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.     

Entrainment of juvenile horseshoe crab activity to artificial tides

Juvenile American horseshoe crabs, Limulus polyphemus, express both daily and tidal rhythms. To determine if, and how, tidal cues influence the expression of these rhythms, we exposed 25 animals to artificial tides, and 17 to artificial tides with inundation, both with a 12:12 LD cycle. In the first experiment, 24% expressed daily rhythms of activity, 24% tidal rhythms, 12% a combination of the two, and the rest were arrhythmic. Under subsequent atidal conditions some expressed daily rhythms, but more were circatidal. In the second experiment, 6% expressed daily rhythms, 71% tidal, 12% a combination, and 12% were arrhythmic. Those expressing tidal rhythms were more active during flood/high tide, while daily animals tended to be nocturnal. Under subsequent constant conditions, the majority exhibited circatidal activity, with some expressing one activity bout per day. We conclude that juvenile horseshoe crabs entrain to artificial tides, with inundation cycles providing stronger cues than water depth changes.     

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.     

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.     

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.     

Endogenous rhythms and entrainment cues of larval activity in the horseshoe crab Limulus polyphemus

The American horseshoe crab, Limulus polyphemus (Linnaeus), typically inhabits estuaries and coastal areas with pronounced semi-diurnal and diurnal tides that are used to synchronize the timing of spawning, larval hatching, and emergence. Horseshoe crabs spawn in the intertidal zone of sandy beaches and larval emergence occurs when the larvae exit the sediments and enter the plankton. However, L. polyphemus populations also occur in areas that lack significant tidal changes and associated synchronization cues. Endogenous activity rhythms that match predictable environmental cycles may enable larval horseshoe crabs to time swimming activity to prevent stranding on the beach. To determine if L. polyphemus larvae possess a circatidal rhythm in vertical swimming, larvae collected from beach nests and the plankton were placed under constant conditions and their activity monitored for 72 h. Time-series analyses of the activity records revealed a circatidal rhythm with a free-running period of ≈ 12.5 h. Maximum swimming activity consistently occurred during the time of expected falling tides, which may serve to reduce the chance of larvae being stranded on the beach and aid in seaward transport by ebb currents (i.e., ebb-tide transport). To determine if agitation serves as the entrainment cue, larvae were shaken on a 12.4 h cycle to simulate conditions during high tide in areas with semi-diurnal tides. When placed under constant conditions, larval swimming increased near the expected times of agitation. Thus, endogenous rhythms of swimming activity of L. polyphemus larvae in both tidal and nontidal systems may help synchronize swimming activity with periods of high water and inundation.     

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 24.8 h. 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.     

An entrainment model for semilunar rhythmic swimming behaviour in the marine isopod Eurydice pulchra Leach

Entrainment experiments have been carried out with geographically widely separated populations of the sand beach isopod Eurydice pulchra Leach subjected to periods of simulated tidal agitation imposed concurrently with a 24-h light: dark (L: D) cycle. Circatidal swimming rhythms of greatest amplitude were induced when agitation was applied with the subjective timing, within the L: D cycle, of local spring high tides. This occurred in a normal L: D regime and also when the L: D regime was phase shifted through 90°. Animals previously maintained in constant darkness (D: D) and subsequently exposed to simulated tidal disturbance at various times in constant darkness were unable to modulate the amplitude of circatidal swimming activity. Isopods previously maintained in a normal L: D cycle and subsequently subjected to artificial tidal agitation in constant darkness were, however, able to modulate circatidal activity. This indicates that E. pulchra is capable of detecting tidal agitation and daily light cues and using them in conjunction with its circadian “clock” to modulate its endogenous circatidal rhythmicity. The free-running semilunar rhythm of swimming activity entrained only when the timing of agitation within the day/night cycle mimicked the pattern of local spring high tides. Agitation with the timing of neap high tides entrained no free-running circa-semilunar activity pattern.     

