295

STUDIES IN DIURNAL RHYTHMS

II. CHANGES IN THE PHYSIOLOGICAL RESPONSES OF THE WOOD-LOUSE ONISCUS ASELLUS TO ENVIRONMENTAL STIMULI

BY J. L. CLOUDSLEY-THOMPSON

From the Department of Zoology, King's College, University of London

{Received 19 July 1951)

(With Three Text-figures)

INTRODUCTIONIn an earlier paper in this series, it was pointed out that diurnal rhythms are basicallyof two kinds: exogenous, a direct response to environmental changes, and endo-genous rhythms which persist under constant conditions. For example, the WestAfrican millipede Ophistreptus sp. exhibits a 24 hr. periodicity under constantconditions for 19 days (Cloudsley-Thompson, 1951a). Many species of animalsshow a combination of both types and such rhythms are termed 'composite' byPark (1949).

Endogenous rhythms are frequently correlated with environmental changes suchas light, temperature and relative humidity. As they are not a direct response tothese changes, however, Dr D. L. Gunn has suggested that it is preferable to referto them as 'clues' rather than 'stimuli'.

Another factor to be considered is that although many species are active duringa certain period of the day or night and are quiescent for the remainder of the24 hours, some exhibit different kinds of activity at different times. Thus thewater-skater Gerris spends the daytime on the surface of ponds and streams, butflies from one locality to another at night (Riley, 1925); many other aquatic insectssuch as beetles, fly mostly at night, but swim actively throughout the day.

Most of the work on rhythmic behaviour in animals has, in the past, been con-cerned with establishing the existence of 24 hr. periodicities, and determining the'clues' with which they are correlated (Calhoun, 1944). In the case of locomotoryrhythms, little attention has been directed to the changes in physiological responsewhich may be correlated with outbursts of activity. An example of this kind ofchange is afforded by the pill-woodlouse Armadillidium vulgare. This species ismore resistant to desiccation than other species of Isopoda (Edney, 1951), andshows locomotory activity principally in the morning when it is often to be seenwalking about in the sunlight (Cloudsley-Thompson, 1951c). This is probablycorrelated with the fact that in this species some individuals exhibit positive photo-taxis when the temperature rises (Henke, 1931).

The majority of woodlice such as Oniscus and Porcellio spp. are nocturnal, how-ever, and come out at night from their hiding places under stones, logs and bark,

J. L. CLOUDSLEY-THOMPSON

and wander in dry places where they are not found during the day. For example,they are often to be seen climbing up walls after dark. Atmospheric humidity isgreater during the night, the temperature drops and light is absent. In this paperan attempt has been made to indicate how nocturnal changes in environmentalconditions may alter the daytime reactions of Oniscus asellus, and thus engender theobserved locomotory activity.

MATERIALThe species used in this investigation was Oniscus asellus L. collected from my gardenin Esher, Surrey. Adult animals of both sexes were used, but these were notseparated.

ESTABLISHING THE RHYTHMDiurnal periodic locomotory activity was investigated using the Aktograph apparatusdescribed by Gunn & Kennedy (1936). Instead of the original long writing lever,however, a short rod was connected with a gymbal lever, and a clockwork baro-graph drum was employed as a kymograph in place of a 12 inch motor-driven drum.

20

15

10

5

I 201

151

10

J JIII 1

30

20

10

30

20

10

10Days

Fig. 1. Activity of Oniscui, in damp surroundings under natural lighting and in darkness at roomtemperature; and later in darkness at room temperature followed by artificially fluctuatingtemperature reaching a minimum during the day.

The object of these modifications was to reduce the overall size of the apparatus sothat it could be placed in a large incubator, and light and temperature controlledartificially.

The floor of the arena was lined with damp filter paper so that the humiditywas maintained at a high level, and did not fluctuate with temperature.

The results obtained are plotted as block histograms (Fig. 1), and show thatlocomotory activity at room temperature is mostly confined to the hours of darkness.

Studies in diurnal rhythms 297

Th preliminary experiments the arena was exposed to normal daylight and darkness,and fluctuating temperatures, the latter being registered on a recording thermometer.When, however, light was excluded, the periodicity disappeared after a few days,suggesting that the rhythm is 'composite', the 'clues' fluctuation in light ratherthan in temperature.

Fluctuations in temperature alone did not maintain the rhythm, but it wasimmediately resumed in alternating light and darkness.

Throughout the work it has been assumed therefore, that if woodlice are keptfor several days in darkness, their normal periodicity is eliminated and the behaviourof such animals resembles that of control animals at night: check tests have con-firmed this.

