Busy bees need rest, too

J Comp Physiol A (1988) 163:565-584 Journal of Comparative s o . ~ , Neural,

and Physiology A Behavioral Physiology

�9 Springer-Verlag 1988

Busy bees need rest, too * Behavioural and electromyographical sleep signs in honeybees

Walter Kaiser Institut fiir Zoologie der Technischen Hochschule Darmstadt, Schnittspahnstrasse 3, D-6100 Darmstadt, Federal Republic of Germany

Accepted March 28, 1988

". . . how very remarkable.., the psychic energy and the activity of these agile.., insects.., whose 'manual' abilities and 'intellec- tual' capacities are a never-ending source of amazement for us. In view of these intense expressions of existence, the industrious workers have earned a few hours of rest, and we should be able to understand that they, too, have a more intense requirement for rest, that insects also 'become weary and sleep'."

(Fiebrig 1912)

Summary. 1. The behaviour of isolated individual forager honeybees during the night has been investigated with a variety of experimental methods. Prolonged rest in these diurnal insects is accompanied by: reduced muscle tone (Figs. 1, 6, 10-12), decreased motility (Figs. 2, 3, Table 1), lowered body temperature (Figs. 7, 8) and raised reaction threshold (Fig. 9). These phenomena strongly re- semble four characteristic features of sleep in humans, mammals and birds. It is thus very likely that the profound rest which forager bees experience at night is sleep. This assumption is further supported by the results of previous investigations of visual interneurones in the bee.

2. The antennae of sleeping bees manifest characteristic postural constellations (Fig. 6). High reaction thresholds are associated with particular antennal positions.

3. The total sleep time (duration of antennal immobility plus durat ion of small antennal move- merits) in 24 h for two bees was 7.6 h and 4.9 h (Table 1).

4. Bees which rest in a hive at night also display phenomena which have been encountered during the laboratory investigations.

Abbreviations: EEG electroencephalogram; EMG electromyogram; R E M rapid eye movement; IR infrared radiation; LD alternating light (L) and darkness (D); LL continuous light; DD continuous darkness; S W S slow wave sleep

* Dedicated to Prof. Dr. D. Burkhardt on the occasion of his 60th birthday

5. Sleep in mammals is an active, controlled process; the same seems to be true of sleep in honeybees (Figs. 3, 4). Unlike mammals, bees experience their deepest sleep towards the end of the sleep phase (Figs. 3, 9, 10, 12).

Introduction

The possibility that forager bees, which are diurnal animals, experience sleep-like states first emerged during the course of electrophysiological recordings from single, higher-order interneurones in the third optic ganglion (lobula) of the honeybee's brain (Kaiser and Steiner-Kaiser 1983; Kaiser 1983). The key result of these experiments was that, during episodes of reduced neuronal respon- siveness at night, the sensitivity of the neurones could be rapidly, albeit temporarily, restored by mechanical or strong visual stimulation of the animals. These data were surprisingly similar to results obtained by Livingstone and Hubel (1981) during investigations of the properties of visual interneurones in sleeping and waking cats. It thus seemed promising to search for further evidence for the presence of sleep-like states in the honeybee.

It is now accepted that some of the daily, regu- larly recurring inactivity (rest) seen in mammals and birds is comparable to human sleep. This infer- ence is based on the many similarities between sleep signs in humans and the phenomena accompanying rest in birds and mammals (Borb61y 1984; Mayes 1983). Sleep signs are behavioural and physiological parameters which are used to objec- tively define the state we call sleep (Tobler 1984). In bees, EEG-data are not available. The present paper therefore compares the phenomena accom-

566 W. Kaiser: Behavioural and electromyographical sleep signs in honeybees

panying rest in these insects with other characteristic features of sleep in humans and other homeotherms. On the basis of the data presented here, I believe that it is justifiable to apply the term sleep to bees, too.

Attempts to interpret rest in non-homeotherms as sleep may lead to an inadvertent neglect of significant, species-specific phenomena, since the term sleep is anthropocentric. Intuitive deductions, common at the start of this century and exempli- fied by the citation at the beginning of this paper, are not compatible with modern, experimental sleep research. However, in the case of the work reported here, the term sleep was helpful because it provided the framework for an experimental analysis of events occurring during rest in bees and did not restrict our attention solely to the rest/ activity rhythm.

The results presented below have been obtained with the aid of a variety of experimental approaches. Accordingly, this paper is divided into three sections (I-III) and a general discussion of the most significant results.

Preliminary reports of part of this work have appeared elsewhere (Kaiser 1984, 1985).

