J. Exp. Biol. (197a), 57. 375-391 375With iz text-figures

Printed in Great Britain

THE LOCOMOTOR ACTIVITY RHYTHM OF TALITRUSSALTATOR (MONTAGU) (CRUSTACEA, AMPHIPODA)

BY P. K. BREGAZZI* AND E. NAYLORfDepartment of Zoology, University College of Swansea

{Received 8 February 1972)

INTRODUCTION

Previous studies on the sun-orientation of Mediterranean Talitrus saltator (Mon-tagu) have revealed the presence of an internal timing mechanism which operates atsolar frequency (Papi & Pardi, 1953, 1959; Pardi, i960). Transfer of animals to alongitude different from that where they originated showed that the angle betweenthe sun and the direction of escape at a given time is fixed for a given population(Papi, 1955). The timing basis of the navigational mechanisms in Talitrus and inTalorchestia deshayesi (Audouin) can be re-set by artificially changing the light/darkcycles. Thus animals subjected to a light/dark cycle delayed by 6 h show an angle ofescape which is at approximately 900 to that of controls (Pardi & Grassi, 1955).These authors also showed in Talitrus that raising the temperature from 35 to 37 °Cslightly accelerated the timing process which controls the angle of orientation, andsimilar effects are reported in Orchestia platensis Kroyer, even in regular light/darkconditions (Jankowsky, 1969). There is therefore a great deal of evidence to suggestthat talitrids which navigate using a sun-compass mechanism have an endogeneoustime-sense.

In British localities there is so far little evidence of a time-sense in Talitrus since itsnavigational ability seems more largely based on visual orientation to the silhouettesof sand-dunes (Williamson, 1951). It therefore seemed worth while to investigatelocomotor rhythmicity in this species and to assess the persistence of any endogeneouscomponent of the rhythm.

MATERIAL AND METHODS

Talitrus saltator is a semi-terrestrial amphipod inhabiting non-permanent burrowsin sand somewhat above high-water mark, so that burrows are found lower down theshore during neap tides than on springs. The animals emerge at dusk and foragebetween the level of the burrows and the water's edge. When seaweed, upon whichthey largely feed, has been washed up in sufficient quantity, they do not move sofar, most remaining around the strand-line. Geppetti & Tongiori (1967) also reportvariability in nocturnal migrations in Mediterranean Talitrus, depending uponavailability of food at the water's edge. The same authors report that high temperatureand low humidity may inhibit migration. Present observations suggest that surfaceactivity is also reduced during rain. Around dawn, animals return to the burrow region,

• Present address: The College, Cheltenham, Gloucestershire.t Present address: Marine Biological Station (University of Liverpool), Port Erin, Isle of Man.

376 P. K. BREGAZZI AND E. NAYLOR

perhaps partly guided by solar navigation, but mainly by visual stimuli from silhouettesof sand-dunes (Williamson, 1951).

On the coast of South Wales animals overwinter in sand well above high water,at depths from 30 to 100 cm. They appear on the shore during nights about the end ofApril and remain active at night until about late October. Pallault (1954) considers atemperature of 10 °C to be critical for the seasonal activity of Normandy populations,and present observations suggest that this is also true for South Wales populations.Animals maintained throughout the winter in the laboratory at room temperature andunder natural light regimes displayed normal activity patterns. Normal behaviourwas also shown by animals collected from the field during the winter and broughtinto the laboratory.

Experimental animals were collected by night at Oxwich Bay, near Swansea.They were located with a torch and picked up by hand. They survived well in thelaboratory in glass tanks containing moist sand and under normal light conditions.They were fed occasionally with seaweed and pieces of apple. Large males, easilydistinguished by long, orange first antennae, were used in all experiments unless other-wise stated.