DIURNAL FLUCTUATIONS IN THE HUMIDITY RESPONSEExperiments were carried out on Onucus asellus by a modification of the methoddescribed by Gunn (1937), using two choice-chambers identical with those em-ployed in the investigation of the humidity responses of millipedes (Cloudsley-Thompson, 1951 b). One choice-chamber was kept in light, the other was darkenedwith a wooden cover. The air in one hah0 of each arena was kept dry, using a mixtureof sulphuric acid and distilled water calculated to give a relative humidity of 50 %(Buxton & Mellanby, 1934). On the other side, distilled water was placed beneaththe perforated zinc gauze. A paper hygrometer recorded 60 and 90 % relativehumidity respectively on the two sides of the arena. All experiments were carriedout at room temperature 18 ±2° C. in subdued daylight during March and April

The apparatus was set up overnight, and on the following morning five woodlicewere placed in each arena. Their positions were noted at intervals of 15 min.Animals moving were counted separately, as were any within 1*5 cm. of the boun-dary. After each reading, the animals were stirred up with a glass rod so that aftereach stirring there were either two animals on each side and one in the middle, orthree on the side which had previously had two.

After ten readings giving fifty position records, the animals were returned to thecultures. The apparatus was rotated between experiments so that external factorswere cancelled out, and the positions of the perforated zinc platforms, and of thesides and lids were interchanged. The intensity of the reaction was so marked thaton one occasion a slight leak of acid into the distilled water was immediatelydetected through the abnormal behaviour of the woodlice in the arena.

Two series of experiments were carried out. In the first, the responses in lightof animals from a control culture exposed to daylight were compared with those ofsimilar animals in the darkened choice-chamber. In the second series, the responsesin light of control animals were compared with those of animals in the darkenedchoice-chamber which had previously been kept in darkness at room temperaturefor several weeks (Table 1).

The intensity of the reaction was calculated in each case by dividing the numberof stationary woodlice on the moist side of the arena plus half the number in the

298 J. L. CLOUDSLEY-THOMPSON

middle, by the number on the dry side plus half the number in the middle. It wasalso calculated using Gunn's (1937) method in which those in the middle areomitted; but since they were considerably more numerous in the experimentscarried out in darkness, they cannot in this case justifiably be ignored.

Table 1. Number of looodtice moving or stationary on the dry (50 % R.H.), middle ormoist (100 % R.H.) sides of humidity choice-chamber apparatus.

A. MovingOn dry sideIn middleOn moist side

TotalIntensity of reaction(Gunn'8 method)

Intensity of reaction(alternative method)

B. MovingOn dry sideIn middleOn moist side

TotalIntensity of reaction(Gunn's method)

Intensity of reaction(alternative method)

C. MovingOn dry sideIn middleOn moist side

TotalIntensity of reaction(Gunn's method)

Intensity of reaction(alternative method)

Totalsof first

halves ofexperiments

" 32 1

49317

5°o—

—

233231

164

250—

—

74654

143

250—

—

Percentagesof first

halves ofexperiments

2 2 64-29-8

63-4

1 0 0

i 5 - I

7 5

9 21 2 81 2 4656

1 0 0

S-I

3-8

2 818-32 1 657-3

1 0 0

3 - i

3-3

Totalsof secondhalves of

experiments

871324

376

500

—

—

173

16214

250—

—

97

332 0 1

250—

—

Percentagesof secondhalves of

experiments

17-42 64 8

75-2

1 0 0

28-9

6-81-2

6-485-6

1 0 0

7i-3

2O'3

3 62 8

1 3 280-4

1 0 0

2 8 7

9 3

Grandtotals

2 0 0

3473

693

1000

—

—

403547

378

500—

—

i 75386

344

5°o—

—

Percentagiof grand

totals

2O-O

3-47-3

6 9 3

1 0 0

2 0 4

1 0 4

8 07 09-4

75-6

1 0 0

io-8

6 9

3-4io-617-268-8

1 0 0

6-5

4 a

A, controls in light; B, controls in darkness; C, animals in darkness from a culture kept in darkness for sevedays previously. Room temperatures 18 ±2° C.

From the results (Table 1) it can be seen that not only was the intensity of thehumidity reaction of woodlice in darkness (B) considerably less than in controls (A),but it was still further reduced in animals (C) which had been kept in darknessfor some days prior to the experiments. The larger number moving about in thechoice-chamber in daylight (A) is due to their kinetic and tactic responses to light(Abbott, 1918; Dietrich, 1931; Henke, 1930), and the figures in the second andfourth columns show that the intensity of the reaction increased as the animalsbecame progressively desiccated. In addition, this increase was considerablygreater in controls in darkness (B) than it was in the animals from the dark culturein darkness (C).

