Sesarma Erythrodactylum Masayuki Saigusa Department of Biology

Benthos Research Vol.52, No.1: 21-33 (1997) BENTHOS RESEARCH

The Japanese Association of Benthology

The circatidal clock of an estuarine semi-terrestrial crab, Sesarma erythrodactylum

Masayuki Saigusa Department of Biology, Faculty of Science, Okayama University

ABSTRACT The influence of the tidal cycle decreases with the distance from the sea, and this may affect the behavioral timing systems in estuarine animals. In addition, the circatidal rhythm of these animals may be controlled by light-sensitive systems. To investigate the timing systems in estuarine animals, the larval release activity of a semi-terrestrial crab, Sesarma erythrodactylum, was monitored in the laboratory without any tidal influence. The larval release rhythm free-ran under constant dim light conditions, which suggests that the timing of release is under the control of an endogenous clock. The free-running period was somewhat different for each individual. Under an artificial 24-h light-dark (LD) cycle in phase with that in the field, the timing of release coincided with high tides at night. In contrast, the rhythm changed to match a phase-shifted 24-h cyclic light regimen. These results demonstrate that a light-sensitive mechanism is certainly involved in the circatidal timing systems of S. erythrodactylum. The role of the 24-h LD cycle is not only to shift the synchrony of the timing of release onto the other high tide when necessary for maintaining a nocturnal schedule, but also to drive the phase of the circatidal rhythm. While the tidal behavioral rhythms of intertidal animals reflect two parameters of the tidal cycle, i, e., the 12.4-h period and the tidal amplitude, those of estuarine crabs lose synchrony with the tidal amplitude and show a nocturnal pattern instead. These properties of the circatidal rhythm of larval release in S. erythrodactylum and other intertidal and estuarine crabs can be explained by a coupled oscillator hypothesis.

Key words: larval release, estuary, crab, Sesarma erythrodactylum, nocturnal, circatidal rhythm, zeitgeber, coupled oscillator

INTRODUCTION Barlow et al. 1986; Saigusa & Akiyama 1995). Some of these patterns are clearly under the In the intertidal and estuarine environments, control of endogenous clocks (Klapow 1972; the lives of organisms are strongly restricted Enright 1976; Hastings 1981), but other pat- spacially and temporally by the tidal cycle. A terns are caused by the direct response of the number of organisms synchronize their activi- animals to cyclical fluctuations of physical fac- ties with this predictable cycle in the environ- tors correlated with tides (e.g., Honegger 1973; ment and show complex activity patterns syn- Lehmann 1976; Reid & Naylor 1990; Northcott chronized with not only the day-night cyle but et al. 1991). also the tidal cycle (Saigusa 1981, 1982, 1985; One of the main problems in tidal rhythm research is the relationship between the rhythmic Received September 13 , 1996 : Accepted January 27, 1997 pattern and the tidal influence. The tidal cycle

21 Vol.52,No.1 Benthos Research June,1997 is characterized by two kinds of physical pa- rhythm in each female. The 12.4-h tidal rhythm rameter; i.e., the mean period length of 12.4-h, for larval release in the coastal terrestrial crab and the semidiurnal inequality of the tidal Sesarma pictum also exhibits the properties height. Intertidal animals may possess timing very similar to those of S. haematochier systems that can respond to both parameters, (Saigusa 1992) . Even in the intertidal crab as exemplified by the swimming activity of Hemigrapsus sanguineus, the circatidal rhythm some amphipods and isopods (Enright 1963; responds to 24-h light cycles with phase shifts Klapow 1972), the mating behavior of a horse- (Saigusa & Kawagoye 1997). shoe crab (Barlow et al. 1986), emergence in a Thus, the circatidal timing systems clearly midge (Saigusa & Akiyama 1995), and larval contain a light-sensitive mechanism. Further release of a grapsid crab (Saigusa & Kawagoye evidence for this would be useful for under- 1997). In contrast, synchrony with the inequal- standing circatidal timing systems. This study ity of the tidal amplitude may not be of great reports the larval release activity of an advantage to the estuarine animals, including estuarine semi-terrestrial crab, Sesarma those living in the terrestrial habitat. Accord- erythrodactylum, monitored in the laboratory, ingly, the animal's tidal rhythm of behavior and discusses the tidal timing systems in may reflect only the 12.4-h (or 24.8-h) interval estuarine crabs. of the tides. Another problem is the light-sensitive sys- MATERIALS AND METHODS tems that are involved in the circatidal rhythms. Palmer (1995) supposed that day- Distribution of estuarine crabs in relation to night cycles can entrain only the circadian salinity fluctuations and tidal amplitude rhythms, and that they cannot be the entrain- The distribution of estuarine crabs is closely ing agent for circatidal rhythms. Most tidal related to salinity, substrate, and fluctuations rhythms reviewed by Palmer (1995) are related in water level (e.g., Ono 1965) . The habitats of to activities in which internal timing systems Sesarma erythrodactylum and other species of are not clearly participating, e.g., locomotor crabs were surveyed along a small river at activity patterns of fiddler crabs (Honegger Kasaoka, Okayama Prefecture, Japan (see 1973; Lehmann 1976), those of a shore crab also Saigusa 1982) . This investigation was made (Bolt et al. 1989; Reid & Naylor 1990), and the by visual inspections many times over the last swimming activity patterns of a fish (Northcott 15 years. et al. 1991) . Such activity patterns make it difficult to evaluate how an environmental cycle en- Collection of ovigerous females and mainte- trains the internal timing systems. nance in the laboratory