The actographs were rectangular boxes 75 x 150 x 250 mm constructed of 3-2 mmperspex and painted black on the outer surface. Each box contained two wells75 x 75 mm filled with moist sand to a depth of 75 mm and into which the experi-mental animals were able to burrow. The two wells were separated by a perspexplatform at the same level as the sand surface. A dim red light beam passed across theplatform and was focused sharply by a lens (focal length 10 cm) on to a photo-transistor (OCP 71) taped to the outside of the box. The perspex platform was neces-sary to prevent animals throwing sand against the photo-transistor while burrowing.The light source was a 6-5 V, 0-3 A bulb, painted black except for a small slit andfitted with a red filter. The intensity of the light was controlled by a variable resistorof 100 fi and a very dull light was found to be adequate.

The photo-transistor was connected to a circuit slightly modified from that ofSouthward & Crisp (1965), so that when an animal crossed the platform and inter-rupted the light beam, the resulting change in resistance of the photo-transistor wasrecorded as a mark upon the moving chart-paper of a 'Rustrak* event recorder.Power for the circuit was provided by a mains-operated transformer producing arectified current at 12 V. Greatest sensitivity of the photo-transistor was found to occurwith minimum power supply, achieved by use of a variable resistor of 1 kQ.

Each actograph had a well-fitting clear perspex lid, and the experiments werecarried out in a constant temperature room, at 15 °C unless otherwise stated, usuallywith a dim red background illumination of immeasurably low intensity.

Preliminary experiments showed that the most consistent results were obtainedusing groups of four animals in each actograph. Activity is expressed as number ofevents for each hour, usually in histogram form. Also, on account of the clear-cutand unimodal nature of the activity rhythm, it is possible to calculate the time of themid-point of an activity period, and this is given, when appropriate, to the nearesto-oi h. All times are expressed as G.M.T.

M

1

t

3 22•a

43

NII

Time of day (t>

M N M

I

, X» Jjwfcmotor activity of a group of four Talitrus over 46 days in continuous dim red background illumination. The densityof dots is proportional to the activity recorded for each hourly period. M = midnight, N = noon.

378 P. K. BREGAZZI AND E. NAYLOR

15 -

8

3O

5(2

06-

Time of day (h)12

Fig. 2. Percentage total hourly activity for the first 24 h period of 20 groups of fourTalitrus in Him red background illumination.

RESULTSEffects of light

Many experiments were conducted in dim red light, and the animals showed a clear-cut unimodal circadian rhythm which persisted for as long as the experiments were run(up to 46 days). In the longer experiments there was some diminution of activityafter the first 2 weeks or so. In most cases the interval between successive activitypeaks was slightly more than 24 h, and there was no sign of a twice daily or tidalcomponent.

Fig. 1 shows the activity of a group of four animals over 46 days. For the first 21days the mean period is 24-49 n (S-D- ± °'49)- After about this time there is a clearincrease in mean period (25-49 n> s.D. ±2-10) and the rhythm is not so precise inappearance or regular in period.

The percentage total hourly activity for each hour of the first activity period of20 groups of four animals in constant dim red light is given in Fig. 2. Maximumactivity occurs between midnight and 01.00 h. These experiments were carried outbetween late May and late November, and no seasonal differences in activity patternswere noted.

To test the effects of continuous white light three groups of four freshly collectedanimals were placed in actographs at 15 °C and 0-9 m vertically below a 100 W lightbulb (about 200 lux). Activity was recorded for 5 days and then for a further 3 days indim red background illumination. A control actograph was maintained in continuousdarkness throughout by means of light-proof covers. The results are given in Fig. 3,in which the control animals show a normal activity rhythm, with a period of slightlymore than 24 h (Fig. 3 a). The activity of those under constant illumination, however,is almost entirely suppressed in two cases. In the third actograph (Fig. 3 d) durationof activity is restricted and its onset is delayed by an hour or two, but a circadianrhythm is still apparent. In the 3 days under dim red illumination following continuous

Locomotor activity rhythm of T. saltator 379

50 -(a)

(b)

(0

fc-(d)

i

i *M M M M M

Time (h)M

Fig. 3. Activity of groups of four Talitrui: (a) in continuous darkness, (b)-(d) in continuouslight (about 200 lux) for 5 days, followed by dim red background illumination. Shadedarea = continuous dnrlrnem or dim red background illumination, M = midnight.

light the period and approximate phase of the rhythm are seen to have been main-tained, despite the previous suppression of activity by light. The inactivity of animalsunder continuous light is probaby accounted for by there being a threshold value oflight intensity, above which the animals remain in their burrows. Moreover, sincethere was a little surface activity during the light-on phase of these experiments, it ispossible that the threshold may be in the region of the light intensity used, i.e. 200 lux.