Studies in diurnal rhythms 299

DIURNAL FLUCTUATIONS IN THE RESPONSE TO LIGHTIn a study of the reactions to light of Ontscus aseUus and two species of Porcellio,Abbott (1918) showed that the animals react negatively by means of both photo-kinesis and phototaxis, and that the response is the same at all intensities. Inconsequence in the experiments described below, no attempt was made to stan-dardize the light intensity, diffuse daylight or darkness being offered on either sideof the choice-chambers. In addition, Abbott suggested that Porcellio becamesomewhat less negative after living in a dry habitat, but that the reaction of Ontscuswas essentially the same whether the animals had previously been exposed to stronglight or to dark, and whether kept in a maximum or minimum of moisture. Waloff(1941), however, found a reversal from negative to positive phototaxis in Ontscuscorrelated with water loss by evaporation, and this was confirmed in the presentwork. Fraenkel & Gunn (1940) have shown that there is no need to postulateskototaxis in interpreting the results of Dietrich (1931) and Henke (1930) on thereactions of woodlice to light, since their orientation can be explained adequatelyin terms of negative phototaxis.

The responses of Ontscus from control cultures exposed to daylight were com-pared, as before, with those of animals from a culture kept in darkness. One halfof each choice-chamber was darkened with a cover, the other was exposed to diffusedaylight. Ten woodlice were placed in each arena, and the number on the light sidewas noted at intervals of 15 min. The animals were stirred up after each reading,the same precautions being taken as before, and the dark cover was moved to theother half of the chamber.

In one set of experiments the floor of the arena was of damp filter-paper, inanother the floor was of voile on zinc gauze covering a mixture of sulphuric acidand distilled water calculated to produce a relative humidity of 50 % (Buxton &Mellanby, 1934). The experiments were carried out at room temperature,19-5 ± 20 C, during June and July 1951. At higher temperatures the differences inthe responses seemed to be less clear. The results obtained are given in Fig. 2 fromwhich it can be seen that animals from the dark cultures were more strongly photo-negative than the controls, and they tended to remain photo-negative at 50 %relative humidity whereas the controls became more positive in dry air.

DIURNAL FLUCTUATIONS IN THE RESPONSE TO CARBON DIOXIDEThe response to carbon dioxide of woodlice from control cultures exposed to day-light was compared with that of animals kept in darkness for a week or more.Groups of ten woodlice were placed in a crystallizing dish lined with damp filter-paper and covered with a lid of Perspex having an inlet and outlet for air. The airwas pumped first through a concentrated solution of potassium hydroxide to removeall traces of carbon dioxide, then bubbled through water and passed into thecrystallizing dish from 15 to 30 min. until all the animals had come to rest. Carbondioxide from a cylinder was then bubbled through water, and allowed to enter theair stream in controlled amounts. The approximate concentration entering the

300 J. L. CLOUDSLEY-THOMPSON

crystallizing dish was ascertained by comparing the number of bubbles ofdioxide per minute with the number of air bubbles (300). The maximum number ofwoodlice stimulated into activity (exhibited either by locomotion, or by intensemovements of the antennae) in a period of 5 min. was plotted graphically (Fig. 3).

Two sets of experiments, one with controls, the other with animals from cultureskept in darkness, were carried out concurrently in February and March 1951, atroom temperature, i6+ i°C. , between io-oo and 16-00 hr. daily. The arena was

Dark Light

Animals from

a culture kept

in darkness

Animals froma culture keptin darkness

3, ^ ^ ^ _100 50 0 50

Percentage of woodlice

Fig. 2. Response of Oniscus to light, on a damp surface and at 50 % relative humidity.Controls and animals from a culture kept in darkness for some days previously.

exposed to daylight, but was considerably shaded, for animals from the darkcultures became very active in brighter light and would not otherwise come torest. The woodlice were changed after one or two readings, because it was foundthat after an initial outburst of activity they frequently failed to respond to continuedcarbon dioxide. The response persisted after removal of the antennae.

From the difference between the two regression lines in Fig. 3, it is obvious thatanimals which had been kept in darkness were more sensitive to carbon dioxidethan were the control animals; and check tests after dark showed that the latterwere much more sensitive at night than during the day.

DISCUSSIONThese experimental results are readily interpreted in relation to the ecology of thespecies. Woodlice spend the daytime under stones, logs, and in other damp, darkplaces. If an animal shows such a tendency to aggregate in particular localities asa result of physiological responses, it is reasonable to expect a reversal or modifica-tion of these responses at some time or other to account for the dispersal of thespecies. The experiments described above give an indication of the changes that

Studies in diurnal rhythms 301

at night in the physiological responses of Oniscus aseUus and are responsiblefor the nocturnal behaviour of this species.