In contrast, the larval release activity pat- Ovigerous females were collected from the field terns of many estuarine and intertidal crabs (Fig. 2) in June and July in 1991-1996. They are controlled endogenously, and for some were brought into the laboratory and placed in crabs there are unequivocal evidence that the aquaria (70 cm long, 40 cm wide, and 25 cm circatidal rhythms respond to 24-h light-dark high). Each aquarium held a shallow pool of di-

(LD) cycles. For example, an estuarine terres- luted seawater (salinity about 2O%), and hiding trial crab, Sesarma haematocheir, exhibits a places made of boards were set above the sur- nocturnal tidal rhythm (Saigusa 1981, 1982). face of the water. The crabs were fed every few

This rhythm becomes desynchronized among days. The experimental rooms in the labora- individuals in constant darkness, but is main- tory were equipped with controlled light and tained for at least 3 weeks under a 24-h LD temperature. Temperature was 24•}1•Ž . Light cycle. In addition, this rhythm is easily phase- intensity was about 700-1200 lux at the floor shifted under a 24-h LD cycle changed in phase with the lights-on, and less than 0.01 lux with from that in nature (Saigusa 1986). The close the lights-off. Three experiments were carried correlation between the phase shift in the be- out under continuous light at 0.5-1.0 lux. havioral rhythm and this light cycle suggests The day of hatching is difficult to predict; the that the 24-h LD cycle sets the phase of the tidal only indication that hatching is imminent is the

22 Larval release of an estuarine crab

brownish green color of the embryos (mainly after being confined in the recording appara- caused by yolk consumption). The embryos of tus. each female were checked by eye every day, and The experimental procedures for S. those crabs carrying embryos that seemed erythrodactylum were basically the same as likely to hatch within a few days were each set those for S. haematocheir and S. pictum, and individually in a recording apparatus placed in they have already been described elsewhere the same room (Fig.1A). With this apparatus, (Saigusa 1986, 1992). A 500ml glass beaker (8.5 the time of larval release could be monitored cm in diameter, 12cm high) was used to hold without any change in the ambient lighting con- the crab's plastic cage (Fig.1A). Because the ditions (e.g., without switching on a red light to ovigerous females of S. erythrodactylum have a monitor the release) (Fig.1B). much smaller carapace (0.8-1.5cm) than that of S. pictum, the amount of seawater was re- Monitoring of larval release activity duced to 160-170ml. The number of ovigerous

The larval release recording system consisted females used in each experiment is described in of a sensor unit (infrared source and receiver: the text. Some females were released into the

E3S-2E4, Omron Co. Ltd., Japan) placed inside aquaria before being confined in cages. Fur- the experimental room and a control unit (S3S- thermore, sometimes the photoelectric switch

A10, Omron) and event recorder (R17-H12T, did not operate because the number of hatched

Fuji Electric Co. Ltd., Japan) placed outside. zoeas was very few. Because the time of the re-

Each ovigerous female was confined in a small lease in such cases was not recorded, they were plastic cage (6.5cm in diameter and 6.5cm excluded from the analyses. high) with many holes drilled in the sides and bottom. As shown in Figure 1A, this cage was RESULTS suspended by fine wires from the rim of a glass beaker containing diluted, clean seawater (sa- Habitat of crabs in the estuary linity about 20•ñ). The animals were not fed Sesarma erythrodactylum exclusively inhabits

Fig.1 . System for recording the larval release activity of Sesarma erythrodactylum females. A. The apparatus used to detect the time of day of larval release . w: fine wire. plc: plastic cage to confine an ovigerous female crab. gb: glass beaker. b: board . Is and Ir: infrared source and receiver. B. An example of an original record showing 6 out of 17 females releasing their larvae; small circles indicate the times of larval release . Some pulses before larval release are probably due to the female's legs emerging from the plastic cage , and many pulses after the release (ch. 12 and ch. 13) were gener- ated by the decrease in the number of swimming zoeas .