In another experiment run simultaneously, modified actographs were used in whichthe perspex platform extended throughout the box and there was no sand availablefor burrowing. A few small pieces of moist cotton wool were introduced to maintainhumidity and to provide a substratum on which the animals could rest when inactive.The results are given in Fig. 4. The control group of animals in continuous darknessshow a normal circadian activity rhythm (Fig. 4 a), but the rhythm of the three groupsof animals under continuous illumination is somewhat different. With these groupsthe onset of activity is at first somewhat delayed, but thereafter the activity rhythmhas an average period which is clearly less than 24 h. The mean periods for two setsof experimental animals in Fig. \{b, c) are 22-2 h and 23-08 h, while that for the con-trols (Fig. 4 a) is 24-36 h. In the subsequent continuous dim red light the advancedtiming of the rhythm is maintained relative to the control animals.

The animals in these modified actographs under continuous illumination wereunable to avoid the light by burrowing, and this apparently resulted in the differentperiod of the rhythm. The advance of the timing of the rhythm does not appear to becomplete with one group of animals (Fig. \d), perhaps due to a slower response by oneor two of the four animals tested. The bimodal pattern persists in subsequent constantdim red light.

To test for the effectiveness of light in synchronizing the rhythm four freshly col-lected animals were placed in an actograph with sand 0-9 m below a 100 W lamp(about 200 lux) and subjected to a regime of 12 h light and 12 h darkness with the

380 P. K. BREGAZZI AND E. NAYLOR(e

venl

vity

Act

i

50

0

50

0

50

0

50

0

J•

-

k

M M M MTime (h)

M M M

Fig. 4. Activity of groups of four Talitna in actographs with no sand: (a) in continuous dark-ness, (b-d) in continuous light (about 200 lux) for 5 days, followed by dim red backgroundillumination. Symbols as in Fig. 3.

' Expected'night

' Expected'night

Fig. 5. Mean hourly activity over 24 h, of groups of four Talitna under continuous dim redbackground illumination for 3 days, following artificial cycles of 12 h darkness and 12 h lightfor 3 days: (a) in actographs with sand, (6) in actographs with no sand. Experimental treatment:O •= about 200 lux, © = about 40 lux, • = control animals under continuous darkness.

onset of darkness beginning at 12 noon. The mid-darkness point was thus 6 h inadvance of normal, and the onset of darkness was about 8 h in advance of normal.Another actograph was maintained under the same hght regime, but 1-2 m horizon-tally from the first one, at about 40 lux. A control actograph was kept beneath light-proof covers. After 3 days of experimental light/dark, the actographs were recorded indim red light for 3 days, and the mean hourly activity over 24 h during this time isi

Locomotor activity rhythm of T. saltator

6 7 8 9Days of experiment

10 11 12 13 14 15

Fig. 6. Time of mid-activity on successive days of groups of four Talitrus under dim red back-ground illumination at different temperatures: O = 15 °C, ffl = 20 °C, • = 25 °C;C = 17-22 °C, natural light/darkness ( x =• accidental rise in temperature).

shown in Fig. 5 a. The timing of the rhythm of the animals exposed to the new lightregime using 200 lux is largely rephased to the ' expected' night, but that of theanimals at 40 lux is virtually unchanged and close to that of the controls.

