physiologia Plantarum, Mironov et al. (2020) monitored the growth of Sphagnum riparium for a period of 4 years and revealed three distinct growth rhythms: a seasonal temperature dependent, a circalunar and a third one, synchronized with the circalunar. This synchronised nature of Sphagnum growth could contribute to its position as a key species in the maintenance of peatlands.     

Crab Clocks Rewound

In response to my advocacy of a circatidal/circadian model to explain behavioral rhythms in Carcinus (1), Palmer (2) raises questions concerning my initial premise, the nature of one of my model parameters, and the form of data analysis that I used. These questions are responded to and it is restated that the circatidal/circadian model of clock control adequately explains characteristics of free-running behavioral rhythms in Carcinus and probably in some other coastal animals, purported to be explained only by the recently formulated circalunidian hypothesis (3). (Chronobiology International, 14(4), 427–430, 1997)     

Circadian rhythms from multiple oscillators: lessons from diverse organisms

The organization of biological activities into daily cycles is universal in organisms as diverse as cyanobacteria, fungi, algae, plants, flies, birds and man. Comparisons of circadian clocks in unicellular and multicellular organisms using molecular genetics and genomics have provided new insights into the mechanisms and complexity of clock systems. Whereas unicellular organisms require stand-alone clocks that can generate 24-hour rhythms for diverse processes, organisms with differentiated tissues can partition clock function to generate and coordinate different rhythms. In both cases, the temporal coordination of a multi-oscillator system is essential for producing robust circadian rhythms of gene expression and biological activity.     

Fitness costs of disrupting circadian rhythms in malaria parasites

Circadian biology assumes that biological rhythms maximize fitness by enabling organisms to coordinate with their environment. Despite circadian clocks being such a widespread phenomenon, demonstrating the fitness benefits of temporal coordination is challenging and such studies are rare. Here, we tested the consequences--for parasites--of being temporally mismatched to host circadian rhythms using the rodent malaria parasite, Plasmodium chabaudi. The cyclical nature of malaria infections is well known, as the cell cycles across parasite species last a multiple of approximately 24 h, but the evolutionary explanations for periodicity are poorly understood. We demonstrate that perturbation of parasite rhythms results in a twofold cost to the production of replicating and transmission stages. Thus, synchronization with host rhythms influences in-host survival and between-host transmission potential, revealing a role for circadian rhythms in the evolution of host-parasite interactions. More generally, our results provide a demonstration of the adaptive value of circadian rhythms and the utility of using an evolutionary framework to understand parasite traits.