23 Vol.52,No.1 Benthos Research June,1997

Fig.2. Habitat of Sesarma erythrodactylum and those of other estuarine crabs. a: location where the field work was carried out. b: the field study site at Kasaoka (enclosed by an open rectangle). c: habitats of estuarine crabs at Kasaoka. Hs: Hemigrapsus sanguineus, Mj: Macrophthalmus japonicus, U: Uca lactea, Pb: Ptychognathus barbatus, Sp: Sesarma pictum, Sb: Sesarma bidens, Ht: Helice tridens, Cc: Chasmagnathus convexus, Sd: Sesarma dehaani, and Sh: Sesarma haematocheir. The influence of the tidal cycle extends at most 250m upriver (left) from the habitat of S. erythrodactylum shown by the arrow. d: cross section of the study site in the habitat of S. erythrodactylum.

sand flats covered with reed vegetation (Fig.2). larvae under water as S. pictum does (Saigusa The habitat is exposed to the air for about 5-6 1992). In the laboratory, the females repeatedly h at low water, and is submerged for about 6-7 flexed their abdomens inward and possibly h at high water. The salinity of the river made associated movements of the pleopods changed cyclically with the ebb and flow of the bearing the embryos. These pumping move-

tides (Saigusa 1982). At high water, it was al- ments swept clouds of newly hatched zoeas

ways 29-30•ñ. away from the female. Male and female crabs spend the winter hi- bernating in burrows dug into the bank quite Hatching of zoeas from ovigerous females close to the reeds, and they become active in Ovigerous females were randomly collected in May. In summer the females incubate their em- the field (Fig.2). Some were ready to release bryos beneath their folded abdomens, where larvae within a few days, whereas others car- the embryos are ventilated by movements of the ried embryos that seemed to have commenced pleopods. When embryonic development is com- incubation just a few days before. The latter fe- pleted, the females liberate newly hatched zoeas males always released larvae within 2-4 weeks into the water. For terrestrial species such as after collection (Figs.3-6), which suggests that Sesarma haematocheir, larval release behavior most females incubate their embryos for about is easily observed in the field, because the fe- three weeks. males release their larvae at the water's edge Hatching of Sesarma erythrodactylum is while vigorously fanning the abdomen and body highly synchronized among embryos. Figure 3 (see Saigusa 1982). However, the larval release shows two instances of the hatching profile. In behavior of S. erythrodactylum has not been one female (Fig.3a) almost all embryos observed in the field; the females may release hatched within 30min, and in another female

24 Larval release of an estuarine crab

the larval release was closely correlated with the tidal cycles (high water) for at least four weeks (Fig.4). Almost all females released their larvae at night. The timing of release coincided with high tide (HW2) for the first 10 days. By the natural progression of the tides, this high tide eventu- ally began to occur during lights-on, but then the release took place at the other high tide after sunset MW,) (Fig.4). The least squares regression line fitted to the data of larval release (RL) showed that the timing of release in the laboratory was delayed just 1-2h from the predicted times of high water in the habitat. The timing of release occurred within a few hours on either side of the regression line, indi- cating that larval release was highly synchronized among individuals. The period of the population's rhythm (based on the release data enclosed with in the ellipse in Fig.4) was estimated at 24.5h by Enright's periodogram (for this method, see Enright 1965a).

Larval release rhythm under constant conditions To determine whether the timing of hatching and larval release is controlled endogenously in each female, the larval release activity of the population was monitored under constant dim Fig.3. Hatching profiles of female-attached embryos light (LL) after collection, and this experiment (data on 9-10 July, 1994). The plastic cage holding the was repeated three times. In one xperiment ovigerous female was placed in a new beaker every 30 (Fig.5a; 33 specimens), the release coincided min, and the number of zoeas that had been released by with the time of high tide (HW2) at night only the female was counted. N: total number of released larvae. Dotted area shows the dark period. Arrows indicate for the first four days (28-31 July). In contrast the time of the high tide (0:20 on 10 July). to Figure 4, a large variability appeared among individuals thereafter, suggesting a desynchronization of the population's rhythm. Nev- (Fig.3b) hatching occurred within 1.5h. Pro- ertheless, the regression line (RL) showed that files for other females were similar. the releases were delayed only 1-3h past high tide. The free-running period (FRP) was esti- Timing of larval release under an artificial 24-h mated at 25.0h on the periodogram. day-night cycle in phase with that in the field Similar results were obtained in the other two To determine whether the timing of hatching experiments under constant dim light. In Figure and larval release corresponds to the 24-h day- 5b (35 specimens) a free-running rhythm is night cycle or to the tidal cycle at the local clear. This rhythm persisted for ten days after habitat, the larval release activity of the popu- collection. Because only two females released lation (45 specimens) was monitored under a larvae in the latter half of the experiment, the 24-h day-night (LD) cycle (15-h light: 9-h dark degree of the variability among individuals is photoperiod; LD 15:9), the phase of which was not known for this period. The FRP was 26.0h, much the same as that in the field (i .e., lights- with these two females included in the analysis. on at 5:00 and lights-off at 20:00). The timing of Forty-three specimens were used for the