In another experiment run simultaneously, modified actographs were used. Theseprovided no sand in which the animals could burrow but small pieces of moist cottonwool were introduced to maintain humidity. Mean activity over 24 h for the 3 daysof constant dim red background light, following the experimental light/dark treatmentas described above, is shown in Fig. 5(6). Animals in the actographs at 200 lux and40 lux both re-synchronized their activity rhythms to match the new light regime. Boththese experiments were repeated twice, with similar results. Clearly light is a majorentraining factor for Talitrus. The failure of the animals to re-synchronize when insand under intermittent dim light at 40 lux is no doubt accounted for by their beingin burrows. They were probably unable to detect the light-intensity difference duringthe change from light to darkness. All these experiments suggest that under normalconditions Talitrus relies on a light/dark cycle with a light-phase threshold of between40 and 200 lux.

Effects of temperatureIn initial experiments to test the effects of temperature on the rhythm three acto-

graphs were maintained in three separate constant-temperature rooms at 15, 20 and25 °C, and in continuous dim red light. The activity of four animals in each actographwas recorded simultaneously for 15 days. A further actograph was maintained undernatural light conditions at temperatures which fluctuated between 17 and 22 °C.At the time of collection of the animals in the field the air temperature was 10-5 °C,and they were introduced into the actographs within 3 h of this time.

23 EXB 57

382 P. K. BREGAZZI AND E. NAYLOR

- 4

•o 3'g"3 2U

*- 1

24

4 5 6 7 8Days of experiment

10 11

Fig. 7. Time of mid-activity upon successive days of two groups of four Talitrus in dim redbackground illumination at 15 °C. x , One group (closed circles) transferred to 23-5 °C from13.00-19.00 h.

The results, expressed as time of mid-activity points on successive days, are given inFig. 6. The animals at fluctuating temperatures and in normal light/dark conditionsare seen to maintain the timing of their rhythm, with mid-activity around midnight.The animals at 15 °C likewise maintain the timing of their rhythm, showing, in thiscase, no appreciable drift, probably because they were subjected to a 4-5 °C rise intemperature on being brought in from the field. Both groups of animals at the highertemperatures have an activity mid-point which is advanced to well before midnightduring the first few days of the experiment, but after 10 days there is very little dif-ference between them and the other groups of animals. On day 8 the 15 °C constant-temperature room suffered a mechanical failure, and between 03.00 h and 15.00 h thetemperature rose to 20 °C. An advance of about 3 h occurred in the succeeding mid-activity point, but this again was compensated for within two days.

In another experiment at 15 °C in continuous dim red light, a group of animals wastransferred to a constant-temperature room at 22-5 °C from 13.00 -19.00 h on day 4and then returned to 15 °C. An advance of about 3 h occurred in the succeedingmid-activity point (Fig. 7), but after 4 days that of both control and experimentalgroups of animals occurred at about the same time. These results seem to suggestthat an increase in temperature results in a temporary shortening of the period of therhythm, when an apparently temperature-sensitive part of the timing mechanismruns fast for a few days. Compensation occurs possibly by a more basic temperature-independent timing mechanism, and this seems to take somewhat longer if the animalsare maintained at the increased temperature.

To assess the role of temperature as a synchronizer of the rhythm the effects ofregular temperature fluctuations in constant dim red light were tested by subjectingtwo actographs, each with four animals to alternating temperatures at 15 and 25 °C at12 h intervals, so that one actograph was out of phase with the other. This involvedcarrying the actographs a few yards between two constant-temperature rooms. A

Locomotor activity rhythm of T. saltator 383

fa

p

M

Fig. 8. Activity of three groups of four Talitrus in continuous Him red background illumina-tion, under I 2 : i a h temperature fluctuations of 15:25 °C (a, b) and continuous 15 °C(c). M = midnight.

control actograph was maintained at 15 °C and was disturbed in the same way as theexperimental actographs at 12 h intervals. The changes took place at 08.00 and 20.00 hfor 4 days. The animals exposed to the higher temperature during the day (Fig. 8a)do not show an advance in the timing of their rhythm to the same extent as has beenreported above. However, the timing of the rhythm remains nearly constant for the4 days, unlike that of the control animals whose rhythm drifted by about 4 h (Fig. 8 c).There is no difference between the control animals and those exposed to a reversedtemperature cycle, with higher temperatures during the expected night. It seems thata temperature cycle with higher temperatures during the day may help to maintain thetiming of the locomotor rhythm, but a temperature cycle which is the reverse of this,even with a difference of 10 °C, does not cause re-synchronization of the rhythm.Longer-term experiments may prove otherwise.