Constant light disrupts the circadian but not the circatidal rhythm in mangrove crickets

Mangrove crickets have a circatidal activity rhythm (~12.6 h cycles) with a circadian modulation under constant darkness (DD), whereby activity levels are higher during subjective night low tides than subjective day low tides. This study explored the locomotor activity rhythm of mangrove crickets under constant light (LL). Under LL, the crickets also exhibited a clear circatidal activity rhythm with a free-running period of 12.6 ± 0.26 h (mean ± SD, n = 6), which was not significantly different from that observed under DD. In contrast, activity levels were almost the same between subjective day and night, unlike those under DD, which were greater during subjective night. The loss of circadian modulation under LL may be explained by the suspension of the circadian clock in these conditions. These results strongly suggest that the circatidal activity rhythm is driven by its own clock system, distinct from 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.     

Endogenous rhythms of locomotor activity in the high-shore limpet, Helcion pectunculus (Patellogastropoda)

Helcion pectunculus, a high-shore, crevice-dwelling limpet, is active during nocturnal low tides and during daytime low tides whilst in the shade. We examined whether this activity is controlled by an internal clock or purely by exogenous stimuli, such as light levels and tidal phase. Maximum entropy spectral analysis (MESA) revealed that the limpets possess a free-running endogenous rhythm of locomotor activity with both circadian (period 28.1 h) and circatidal (period 13.8 h) components. We suggest that this rhythm plays a role in allowing individuals to avoid unfavourable environmental conditions. The exogenous entrainment factor of the endogenous circatidal rhythm in H. pectunculus is the time of exposure to air, whilst the zeitgeber for the circadian component is not yet known. Copyright 1999 The Association for the Study of Animal Behaviour.     

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.     

The diurnal modulation of the circatidal activity rhythm by feeding in the isopod eurydice pulchra

The diurnal modulation of circatidal activity was studied in freshly caught Eurydice pulchra of different feeding states. Apparently fully fed specimens displayed definite circadian modulation, with greatest tidal activity at the time of expected night‐time high tides, and apparently starved animals showed no circadian variation. Circatidal activity was also entrained in the laboratory using cycles of artificial agitation and the subsequent pattern of free‐running rhythmicity was studied. Animals fed prior to entrainment displayed definite diurnal modulation of the pattern of ciractidal swimming activity. Those fed after the entrainment regime, or not at all, displayed no apparent diurnal inequality of tidal activity peaks. No relationship between diurnal inequality and phototactic behaviour of freshly collected specimens was detected.     

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.     

Contact with water functions as a Zeitgeber for the circatidal rhythm in the mangrove cricket Apteronemobius asahinai

The mangrove cricket Apteronemobius asahinai shows a circatidal rhythm in its locomotor activity, and this rhythm was shown to be entrained to artificial tidal cycles in the laboratory. To examine the Zeitgeber for this rhythm, in the present study, crickets were fixed with insect pins to prevent their body locomotion and a water stimulus was given to them by soaking in water, while recording their locomotor activities. A single water stimulus delayed the phase when given in the middle subjective low tide and advanced the phase when given in the later subjective low tide, whereas it had only a slight effect in the subjective high tide. We conclude that contact with water functions as a Zeitgeber for the circatidal rhythm.     