25 Vol.52,No.1 Benthos Research June,1997

Fig.4. Daily timing of larval release by Sesarma erythrodactylum monitored under a 24-h light-dark (LD) cycle in the laboratory. Date of collection: 14 July, 1996. Forty-five crabs were used (black dots indicate the time of day of larval release by those females). For comparison, environmental cycles in the field are characterized by the times of sunset (SS) and sunrise (SR) (broken lines), by curves connecting the predicted times of day of high tides (HW1 and HW2), and by the phase of the moon(•›: full moon, •¬ and •¬: first and last quarters of the moon, •œ: new moon). The entire record is du- plicated on the right and displaced upwards one day, so that each day's data can be matched with those of the following day. Vertical lines indicate the times of lights-off and lights-on in the experimental room. A diagonal line (RL) is the least squares regression line fitted to the data of larval release (enclosed within an ellipse). The right panel shows the sta- tistical treatments of the data enclosed by the ellipse. Upper diagram: analysis by correlogram. Dotted line indicates

95% confidence limit. Lower diagram: analysis by periodogram. experiment shown in Figure 5c. A free-running periodograms (Figs.5a-c), but these data rhythm is also clear here, but desynchronization clearly indicate nonetheless that the internally appeared among individuals and increased fur- controlled period in each female is close to 24.8 ther during the latter half of the experiment. h, not 12.4h. Nevertheless, the FRP was easily estimated at 26 h, even with the inclusion in the analysis of Phase shift of the circatidal rhythm by an arti- the 8 females that released larvae during the ficial 24-h LD cycle changed in phase from that latter half of the period. in the field As shown in Figures 5a-5c, the rhythm of lar- As shown in Figure 4, almost all females re- val release free-ran in constant dim light, sug- leased their larvae at night. So the 24-h LD gesting that the timing of release is controlled cycle may mash the 24.8-h tidal rhythm during by an endogenous clock. Since this rhythm the light phase. To test this hypothesis, larval clearly coincided with the tidal cycle under a 24- release activity was monitored under h LD cycle (see Fig.4), the rhythm shown in photoperiods with the same LD-ratio (LD Figures 5a-5c should be regarded as the free- 15:9), but with different phases. In the control run of a tidal rhythm, and not a daily rhythm. population (47 females), the phase of the The FRP was somewhat different in each ex- photoperiod was similar to that in the field ( periment: i.e., between 25 and 26h on the i.e., lights-on at 5:00; lights-off at 20:00). In the

26 Larval release of an estuarine crab

Fig.5 . Time of day of larval release monitored under a regime of continuous light (LL: 0.5-1.0 lux) and no tidal influence in the laboratory . Date of collection: 27 July, 1992 (a), 21 July, 1995 (b), and 7 July, 1996 (c). Crabs used for each experiment: 33 specimens (a) , 35 specimens (b), and 43 specimens (c). Symbols are the same as in Figure 4.