Effects of density of animals

Single animals in the actograph displayed locomotor rhythms whose period andduration often differed markedly from one another. Activity was reduced, and some-times even absent, for the entire duration of the experiment. In all these cases theanimals were healthy at the end of the experiment. On the other hand, groups of fouranimals gave consistent results. The mid-activity points of 15 single animals and 8groups of four animals in experiments of 10 days' duration in constant dim red lightare given in Fig. 9. In each case the mid-activity points follow a pattern to which alinear regression line can be fitted to confirm a constant rate of drift of activity peaks.The two regression coefficients are significantly different (P < o-ooi) with the singleanimals losing time (0-87 h/day) twice as fast as the groups of four animals (0*39 h/day). It seems likely that the timing mechanisms of single animals have periods whichdiffer between individuals, and that the animals tend to influence one another so that

25-a

P. K. BREGAZZI AND E. NAYLOR

0O

I24

23

2210

Days of experiment

Fig. 9. Time of mid-activity of eight groups of four animals (O) and 15 single animals(•) on successive days of 10-day experiments. Regression equations are y = 1-53 + 0-87*(single animals) and y = 3-49 + 0-30* (groups).

Time (h)

Fig. 10. Total activity of a group of four and a group of 32 Talitnu in continuousHim red background illumination. M = midnight.

as a group they phase their activity peaks to the timing of those animals showing leastdrift.

Groups of 8, 16 and 32 animals in the usual actographs all show much less driftof activity peaks than single animals. The results of an experiment with a group of4 and a group of 32 animals is shown in Fig. 10. The phasing of the two activity pat-terns are very similar, each with a period of only slightly more than 24 h. A furtherpoint emerges from these results in that the amount of activity recorded by the 32-(

Locomotor activity rhythm of T. saltator

1000

-

/ J

J

MI

M

w L• . i

IM M

Time (h)

c

7 I

Q

10

11

12

13

14

Fig. 11. Activity of four groups of six Talitrus in continuous dim red background illumination,(a) Six untreated animals, (6, c) three untreated + three chilled animals, (d) six chilled animals(chilled animals previously exposed to 3 °C so that timing of activity rhythm is about 12 h outof phase with untreated animals). M = midnight.

animal group in each spell of activity is much less than eight times that of the 4-animal group, so it is reasonable to suggest that a high density of animals will to someextent inhibit the activity of individuals. In most cases the time at which activity ceasesis somewhat later in the 32-animal group, and this is probably because it takes indivi-duals longer to find an unoccupied burrow or find space to make a new one.

386 P. K. BREGAZZI AND E. NAYLOR

Table 1. Summary of mutual entrainment experiments

Numbers ofanimals of

different phase

4 : 12 : 22 : 2

2 : 2

3 :15:1

10:10

Phasedifference (h)

812

1 2

1 2

1 0

87

Number ofexperimental

dishes

1622

4441

Duration ofexperimental

contact (days)

536

17649

In view of the differences in period recorded between the activity rhythms of singleanimals and groups of animals, and the inference that individuals are able to influencethe time-keeping abilities of one another, attempts were made to re-synchronize thelocomotor rhythm of Talitrus by contact with other animals possessing rhythms thatwere out of phase. The fact that chilling can apparently retard or even stop the timingmechanism of Talitrus (Bregazzi, 1972) makes it reasonably easy to obtain two groupsof animals, each possessing a rhythm which is out of phase with the other. In pre-liminary experiments, differences in phase between 'chilled' and 'normal' animalswere maintained in separate actographs for up to 19 days.