I. The natural behaviour of bees at night

Methods All the members of a colony of honeybees (Apis mellifera L.) normally remain in the hive at night. The behaviour of bees in the hive can be observed with the aid of a single-frame observation hive (von Frisch 1965). Our observation hive consisted of an upper and a lower comb and contained several thousand animals. It was located in a small t imber house at the edge of a patch of forest. The hive entrance was connected to the surroundings outside via a short piece of tubing. The room containing the hive was heated up to 25 ~ in order to prevent excessive cooling of the colony when the insulating outer covers of the hive were removed. Bees were observed on warm summer nights, during stable periods of good weather. Observations were made for brief periods under dim incandescent illumination,

During one night, a thermovision camera with display screen (Thermovision 782, A G E M A Infrared Systems) was kindly put at my disposal. The brightness of the points on the screen is a measure of the relative temperature of the objects imaged by the camera. The camera viewed the whole of one side of the observation hive. The glass window of the hive had to be replaced with transparent wrapping foil, since window glass is fairly impermeable to infrared radiation.

brood is absent, as well as on the wooden walls of the hive and on the glass panes. Only a small number of bees could be seen occupying empty cells, although these were available in large numbers. These bees' heads were always directed towards the closed ends of the cells. Animals which rested on horizontal surfaces provided the first behavioural evidence for the existence of a sleep-like state: the bodies of several of these bees had sunk- en down so close to the substrate that the mandi- bles made contact with the latter. The tone in the leg muscles had obviously decreased. The antennae of these animals were observed to occupy a particular position. This postural constellation was subsequently also observed in the laboratory and ex- amined there in detail (see Fig. 6 B, a-d). The same antennal position could be seen in many of the bees which rested on vertical surfaces in the hive. Most of these bees rested with their heads pointing upwards. The majority of the stationary animals maintained body contact; some of them held each other's legs. Resting bees showed very few or no movements of the head, body or legs; the antennae were seen to move more frequently. Increasing the brightness of the lighting had very little observable effect on the behaviour of the bees.

Most of the active bees were concentrated around the brood area, in the central portion of the comb. The queen bee could often be observed laying eggs in this area.

Since honeybees are obliged to maintain a temperature of about 35 ~ in the brood area, it was conceivable that the bees which were apparently motionless were, in fact, engaged in producing heat by activation of the thoracic musculature. This possibility could be ruled out, however, with the help of relative temperature measurements made with the thermovision camera. The motionless bees at the outer edges of the comb were indistinguish- able from the background i.e., both bees and background were at the same temperature. Only one large bright patch was visible on the screen. This patch corresponded to the brood zone. A few bees which were engaged in comb-building in the 'dark ' periphery of the comb appeared as bright spots on the screen. These active bees were thus producing heat, in contrast to the resting bees around them.

Results

The first impression obtained was that many bees were moving around in the hive. Closer examina- tion revealed, however, that there were many animals sitting still at the edges of the comb, where

Discussion

The phenomena which can be observed when bees remain stationary in an observation hive at night can be interpreted as sleep signs (Tobler 1984) in these insects. The sleep signs which resting hive

W. Kaiser: Behavioural and electromyographical sleep signs in honeybees 567

bees display include decreased body temperature and muscle tone, reduced motility, relative in- sensitivity to strong (visual) stimuli and a tendency to adopt a preferred resting posture and to rest at a preferred location in the hive. Since no information about the social 'status ' (e.g. foragers, nurses, cell-cleaners etc.) was available - only un- marked bees were observed - it was not possible to correlate any sleep signs with social r61e in the colony.

Lindauer (1952) observed a marked forager bee, in an observation hive, without interruption over 2 days and nights. This bee (Nr. 16) engaged in no observable activities ('idling') for, on average, approx. 78% of the nighttime hours and approx. 48 % of the daytime hours. The amount of inactivity during the day was probably atypically high, since unfavourable foraging conditions prevailed at the time. Nevertheless, this bee displayed more resting behaviour at night than during the day. Forager bees have been used exclusively in all the laboratory experiments described below.

Our observations with the thermovision camera showed that bees which rested on the outer regions of the combs during the night were not engaged in heat production. This finding is in agreement with results obtained with different techniques by Esch (1960).

II. Observations and experiments on single, intact bees in the laboratory

The behaviour of individual honeybees in a hive can be influenced by a variety of factors (e.g. age, weather, food availability, etc.) which are not amenable to experimental control. Detailed observations on single forager bees were therefore performed under controlled conditions in the laboratory. The data presented in this section were obtained either from unrestrained honeybees which could move around in a chamber (Part 1) or from fixed animals mounted above a lightweight treadwheel (Part 2), Methodological details pertaining to each procedure are described under the appropriate sub-headings.

A necessary precondition for statistical analysis - pooling of data - could not be fulfilled in the experiments described below because the animal samples were not homogeneous. This inhomogen- city derives from two major sources. (1) Most of the bees came from outdoor hives. Under normal conditions, any one bee forages at only one source. Since flowers-species differ with respect to the times of day at which they offer nectar/pollen, different bees undertake foraging flights at various

times during the day (von Frisch 1965). (2) Bees were collected at different times of the year and were thus adapted to various day-lengths. Data analysis was therefore performed by first arranging the data from each individual into appropriate graphical form. Similarities and differences between the graphs were then investigated. The data clearly displayed uniform trends within each experimental series. In addition, there was considerable conformity between key results obtained with different experimental approaches.