27 Vol.52,No.1 Benthos Research June,1997 experimental population (51 females), the Endogenously controlled timing of larval re- phase was shifted 5-6h from that in the field lease (i.e., lights-on at 10:00; lights-off at 1:00) . If the The larval release activity of several estuarine rhythm is simply masked by the dark phase, and intertidal crabs has been monitored in the larval release should take place 1-2h after laboratory away from any influence of the tidal HW1 or HW2 during the 'dark period' (i.e., just cycle (e.g., Bergin 1981; Forward et al. 1982; De after high tides between 01: 00 and 10:00). Vries & Forward 1989, 1991). In these studies, As shown in Figure 6a, the phase of the tidal larval release was correlated with the high tides rhythm in the control population was delayed at the natural habitat. The present study offers only 1h from the tidal cycle at the habitat additional clear evidence that the timing of re- (compare HW2 with RL). The period of the lease is under the control of an endogenous population's rhythm (calculated from the data clock (Figs.5a-5c). points enclosed within the ellipse) was 24.6h. In In this work, each female released larvae just the experimental population (Fig.6b) the tidal once during the 2-4 week experimental period. rhythm was clearly phase-shifted (compare Since the free-running period of each individual HW1 with RL), and the release occurred at is somewhat different, we can probably assume night. The magnitude of the phase-shift (i,e., that constant light increases the variability of about 5h) corresponded to that of the light the internal period among females, desynchro- cycle between the field and experimental room nizine the population's rhythm (Figs.5a-5c). (6h for the shift between SS and lights-off, and Although the variability certainly increased 4.8h for the shift between SR and lights-on), for 3-4 weeks in continuous darkness, the free- and this indicates that the 24-h LD cycle does running rhythm of S. erythrodactylum was al- not mask the daytime expression of the tidal ways evident. On the other hand, in the coastal rhythm. terrestrial crab Sesarma pictum (Saigusa 1992) The period of the population's rhythm (Fig. and the intertidal crab Hemigrapsus 6b) was the same as in Figure 6a, and larval re- sanguineus (Saigusa & Kawagoye 1997), the lease occurred within a few hours on either side free-running period was evident for only the of the regression line (RL). This indicates that first half of the experiment (about 10 days) and the shifted rhythm is not desynchronized at all. was not clear thereafter. In S. erythrodactylum The 24-h LD cycle thus drives the phase of the the tidal rhythm is synchronized with either the circatidal rhythm, but this study could not de- two high tides, more exactly with the high tides termine whether the lights-on or lights-off in at night, showing a 24.5-24.6h periodicity (Figs. the 24-h LD cycle actually sets the phase of the 4, 6a & 6b) . In contrast, the circatidal rhythms tidal rhythm. of S. pictum and H. sanguineus are synchronized with both high tides, showing a 12.4h pe- DISCUSSION riodicity. Since the variablity of internal period increases as a function of day elapsed after the Sesarma erythrodactylum is a small crab in- crabs were kept under constant darkness, the habiting reed vegetation in estaurins. Females free-running rhythm of those crabs would have release their larvae at high tide at night. The become ambiguous apparently much earlier timing of release is clearly under the control of than that of S. erythrodactylum (Figs.5a-5c). an endogenous clock, because the tidal rhythm of a population free-ran in constant dim light. Loss of synchrony with tidal amplitude in On the other hand, the tidal rhythm was main- estuarine species and their nocturnal pattern tained at least for a few weeks under a 24-h LD The tidal cycle involves two major parameters, cycle the phase of which was similar to that in i, e., the mean period length of 12.4h, and the the filed. Furthermore, it was phase-shifted by semidiurnal inequality in the tidal amplitude. In an altered 24-h LD cycle. The 24-h LD cycle thus the Inland Sea of Japan, the two high and two participates in controlling the the endogenous low tides on a given day are usually of different rhythm in this species. These results raise the heights, and they recur at asymmetrical inter- following six topics for discussion. vals every two weeks. The influence of these

28 Larval release of an estuarine crab

Fig.6. Daily timing of larval release monitored under 24-h light-dark cycles in the laboratory. a (upper panel): the light cycle the phase of which was similar to that in the field (lights-off at 20:00, lights-on at 5:00). b (lower panel): light cycle changed in phase by 5-6h with respect to natural conditions. Times of lights-off and lights-on in the artificial light cyles are shown by vertical lines (lights-off at 1:00, lights-on at 10:00), and times of sunset and sunrise are marked by broken lines. For other symbols, see Figure 4. parameters is strongest in the intertidal zone. 1972; Enright 1972). Mating activity of the Intertidal animals presumably show activity horseshoe crab Limulus polyphemus is also patterns marked by these two parameters. highly dependent on the inequality of the high Enright (1963) first reported that the swimming tides (Barlow et al. 1986). Emergence of the activity rhythm of an amphipod population intertidal midge Clunio tsushimensis occurs (Synchelidium sp.) freshly collected on the during the lower low tides. In the Inland Sea of beach reflects the inequality of the tides; i.e., Japan morning low waters recede much further with greater activity during the higher high than afternoon low waters in winter, and the tides and lesser activity during the lower high tidal pattern is revesed in winter. Therefore, tides during the first few days after collection. emergence occurs in the early morning in win- The swimming activity of the isopod Excirolana ter, and in the afternoon in summer (Saigusa & chiltoni also shows a similar pattern (Klapow Akiyama 1995). Furthermore, larval release in