The term ' mutual entrainment' has been used previously to denote the entrainmentof more than one supposed internal timing mechanism within a single animal bymutual interaction (Harker, 1964). In the present account the term is used to denotethe entrainment of individual animal's rhythms by contact with other rhythmicallyactive individuals. A number of different experiments failed to reveal an unmistakablecase of mutual entrainment. In the first experiment three animals, entrained to naturallight conditions, were introduced into an actograph together with three animals thathad been chilled so that their locomotor activity pattern was 12 h out of phase withthe first group. Two such actographs, together with controls of normal and chilledanimals, were monitored in dim red background illumination for 15 days (Fig. n ) .At first there are two distinct peaks per day, each following closely the phase of thecontrol animals as indicated by the subjective shading on the histograms. Thereafter,activity declines and in one case is completely absent for a few days. It is then resumedat the previous level, but with each peak in an intermediate position between thetwo controls and difficult to relate by shading. It seems that two groups of animalsthat are sufficiently out of phase with one another can maintain the asynchrony of theirlocomotor rhythms, even in the same actograph. While one group was active, the otherwas buried in the sand, and the two groups need not necessarily have come into con-tact at all. It remains to investigate the possibility that the two groups of animalsinteracted to result in an overall reduction of activity, while the subsequent reappear-ance of a phased-shifted rhythm also requires investigation.

Since in the experiments so far described the animals could avoid each other insand, additional experiments were carried out using 12 cm glass dishes fitted withperspex lids and containing only about 3 mm of damp sand, into which animals couldpartly burrow when inactive, but which was not deep enough for them to disappearentirely beneath the surface. Normal and chilled animals in the same dish were(

Locomotor activity rhythm of T. saltator 387

M M M

Time(h)

Fig. 12. Activity of four groups of Talitrus in continuous dim red background light, usingmixtures of chilled animals 8 h out of phase with unchilled animals, (a) Six unchilled animals(controls). (6) Six unchilled animals, having been in contact with chilled animals for 4 days.(c) Six chilled animals, each having been in contact with five unchilled animals for 4 days.(d) Six chilled animals (controls). M = midnight.

identified by being of different sex, or by the tip of one first antennal flagellum beingremoved. Animals so treated were allowed 3 or 4 days to recover from wounding,and behaved normally. The animals were tested for 3 days in actographs followingsome days in the experimental dishes. All experiments were conducted under dimred background illumination.

The number of experiments carried out, together with their duration, the numbersof animals used and the differences in phase are given in Table 1. In no case did anyanimal obviously alter its phase to that of the group in contact. A typical result, inwhich groups of ' normal' males were in contact for 4 days with chilled males 8 h outof phase, is given in Fig. 12.

An additional point of interest has emerged from these experiments. It was foundthat the rate at which the locomotor activity peaks of animals in the dishes with littlesand drifted across natural time varied considerably and was usually at a greater ratethan in those animals kept in sand into which complete burrowing was possible.kThe difference in period between different groups of animals was not great enough to

388 P. K. BREGAZZI AND E. NAYLOR

affect the interpretation of the results if the time spent in the dishes was not mor«than a few days, but it imposed a serious limitation upon this method for longer-terminvestigations of mutual entrainment. In one experiment two control dishes, bothcontaining groups of animals with naturally synchronized rhythms to begin with, were8 h out of phase with each other after 17 days.

DISCUSSION

The locomotor activity rhythm of Talitrus under conditions of constant temperatureand dim red background illumination has a regular circadian period of somewhatmore than 24 h. It persists for several weeks, which indicates that it has a very signifi-cant endogeneous component. The shortening of the period of the rhythm underconstant illumination (Fig. 4) is contrary to Aschoff's rule (Pittendrigh, i960), whichstates, in part, that the rhythms of dark-active species have a shorter period in constantdarkness than in constant light and that the period of the rhythms in constant lightincrease with increasing intensity of illumination. Another probable reversal ofAschoff's rule has been reported for Aedes aegypti (L.) (Taylor & Jones, 1969).

