Studies in Diurnal Rhythms: II. Changes in the Physiological Responses of the Wood-Louse Oniscus Asellus to Environmental Stimuli

In an earlier paper in this series, it was pointed out that diurnal rhythms are basically of two kinds: exogenous, a direct response to environmental changes, and endogenous rhythms which persist under constant conditions. For example, the West African millipede Ophistreptus sp. exhibits a 24 hr. periodicity under constant conditions for 19 days (Cloudsley-Thompson, 1951a). Many species of animals show a combination of both types and such rhythms are termed ‘composite’ by Park (1949).

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

Another factor to be considered is that although many species are active during a certain period of the day or night and are quiescent for the remainder of the 24 hours, some exhibit different kinds of activity at different times. Thus the water-skater Gerris spends the daytime on the surface of ponds and streams, but flies from one locality to another at night (Riley, 1925); many other aquatic insects such 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 concerned with establishing the existence of 24 hr. periodicities, and determining the ‘clues’ with which they are correlated (Calhoun, 1944). In the case of locomotory rhythms, little attention has been directed to the changes in physiological response which may be correlated with outbursts of activity. An example of this kind of change is afforded by the pill-woodlouse Armadillidium vulgare. This species is more resistant to desiccation than other species of Isopoda (Edney, 1951), and shows locomotory activity principally in the morning when it is often to be seen walking about in the sunlight (Cloudsley-Thompson, 1951c). This is probably correlated 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, however, and come out at night from their hiding places under stones, logs and bark, 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 is greater during the night, the temperature drops and light is absent. In this paper an attempt has been made to indicate how nocturnal changes in environmental conditions may alter the daytime reactions of Oniscus asellus, and thus engender the observed locomotory activity.

The species used in this investigation was Oniscus asellus L. collected from my garden in Esher, Surrey. Adult animals of both sexes were used, but these were not separated.

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

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

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

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

In 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 rather than in temperature.

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

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

Experiments were carried out on Oniscus asellus by a modification of the method described by Gunn (1937), using two choice-chambers identical with those employed in the investigation of the humidity responses of millipedes (Cloudsley-Thompson, 1951b). One choice-chamber was kept in fight, the other was darkened with a wooden cover. The air in one half of each arena was kept dry, using a mixture of sulphuric acid and distilled water calculated to give a relative humidity of 50 % (Buxton & Mellanby, 1934). On the other side, distilled water was placed beneath the perforated zinc gauze. A paper hygrometer recorded 60 and 90 % relative humidity respectively on the two sides of the arena. All experiments were carried out at room temperature 18 ±2 ° C. in subdued daylight during March and April I95I-

The apparatus was set up overnight, and on the following morning five woodlice were 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 boundary. After each reading, the animals were stirred up with a glass rod so that after each stirring there were either two animals on each side and one in the middle, or three on the side which had previously had two.

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

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

The intensity of the reaction was calculated in each case by dividing the number of stationary woodlice on the moist side of the arena plus half the number in the middle, by the number on the dry side plus half the number in the middle. It was also calculated using Gunn’s (1937) method in which those in the middle are omitted; but since they were considerably more numerous in the experiments carried out in darkness, they cannot in this case justifiably be ignored.

From the results (Table 1) it can be seen that not only was the intensity of the humidity 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 darkness for some days prior to the experiments. The larger number moving about in the choice-chamber in daylight (A) is due to their kinetic and tactic responses to fight (Abbott, 1918; Dietrich, 1931; Henke, 1930), and the figures in the second and fourth columns show that the intensity of the reaction increased as the animals became progressively desiccated. In addition, this increase was considerably greater in controls in darkness (B) than it was in the animals from the dark culture in darkness (C).

In a study of the reactions to light of Oniscus aseUus and two species of Porcellio, Abbott (1918) showed that the animals react negatively by means of both photokinesis and phototaxis, and that the response is the same at all intensities. In consequence in the experiments described below, no attempt was made to standardize the light intensity, diffuse daylight or darkness being offered on either side of the choice-chambers. In addition, Abbott suggested that Porcellio became somewhat less negative after living in a dry habitat, but that the reaction of Oniscus was essentially the same whether the animals had previously been exposed to strong light 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 Oniscus correlated with water loss by evaporation, and this was confirmed in the present work. Fraenkel & Gunn (1940) have shown that there is no need to postulate skototaxis in interpreting the results of Dietrich (1931) and Henke (1930) on the reactions of woodlice to light, since their orientation can be explained adequately in terms of negative phototaxis.

The responses of Oniscus from control cultures exposed to daylight were compared, as before, with those of animals from a culture kept in darkness. One half of each choice-chamber was darkened with a cover, the other was exposed to diffuse daylight. Ten woodlice were placed in each arena, and the number on the light side was 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 the other half of the chamber.

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

The response to carbon dioxide of woodlice from control cultures exposed to daylight 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 filterpaper and covered with a lid of Perspex having an inlet and outlet for air. The air was pumped first through a concentrated solution of potassium hydroxide to remove all traces of carbon dioxide, then bubbled through water and passed into the crystallizing dish from 15 to 30 min. until all the animals had come to rest. Carbon dioxide from a cylinder was then bubbled through water, and allowed to enter the air stream in controlled amounts. The approximate concentration entering the crystallizing dish was ascertained by comparing the number of bubbles of carbon dioxide per minute with the number of air bubbles (300). The maximum number of woodlice stimulated into activity (exhibited either by locomotion, or by intense movements 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 cultures kept in darkness, were carried out concurrently in February and March 1951, at room temperature, 16±1° C., between 10· 00 and 16· 00 hr. daily. The arena was exposed to daylight, but was considerably shaded, for animals from the dark cultures became very active in brighter light and would not otherwise come to rest. The woodlice were changed after one or two readings, because it was found that after an initial outburst of activity they frequently failed to respond to continued carbon dioxide. The response persisted after removal of the antennae.

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

These experimental results are readily interpreted in relation to the ecology of the species. Woodlice spend the daytime under stones, logs, and in other damp, dark places. If an animal shows such a tendency to aggregate in particular localities as a result of physiological responses, it is reasonable to expect a reversal or modification of these responses at some time or other to account for the dispersal of the species. The experiments described above give an indication of the changes that occur at night in the physiological responses of Oniscus asellus and are responsible for the nocturnal behaviour of this species.

A composite diurnal rhythm is correlated primarily with alternating light and darkness, and not with fluctuating temperature or humidity. Although none of these environmental factors is likely to vary much in the sheltered places where the animals aggregate during the day, the endogenous component of the rhythm will engender locomotory activity in some individuals at least, at nightfall. Even if the majority are exposed to daylight only occasionally, this ‘clue’ may be sufficient to keep their periodicity in phase with the 24 hr. cycle.

Although the carbon dioxide in the atmosphere does not fluctuate greatly from day to night, the increased sensitivity of the responses to this gas under experimental conditions provides an illustration of the fact that woodlice are more responsive generally at night, are more easily disturbed then, and more readily exhibit locomotory activity.

The intensity of their humidity responses decreases at night, at any rate until the animals become somewhat desiccated, and this permits them to walk in drier places than those in which they pass the day; and the increased photo-negative response in darkness ensures that they get under cover at daybreak. In this way no doubt many potential predators are avoided. Perhaps this increase in the light response 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 not restrained there until they die from desiccation, for they become photo-positive in dry air and thus are able to wander in the open until they find some other damp hiding place when they again become photo-negative.

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