29 Vol.52,No.1 Benthos Research June,1997 the intertidal crab Hemigrapsus sanguineus was phase-shifted in response to an artificial also occurs in synchrony with high tides, but shift in the 24-h LD cycle (Figs.6a & 6b), we the timing of hatching is actually dependent on can assume that the light cycle drives the phase the lower low tides which initiate the 'hatching of the rhythm and sets it to the times of the ap- program' (Saigusa & Kawagoye 1997) . Do propriate high tide. estuarine animals also have activity patterns In S. erythrodactylum, however, the reflecting not only the 12.4-h period but also circatidal rhythm could be entrained by at least tidal amplitude? two kinds of timing cue in the field. The 24-h LD Concerning this question, the larval release cycle is one zeitgeber. Others related to tidal activity of the estuarine terrestrial crab cycles directly or indirectly must participate in Sesarma haematocheir has been extensively in- the phase control of the rhythm. A number of vestigated in both the field and laboratory studies have shown that cyclical or non-cyclical (Saigusa 1982, 1985, 1986, 1988) . The release of changes of hydrostatic pressure, salinity, and zoeas coincides with the high tide at night and, temperature cause behavioral responses (e.g. therefore, displays a tidal rhythm with a 24.5-h Enright 1962; Reid & Naylor 1990; Northcott et period. The higher high tide occurs at night in al. 1991) . Since these cues do not affect the in- the habitat of this species at Kasaoka (Fig. 2), ternal timing systems (see Enright 1962), they so the timing of larval release always coincides could not be zeitgebers of the circatidal rhythm with the higher high tide at this site (Saigusa of Sesarma. On the other hand, the swimming 1982, 1988) . However, the release of a local activity of intertidal isopods is under the con- population in Izu (Shizuoka Prefecture) occurs trol of an the endogenous clock, and the phase during the lower high tide at night (Saigusa responds to cycles of water turbulence (Enright 1981, 1985). This indicates that the larval re- 1965b, 1976; Klapow 1972). These factors might lease in the Kasaoka population (Fig.2) is not thus be the zeitgebers of Sesarma's circatidal dependent on inequality of the tidal pattern. rhythm, or the moonlight cycle might be related The pattern of S. erythrodactylum's tidal to the tidal entrainment. rhythm is similar to that of S. haematocheir's Kasaoka population (Fig.4) . Thus, as shown in Internal timing systems of S. erythrodactylum Figures 4, 6a & 6b, tidal rhythms of estuarine The activity patterns of most terrestrial ani- crabs are nocturnal without the benefit of syn- mals have the period close to 24-h, i, e., a cir- chronization with the semidiurnal amplitude of cadian rhythm, under constant dark or dim the tides. light conditions. Circadian timing systems are explained physiologically in terms of internal Effect of the 24-h LD cycle on the phase-shift, oscillators. For example, Pittendrigh and co- and other zeitgebers workers (Pittendrigh & Bruce 1959; Pittendrigh

As shown in Figures 5a-5c, the circatidal 1960, 1981) assumed two kinds of oscillator,

rhythm of S. erythrodactylum free-runs with a each with a period close to 24-h (A and B oscillators) . In their hypothesis concerning period of 25-26h. On the other hand, Figures 4, 6a & 6b show that the tidal behavioral rhythm Drosophila, while the A-oscillator reacts to the is controlled by the 24-h LD cycle and exhibits 24-h LD cycle and can control the phase of B-

a nocturnal pattern. To synchronize the timing oscillator, the B-oscillator controls directly the

of larval release with the times of high tides at timine of emergence (see also Saigusa 1986).

night, a switchover of synchrony between the As shown in Figures 5a-5c, the timing of lar-

two high tides must occur at intervals of two val release in our crab is also under the control weeks. But this has nothing to do with the inter- of an endogenous clock. Furthermore, the

nal timing systems (Figs.5a-5c) . The 24-h LD phase of the circatidal rhythm is actually con-

cycle functions to switch the synchrony of the trolled by the 24-h LD cycle (Figs.4, 6a & 6b).

larval release to the other high tide (HW1 The properties of Sesarma erythrodactylum's

HW2 or HW2•¨HW,) when the scheduled release circatidal rhythm are very similar to those of a

enters into the light phase in the 24-h LD cycle circadian rhythm in the following respects: 1)

(Fig.4) . Moreover, since the circatidal rhythm the rhythm is under the control of an