A composite diurnal rhythm is correlated primarily with alternating light anddarkness, and not with fluctuating temperature or humidity. Although none ofthese environmental factors is likely to vary much in the sheltered places where the

100

90

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10

t •

I I I I4 5 6 7

Carbon dioxide In the air stream (%)

10

Fig. 3. Response of Oniscus to carbon dioxide. Controls • • (means ©). Animals froma culture kept in darkness for some days previously x x (means (55).

animals aggregate during the day, the endogenous component of the rhythm willengender locomotory activity in some individuals at least, at nightfall. Even if themajority are exposed to daylight only occasionally, this 'clue' may be sufficient tokeep their periodicity in phase with the 24 hr. cycle.

Although the carbon dioxide in the atmosphere does not fluctuate greatly fromday to night, the increased sensitivity of the responses to this gas under experi-mental conditions provides an illustration of the fact that woodlice are more respon-

jEB.29, 2 20

302 J. L. CLOUDSLEY-THOMPSON

sive generally at night, are more easily disturbed then, and more readily exhibrFlocomotory activity.

The intensity of their humidity responses decreases at night, at any rate untilthe animals become somewhat desiccated, and this permits them to walk in drierplaces than those in which they pass the day; and the increased photo-negativeresponse in darkness ensures that they get under cover at daybreak. In this wayno doubt many potential predators are avoided. Perhaps this increase in the lightresponse at night could be regarded as a kind of conditioning to darkness.

On the other hand, if their daytime habitat should dry up, the woodlice are notrestrained there until they die from desiccation, for they become photo-positivein dry air and thus are able to wander in the open until they find some other damphiding place when they again become photo-negative.

SUMMARYA composite diurnal locomotory rhythm has been established in the woodlouseOniscus asellus. This is correlated primarily with alternating light and darkness,and not with fluctuating temperature or humidity.

The intensity of the humidity response of the species is less in darkness thanin light, and less still in darkness when the animals have been kept in darknessfor some days previously. It increases with desiccation. The response to light isgreater, too, in animals which have been kept in darkness for some days, and thesetend to remain photo-negative in dry air whereas controls become photo-positivewith desiccation. The sensitivity to carbon dioxide is much greater in animals froma culture kept in darkness than it is in controls.

The results obtained are discussed in relation to the nocturnal ecology of thespecies.

My thanks are due to Dr D. L. Gunn for the loan of his aktograph apparatusand for a number of stimulating discussions, to Dr G. P. Wells for suggesting theuse of a barograph clockwork motor as a miniature kymograph, and to my wife forher constant help and advice.

REFERENCESABBOTT, C. H. (1918). Reactions of land Isopods to light. J. Exp. Zool. 37, 193-246.BUTTON, P. A. & MELLANBY, K. (1934). The measurement and control of humidity. Bull. Ent. Res.

as, i7i-5-CALHOUN, J. B. (1944). Twenty-four hour periodicities in the animal kingdom. Part 1. The

Invertebrates. J. Term. Acad. Sd. 19, 170-200, 252-62.CLOUDSLEY-THOMPSON, J. L. (1951a). Studies in diurnal rhythms. 1. Rhythmic behaviour in

millipedes. J. Exp. Biol. 38, 165-72.CLOUDSLEY-THOMPSON, J. L. (19516). On the responses to environmental stimuli and the sensory

physiology of millipedes (Diplopoda). Proc. Zool. Soc. Lond. iai, 253—77.CLOUDSLEY-THOMPSON, J. L. (1951c). Rhythmicity in the woodlouse ArmadilUdium vulgare. Ent.

Mon. Mag. 87, 276-7.DIETRICH, W. (1931). Die lokomotorischen Reaktionen der Landasseln auf Licht und Dunkelheit.

Z. toiss. Zool. 138, 187-232.EDNEY, E. B. (1951). The evaporation of water from woodlice and the millipede Glomeris. J. Exp.

Biol. 38, 91-115.

Studies in diurnal rhythms 303L, G. S. & GUNN, D. L. (1940). The Orientation of Animals. Oxford University Press.

GUNN, D. L. (1937). The humidity of the woodlouse, Porcellio tcaber (Latreille). J. Exp. Biol. 14,178-86.

GUNN, D. L. & KENNEDY, J. (1936). Apparatus for investigating the reactions of land arthropods tohumidity. J. Exp. Biol. 13, 450-59.

HENKE, K. (1930). Die Lichtorientierung und die Bedingungen der Lichtstimmung bei der Rollassel,Armadtilidiwn cineraan Zenker. Z. vergl. Physiol. 13, 534-625.

PARK, O. (1949). Ch. 28 in Allee, W. C, Emerson, A. E., Park, O., Park, T. & Schmidt, K. P.Principles of Animal Ecology. Philadelphia & London: W. B. Saunders.

RILEY, C. F. C. (1925). Some aspects of the general ecology of the water-strider, Gerris ntfoscutel-latus Latreille. III. Ent. Rec. 37, 107-14.

WALOFF, N. (1941). The mechanisms of humidity reactions of terrestrial Isopods. J. Exp. Biol.18, H5-35-