Present experiments show that light-intensity cycles are likely to be the chief,and perhaps the only, entraining agent for Talitrus under natural conditions. A changein time of activity to coincide with the dark period, under artificial light conditions6 h out of phase with normal daylight, has also been reported by Pardi & Grassi0955)-

Ercolini (i960) describes the locomotor activity of Mediterranean Talitrus as noc-turnal under normal light/dark conditions and, in some populations, as having pos-sible bimodal characteristics. No such bimodal pattern occurred in the locomotorrhythm in the present study under constant conditions (Fig. 2), and if it does exist inthe field, it may be due to activity representing the search for a suitable burrowing sub-strate as dawn approaches. The difference between Mediterranean and South Walespopulations may also be related to the different ecological situations in these regions.In South Wales, Talitrus is a largely intertidal scavenger; in the Mediterranean thereis a restricted intertidal region and animals move inland to feed, returning to burrowin sand near the water's edge at dawn. Furthermore, due to differences in latitude, thesummer-active South Wales populations experience a shorter night than that experi-enced by the Mediterranean populations, and this may have restricted the develop-ment of a bimodal activity pattern. Geppetti & Tongiori (1967) suggest that sunrisecauses the seaward migration in Mediterranean Talitrus, the animals having movedinland to forage at the time of the previous sunset.

Although some organisms do possess rhythms which can be entrained by regulartemperature fluctuations (Pittendrigh, 1954; Roberts, 1959; Williams & Naylor, 1969),it is not surprising that experimental temperature cycles failed to change significantlythe timing of the locomotor rhythm of Talitrus. This is because the animals lie buriedin sand during the day, where the effect of daily temperature fluctuations is likely tobe small and often irregular. It follows that adaptation to temperature cycles alonecould hardly have arisen. Pardi & Grassi (1955) report that the re-setting of thenavigational timing mechanism of Talitrus by artificial light/dark cycles proceedsin constant temperature just as readily as when there is an imposed cycle of hightemperature during the light period and low temperature during the dark period.

Locomotor activity rhythm of T. saltator 389

Near or complete independence of rhythms from the effects of different ambienttemperatures has been reported for many organisms (e.g. Brown & Webb, 1948;Pittendrigh, 1954; Biinning, 1958; Naylor, 1963). For TaUtrus, it seems that anincrease in temperature causes a temporary advance of the timing mechanism con-trolling locomotor activity. An increase in temperature some time before the expectedtime of activity results in a delay in the timing of the locomotor rhythm and a loweringof the temperature advances of the timing of the rhythm in both Cardnus (Naylor,1963) and in Blattella germamca (L.) (Drei9ig & Nielsen, 1971) but not in TaUtrus.

Compensation for the effects of increase in temperature in Talitrus occurs spon-taneously after a few days (Figs. 6, 7), which suggests a dual control mechanism, partlytemperature-sensitive and partly temperature-independent. A similar coupled dualmechanism has been advanced by Pittendrigh & Bruce (1959) for the control of theeclosion rhythm in Drosophila. Two levels of temperature sensitivity have also beensuggested for Cardnus by Naylor (1963) in view of the different effects of coolingwithin the normal temperature range, which increases locomotor activity, and chillingto temperatures below that normally experienced by the animal, which suppressesactivity and inhibits the timing mechanism which controls locomotor rhythmicity.

In the field, Talitrus is protected to a large extent from fluctuations in air tempera-ture by being several centimetres below the sand surface during daytime whenfluctuations are more likely to occur. The small or gradual temperature changesthat the animal might encounter can probably be accommodated by the endogenoustiming mechanism which is itself, in any case re-synchronized every day by lightchanges.

No clear demonstration of mutual entrainment emerged from the laboratoryexperiments, but this does not necessarily invalidate the inference from the investiga-tions with single animals and groups of animals that it does occur. The experimentsemployed phase differences of between 7 h and 12 h in order that any mutual effectwould be clear. In view of the results, it is likely that mutual entrainment is onlypossible between animals which possess relatively small phase differences, such asare found between single animals after free-running for 24 h. This makes unequivocaldemonstration of mutual entrainment all the more difficult.