30 Larval release of an estuarine crab

well as the larval release rhythm could be en- endogenous clock; 2) the rhythm responds to a trained by the 24-h LD cycle reported here. 24-h LD cycle; and 3) the magnitude of the Furthermore, in Sesarma haematocheir phase-shift corresponds to the time difference between the natural and artificial LD cycles. (Saigusa 1986, 1988), the tidal and semilunar If Pittendrigh's hypothesis were modified a rhythms of larval release are entrained by the little, the circatidal rhythms of larval release in same zeitgebers (i, e., a 24-h LD cycle and a 24.8 intertidal and estuarine animals could be ex- h moonlight cycle), suggesting that semilunar timing may be involved in the circatidal rhythm plained more reasonably. This new model also assumes two kinds of oscillator with different itself. In short, circa-semilunar timing is certainly present in many animals living in the periods, i.e., an ƒ¿-oscillator with a period close to 24-h and a ƒÀ-oscillator with a period intertidal and estuarine environments, but its close to the tidal cycle (about 24.5h) . The ƒ¿- underlying timing mechanism is not known yet. oscillator is light-sensitive and can control the Comparison of the internal timing systems phase of the ƒÀ-oscillator. On the other hand, the ƒÀ-oscillator not only directly controls the among different species of crabs: a diversity of timing of larval release, but also can be en- circatidal clocks trained by tide-correlated or moonlight fac- In the estuarine environment the influence of tors. The ƒÀ-oscillator may also affect the a -o the tidal cycle decreases with the distance from scillator, but the ability to control phase this the ocean. This should affect the internal timing way would be much less than the control of a systems of estuarine organisms. The circatidal by a. rhythms of larval release can be explained

physiologically in terms of both the relative Semilunar timing strength of the light-sensitive driving a -oscilla

A further problem is the occurence of for and the change of the period in the driven semilunar timing in this species. Many females ƒÀ-oscillator. In the intertidal crab released larvae around full and new moons, but Hemigrapsus sanguineus, which is subjected to very few larvae were released around the quar- a strong tidal influence, the light-sensitive ƒ¿- ter moons (Figs.4, 5 & 6). The tidal rhythm of oscillator does not strongly drive the phase of reproduction in marine organisms is often as- the driven ƒÀ-oscillator; as a result, the popu- sociated with semimonthly fluctuations in the lation rhythm shows a large variability among 'number' or 'amount of activity' of individuals individuals, associated with the phase-shift of (e.g., Enright 1972; Neumann 1981; Yoshioka the rhythm (Saigusa & Kawagoye 1997) . On the

1989; Saigusa & Akiyama 1995). The semilunar other hand, the timing systems of estuarine rhythm was explained classically by the 'beat' animals, especially for those living in the upper of two different internal rhythms in an organ- regions of estuary, will be close to the circadian ism (Brown et al. 1953; Banning and Muler timing systems of terrestrial animals. In S.

1961). If an organism possesses two kinds of in- eryhtrodactylum, the driving oscillator could ternal rhythms with slightly different periods control the driven oscillator more strongly, so (e.g., 12h and 12.4h, or 24h and 24.8h), these the circatidal rhythm can be easily phase-set by rhythms would interact together , exhibiting a the 24-h LD cycle. In terrestrial crabs such as S. beat at intervals of 15 days or 30 days . This hy- haematocheir, S. intermdedium, and S. dehaani, pothesis, however, received no support thereaf- under very weak tidal influence, the period of ter (Enright 1972; Saigusa 1986) . Concerning the driven oscillator is much closer to that of the emergence of the intertidal midge Clunio , the circadian rhythm (i.e., less than 24.5h), es- Neumann (1981) considered that the timing of pecially in the Izu population (Saigusa 1988). Pupation is under the control of a circa- Furthermore, the driving oscillator could con-

Semilunar clock and that emergence is regu- trol not only the phase of the driven oscillator, lated by a the circadian clock . However, in the but also the phase jump every 15 days. Hence, intertidal environments the underlying rhythm the shift of the synchrony to the other high tide of emergence is tidal , not daily (Saigusa & would be controlled endogenously in these Akiyama 1995) , and this circatidal rhythm as crabs.