TIDAL RHYTHMS: THE CLOCK CONTROL OF THE RHYTHMIC PHYSIOLOGY OF MARINE ORGANISMS

1. A great number of vital processes are rhythmic and the rhythms quite often persist in constant conditions. The best-known rhythms are circadian; much less is known about circalunadian rhythms, and this review was prepared in an attempt to rectify this deficiency. All through the article comparisons are drawn between circalunadian and circacian rhythms. 2. Activity rhythms. (a) The activity patterns of 28 intertidal animals are discussed. All describe a periodicity with a basic component of 24.8 hours, and this approximate period persists in the laboratory in constant light and temperature and in the absence of the tides. The duration of persistence ranges from a few cycles to months, and is a function of the species studied, the conditions imposed, and individual tenacity. (b) In those few cases where relatively long-term observations have been made, there is a trend for the period of the rhythm to become circatidal, or better, circalunadian. (c) The ‘desired’ phase relationship between rhythm and tidal cycle is species-specific. Geographical translocation experiments have shown that the phase is set by the local tides. (d) In some cases the amplitude of the persistent rhythm mimics the semidiurnal inequality of the tides. (e) In about a third of the species discussed, a circadian component has been found combined with the tidal component. Many of the other studies were of such short duration that a low-amplitude circadian component would have gone unnoticed. (f) The tidal rhythm is innate. However, the rhythm is (i) sometimes lacking in organisms living in non-tidal habitats, or (ii) fades after a spell of incarceration in constant conditions. Various treatments — some aperiodic — can induce the expression of the missing tidal rhythm. (g) In the green crab, removal of the eyestalks destroys the activity rhythm. 3. Vertical migration rhythms. (a) A rather surprisingly large number of intertidal animals have been found to undergo migration rhythms between the upper layers of the substratum and its surface. The movements are synchronized with the tides in nature, but most species have either been shown to be diurnal in constant conditions, or in cases where adequate testing has not been done, suspected of being so. (b) In only one species has confirming work shown that the fundamental frequency is truly tidal. This finding is especially important as it shows that tidal rhythms need only the single-cell level of organization for expression. Even at this level there appears to be a dictatorial override by a circadian clock. 4. Colour change. Low-amplitude tidal rhythms in colour change — superimposed on a more dominant circadian change — have been reported to be intrinsic in four species and inducible in a fifth. 5. Oxygen consumption. Tidal rhythms in oxygen consumption have been described for seven invertebrates and one alga; six of the species have superimposed solar-day rhythmic components also. 6. Translocation. A total of five geographical translocation experiments, in which the organisms were maintained in constant conditions throughout, have been tried. Unequivocally in one case, and possibly in a second, the test organisms rephased spontaneously to the times commensurate with local tidal conditions. In two other cases, the pretranslocation phase was retained. The fifth experiment has not been reproducible. 7. Determination of phase. (a) The tidal cycle on the home shoreline sets the phase of the inhabitant's rhythms. Even the location of a crab's burrow on the beach incline can play a determining role. (b) Paradoxically, the periodic wetting by inundation is not an important entraining factor for most intertidal organisms. Instead, the effective portions of the tidal cycle include one or more of the following. (i) Mechanical agitation, especially for animals living in an uprush zone where they are periodically subjected to the pounding surf, (ii) Temperature cycles, though they have not yet been systematically investigated, have very pronounced entraining roles in crabs. (iii) Pressure is probably not a generally important entraining agent for most intertidal organisms, but it is so for the green crab. (c) Light-dark cycles in general, whether daily or tidal in length, have no effect on the entrainment or phase setting of many tidal rhythms. There are two exceptions: (i) a 24-hour light-dark cycle is known to keep a tidal locomotor rhythm (one that becomes circalunadian in constant conditions) at a strict tidal frequency. (ii) In rhythms with both daily and tidal components, when the former is shifted by light stimuli, the latter is affected in a nearly identical manner. 8. Temperature. (a) The role of temperature on tidal rhythms is compared with its role on circadian rhythms. (b) The effects of different constant temperatures have so far been studied on only four tidal rhythms. All studies indicate a lack of any permanent change in period, which is not so with most circadian rhythms; the latter having temperature coefficients around 1.1. In two of the studies the rhythms under test temperatures were followed for less than a day, and a third study cannot be repeated. (c) Short exposure to very cold temperature pulses produced a response that may be interpreted as a temporary stoppage of the clock. Exposure to relatively less-cold pulses appear simply to reset the hands of the clock. The same responses have been demonstrated with circadian rhythms. (d) In the case of green crabs, which had become arrhythmic during prolongued captivity in the laboratory, a tidal rhythm could be reinitiated by a single short cold treatment. The cold pulse also set the phase of the rhythm. (e) A few superficial studies employing temperature steps or pulses have produced results which suggest that a phase-change sensitivity rhythm — just like that found associated with circadian rhythms — may underlie tidal rhythms. Certainly a determined search for this rhythm should be made in the near future. 9. Clock control of rhythms. (a) An argument is constructed claiming that tidal rhythms have a basic period of about 24–8 hours rather than the more expected tidal interval of 12.4 hours. In constant conditions, a circalunadian period is usually displayed. (b) After speculating that a frequency-transforming coupler may function between the clock and the overt rhythm, reasons are given that lead to the further speculation that both circadian and circalunadian rhythms could be generated by a single clock, via specific coupling mechanisms. (c) Two current hypotheses concerning the nature of the clockworks are reviewed and discussed. (d) Suggestions are made for future investigations.     