The clear-cut and persistent nature of the locomotor timing mechanism of Talitruspoints to a strong adaptive significance, although Ruppell (1967) does not come tothis conclusion for the locomotor rhythm of Orchestia platensis. Aschoff (1964) con-siders that a physiological oscillatory mechanism confers an advantage in its own right,because the periodic increases in amplitude allow, at certain times, higher levels ofenergy expenditure and hence, perhaps, of efficiency (alternating with low-levelquiescent periods) than could be achieved by a non-oscillating system for the sameoverall expenditure of energy. Be this as it may, in view of the fact that the vastmajority of organisms live in a cyclically fluctuating environment of one sort or an-other, there is an obvious advantage in terms of energy conservation in having thephysiological oscillatory system correctly synchronized with the environment. In thecase of Talitrus the best time for feeding and mating contacts is clearly the night-.time, when humidity is high, rate of evaporation low and bird predators are absent.

Although the animals are sometimes disturbed by tidal action, especially duringspring tides and storms, there has been no advantage in developing a timing mechanism

390 P . K. BREGAZZI AND E. NAYLOR

of tidal frequency, for Talitrus is a supra-littoral species, opportunist in feeding habits|and not necessarily dependent upon excursions into the inter-tidal zone for food orshelter. Furthermore, regular synchronization of a timing component of tidal frequencywould clearly be difficult to achieve. Wildish (1970) found that Orchestia gammareUa,which is found beneath wrack and stones around high-water mark, possesses an endo-genous locomotor rhythm of circadian frequency only, but O. mediterranean whichlives somewhat lower down the shore, has endogenous rhythms of both circadianand tidal frequency.

The endogenous timing mechanism is of further importance to a burrowing organ-ism such as Talitrus for it provides a means whereby the fluctuations of the environ-ment can be predicted and so prepared for, without constant reference to them.Although Talitrus burrows may remain intact during daytime and allow informationof light intensity to reach the animal, more often than not the burrows collapse nearthe entrance, particularly when the surface sand dries out. Furthermore, the mannerof burrowing, namely scooping the sand with the gnathopods and first two pairs ofpereieopods, passing it between the last three pairs of pereieopods and then flickingit backwards with the pleon, is such that the entrance is often blocked before theanimal has finished its burrowing activity. On account of the endogenous' clock', excur-sions to the surface need only be made as the time for locomotor activity approaches,and so the animal does not require information about light intensity during the day,and also, should the burrow remain open, the animal is not 'misled' by spurious lightfluctuations.

Another adaptive advantage of the endogenous timing mechanism of Talitrus isseen in the much-studied ability to navigate by reference to the sun. It is not possibleat present to state whether this mechanism is the same as that which controls thetiming of the locomotory rhythm. However, they do have certain features in common.Both are re-synchronized by light/dark changes and both appear to 'run fast'initially when subjected to a temperature rise of more than a few degrees. Rates ofadvance which are similar under free-running conditions have been demonstratedin both the activity and navigational timing mechanisms of starlings by Hoffmann(i960). The lack of 'valuable results' from navigational experiments by Pardi &Grassi (1955) after Talitrus was chilled may be due to the same sort of differentialeffect of chilling upon the navigational timing mechanism as occurs with the loco-motor activity rhythm (Bregazzi, 1972).

SUMMARY

1. Talitrus saltator possesses a clear-cut locomotor activity rhythm, largely underthe control of a persistent endogenous timing mechanism of circadian frequency. Indim red illumination the period of the rhythm is somewhat greater than 24 h; incontinuous white light it is less than 24 h, provided that the animals are unable toavoid the light by burrowing. The rhythm is synchronized by light/dark fluctuations,and the animal is active during the dark period.

2. The timing mechanism is temporarily advanced by increases in temperature,but otherwise it possesses a large measure of temperature independence within thenormal environmental range.

Locomotor activity rhythm of T. saltator 391

3. There is some evidence that, in groups, the animals influence one another andso reduce the rate of drift of the activity rhythm in constant conditions.

4. The adaptive significance of the endogenous timing mechanism of Talitrus isdiscussed.

We are grateful to Mr R. G. James for helpful discussion, Professor E. W. Knight-Jones for the provision of laboratory facilities and the Science Research Council forfinancial support.

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