31 Vol.52,No.1 Benthos Research June,1997

Acknowledgments Enright, J. T. 1976 Plasticity in an isopod's clock- Mr. K. Oishi helped me to make the apparatus works: shaking shapes form and affects phase and for monitoring the larval release activity. Sta- frequency. Journal of Comparative Physiology, 107: tistical programs for personal computers were 13-37. made by Dr. N. Matsumoto (Department of Forward, R. B. Jr., K. Lohmann and T. W. Cronin Physics). Supported by Grants-in-Aid for Sci- (1982) Rhythms in larval release by an estuarine crab entific Research (C) from the Ministry of Edu- (Rhithropanopeus harrisii). Biological Bulletin, 163: cation, Science and Culture, Nos. 06839017 and 287-300. 08833009 (Marine Biology). Hastings, M. H. 1981 The entraining effect of turbulence on the circa-tidal activity rhythm and its semi- REFERENCES lunar modulation in Eurydice pulchra. Journal of the Marine Biological Association of the United King- Barlow, R. B. Jr., M. K. Powers, H. Howard and L. dom, 61: 151-160. Kass 1986 Migration of Limulus for mating: relation Honegger, H.-W. 1973 Rhythmic activity responses of to lunar phase, tide height, and sunlight. Biological the fiddler crab Uca crenulata to artificial tides and Bulletin, 171: 310-329. artificial light. Marine Biology, 21: 196-202. Bergin, M. E. 1981 Hatching rhythms in Uca pugilator Klapow, L. A. 1972 Natural and artificial rephasing of (Decapoda: Brachyura). Marine Biology, 63:151-158. a tidal rhythm. Journal of Comparative Physiology, Bolt, S. R. L., D. G. Reid and E. Naylor 1989 Effects of 79: 233-258. combined temperature and salinity on the Lehmann, U. 1976 Interpretation of entrained and free- entrainment of endogenous rhythms in the shore crab running locomotor activity patterns of Uca. In, Bio- Carcinus maenas. Marine Behaviour and Physiology, logical Rhythms in the Marine Environment, 14: 245-254. DeCoursey P. J. (ed.), Univ. of South Carolina Brown, F. A. Jr., M. Fingerman, M. I. Sandeen and Press, Columbia, pp. 77-91. H.M. Webb 1953 Persistent diurnal and tidal rhythms Neumann, D. 1981 Tidal and lunar rhythms. In, Biologi- of color change in the fiddler crab, Uca pugnax. Jour- cal Rhythms (Handbook of Behavioral Neurobiology, nal of Experimental Zoology, 123: 29-60 Vol.4), Aschoff J. (ed.), Plenum Press, New York, Bunning, E and D. Muller 1961 Wie messen Organismen pp. 351-380. lunare Zyklen? Zeitschrif t fur Naturf orschung, 16 Northcott, S. J., R. N. Gibson and E. Morgan 1991 The (b) : 391-395. effect of tidal cycles of hydrostatic pressure on the De Vries, M. C. and R. B. Forward Jr. 1989 Rhythms in activity of Lipophrys pholis (L.) (Teleostei). Journal larval release of the sublittoral crab Neopanope sayi of Experimental Marine Biology and Ecology, 148: and the supralittoral crab Sesarma cinereum 35-45. (Decapoda: Brachyura). Marine Biology, 100: 241- Ono, Y. 1965 On the ecological distribution of ocypoid 248. crabs in the estuary. Memoirs of the Faculty of Sci- De Vries, M. C. and R.B. Forward Jr. 1991 Control of ence, Kyushu University, Series E (Biology), 4: 1-60 egg-hatching time in crabs from different tidal (plus 5 plate pages). heights. Journal of Crustacean Biology, 11: 29-39. Palmer, J. D. 1995 The Biological Rhythms and Clocks Enright, J. T. 1962 Pressure sensitivity of an amphipod. of Intertidal Animals. Oxford University Press, New Science, 133: 758-760. York, 217 pp. Enright, J. T. 1963 The tidal rhythm of activity of a Pittendrigh, C. S. 1960 Circadian rhythms and the cir- sand-beach amphipod. Zeitschrift fur Vergleichende cadian organization of living systems. Cold Spring Physiologie, 46: 276-313. Harbor Symposia on Quantitative Biology, 25: 159- Enright, J. T. 1965a The search for rhythmicity in bio- 184. logical time-series. Journal of Theoretical Biology, Pittendrigh, C. S. 1981 Circadian systems: general per- 8: 426-468. spective. In, Biological Rhythms (Handbook of Be- Enright, J. T. 1965b Entrainment of a tidal rhythm. havioral Neurobiology, Vol.4), J. Aschoff (ed.), Science, 147: 864-867. Plenum Press, London, pp. 57-80. Enright, J. T. 1972 A virtuoso isopod: circa-lunar Pittendrigh, C. S, and V. G. Bruce 1959 Daily rhythms rhythms and their tidal fine structure. Journal of as coupled oscillator systems and their relation to Comparative Physiology, 77: 141-162. thermoperiodism and photoperiodism. In,

32 Larval release of an estuarine crab

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