MODELING ACTIVITY RHYTHMS IN FIDDLER CRABS

Burrowing crabs of the genus Uca inhabit tidal mudflats and beaches. They feed actively during low tide and remain in their burrows when the tide is high. The timing of this activity has been shown to persist in the absence of external light and tidal cues, indicating the presence of an internal timing mechanism. Researchers report the persistence of several variations in locomotor activity under laboratory conditions that cannot be explained by a single circatidal clock. Previous studies supported two alternative hypotheses: the presence of either two circalunidian clocks, or a circadian and circatidal clock to regulate these activity rhythms. In this paper, we formulate mathematical models to describe and test these hypotheses. The models suggested by the literature contain some important differences beyond the frequency of proposed clocks, and these are reflected in the mathematical formulations and simulation results. One hypothesis suggests independent phase oscillators, while the other hypothesis suggests that they are coupled in anti-phase. Neither model is able to recover all of the variations in locomotor acitivity observed under laboratory conditions. However, we propose a new model that incorporates aspects of both existing hypotheses and is able to reproduce all laboratory observations. (Author correspondence: verzi@math.sdsu.edu)     

Seasonal effects of pinealectomy on the locomotor activity rhythm in the lizard Sceloporus torquatus

Adult lizards (Sceloporus torquatus) were used to test whether seasonal differences in the effects of pinealectomy upon the locomotor activity rhythm exist. Animals were field collected and exposed to artificial light - dark cycles and constant temperature in winter and summer. Free running circadian rhythms under constant temperature and dim red light were monitored using infrared light-crossings. The effects of pinealectomy were assessed by analysing the circadian parameters of free running period and activity - rest ratio in constant darkness or light - dark cycles. Results obtained indicate that pinealectomy changes the free running period of locomotor activity rhythm, irrespective of season, while seasonal differences in activity-rest ratio were detected. Our findings support the hypothesis that seasonal regulation of circadian rhythms in lizards is accomplished, in part, via the output of the pineal gland.     

Entrainment of the activity rhythm of the mole crab Emerita talpoida

The mole crab Emerita talpoida migrates with the tide in the swash zone of sand beaches. A circatidal rhythm in vertical swimming underlies movement, in which mature male crabs show peak swimming activity 1-2 h after the time of high tides at the collection site. In addition, there is a secondary rhythm in activity amplitude, in which crabs are maximally active following low amplitude high tides and minimally active following high amplitude high tides. The present study determined the phase response relationship for entrainment of the circatidal rhythm with mechanical agitation and whether the cycle in activity related to tidal amplitude could be entrained by a cycle in the duration of mechanical agitation at the times of consecutive high tides. After entrainment with mechanical agitation on an orbital shaker, activity of individual crabs was monitored in constant conditions with a video system and quantified as the number of ascents from the sand each 0.5 h. Mechanical agitation at the times of high tide, mid-ebb and low tide reset the timing of the circatidal rhythm according to the timing relationship to high tide. However, mechanical agitation during flood tide had no entrainment effect. In addition, a cycle in duration of mechanical agitation entrained the rhythm in activity amplitude associated with tidal amplitude. Both rhythms and entrainment effectiveness over the tidal cycle may function to reduce the likelihood of stranding above the swash zone.     

Interactions of the circadian and circatidal rhythms of the littoral gastropod melanerita atramentosa Reeve

Melanerita atramentosa Reeve is active during high water and for a period after the ebb, until the substratum dries. A circatidal rhythm with an endogenous inequality between the diurnal and nocturnal high water activity is present in freshly collected snails placed under constant conditions (LL, 30 lux, temperature 21 °C, water level constant). The free-running period of the circatidal rhythm was approximately 24.3 h, slightly less than the tidal period of 24.8 h. This rhythm faded out after 4–6 days of constant conditions. Snails acclimated to non-tidal conditions were active at night. This rhythm persisted under constant conditions for 5–10 days, after which it too faded out. Shock-freezing re-initiated the circatidal rhythm, supporting a ‘multiple-clock’ hypothesis of control. Its limits entrainment are probably narrow, but an entrained periodicity persists for a number of cycles. A model of clock interaction and environmental influence is advanced.