Part ! : Observations on unrestrained bees

Since sleep/rest is characterized by reduced motility, the experiment described below was designed to investigate motility changes in honeybees. In a series of preliminary experiments, a time lapse movie camera recorded the behaviour of individual bees in a small lucite chamber which contained some comb-cells. Five bees were filmed during a total of 310.5 h. The movie records showed that the animals did not rest exclusively in comb-cells. Bees also rested frequently, and for long intervals, outside of comb-cells (Fig. 1). Thus, subsequent observations were always performed using a chamber without comb-cells. This enabled uninterrupted monitoring of all activities. Experimental data were collected with video equipment which offered the advantages of high time-resolution and the possibility of filming in complete darkness (see below). The use of a video camera did not elicit behaviour detectably different from that expressed during observations with movie equipment.

Methods

Experimental animals. All experiments were performed with young forager bees (with pollen baskets) which were captured when they returned to the hive. The hive was located outdoors in the summer and in a flight-room with LD maintained at 25 ~ for the remaining months of the year. Several bees were captured together and this group was kept in a cage in the laboratory until the start of the experimental procedures. The cage consisted of a dark area, warmed up to 33 ~ and a larger, illuminated area maintained at 25 ~ Sugar solution or diluted honey was available ad libitum in the cage and throughout the experiments.

Experimental conditions. A precision thermostat maintained air temperature at 25 + 1 ~ during the experiments. The relative humidity was between 50 and 60%.

In all experiments, the influence of external vibrations was reduced as much as possible by placing foam rubber matt ing under the experimental boxes which housed the equipment. In addition, all parts of the apparatus with which the bees had contact were embedded in fine sand.

Individual bees were viewed in silhouette by the camera. The floor and the narrow sides of the lucite chamber were


Fig. l a d . Single frames from a movie film. The photographs are from the second experimental night; the bee showed normal activity both before and after this night. The lucite chamber was illuminated continuously with dim, orange-coloured light (edge filter, cutoff at 610 nm). The 16 mm camera (Bolex HI6 EL) took one picture every 30 s. The left wall of the chamber consists of a piece of comb which contains 10 empty cells. Clock times are on the right. Exposure 1/50 s, Kodak 4-X reversal film. All clock times Mid-European Summer Time (MEST). Animal from an outdoor hive, July 1983

lined with balsa wood. The free space available to the animal in the chamber measured 25 mm (width)x 13 mm (depth)x 20 mm (height). Ventilation was provided by holes drilled into the lid of the chamber. Diluted honey was available from the open end of a tube which entered the chamber via one of the ventilation holes. Honey flowed into the tube from a reservoir hanging above the chamber.

Time lapse recordings in LD. It was possible to register the animals' activities during the total darkness which prevailed throughout the D-phase by using an IR-sensitive video camera (Panasonic WVI450 with an Ultricon tube) and short-wavelength infrared illumination which bees cannot perceive. The camera's sensitivity for light in the visible region of the spec- trum was suppressed by an edge filter (cutoff at 715 nm) placed in front of the camera. Thus the 'day light' (see below) could not influence the brightness of the video picture.

The IR source was a low-voltage incandescent lamp which burned continuously. It was located behind the following com- bination of filters: a wide-band infrared filter (RG 9, 3 mm thick, bandwidth approx. 72(~1100 nm, Schott) plus a filter (ITO, Schott) which reduced the intensity of the long-wavelength IR transmitted by the RG 9 filter. Employing Maxwel- lian view (bee seen in silhouette) made it possible to keep the operating current of the bulb low and thus reduce the radiation exposure of the animal to a minimum.

The light source for the 'day light' was a second low-volt-

age incandescent bulb which was run at a current somewhat lower than the nominal value. Thus the relative amount of short-wavelength light emitted was slightly reduced. A wide- band filter (BG 39, 3 mm thick, bandwidth approx. 350-600 nm, Schott) was located in front of the light source. The ' day light' (probably ' bee-blue-green'; Daumer 1956) thus did not contain long-wavelength radiation which would have influenced the camera's tube.

The intensity of the 'day light' in the chamber was measured with a radiometer (Model 65A, Yellow Springs Instru- ments). In these experiments, the intensity of illumination during the L-phase of the LD-cycle was 5.5 W-m -a. The times at which the 'day light' was switched on and off matched the local time for sunrise and sunset or corresponded to the illumination cycle prevailing in the flight room.

The video camera was connected to a time lapse recorder (Panasonic NV-8050) with a built-in time/date generator. The time display on the monitor was in hours, minutes and seconds. During recording, the recorder was run at 1/4 normal speed (12.5 half-frames/s). The tapes could be analysed frame by frame.

Results

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W. Kaiser: Behavioural and electromyograpbical sleep signs in honeybees 569

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Fig. 2. Rest/activity cycles of two bees, M1 and M2. Data extracted from video tapes played back at 1-8 times the recording speed. Stippled bars represent time intervals without locomotion which lasted at least 8 min. Behaviour expressed during these intervals has been termed resting behaviour. The small vertical lines in the stippled bars indicate the presence of locomotor behaviour lasting up to 5 rain. Following such interruptions, the bees usually came to rest at a new location in the lucite chamber. Arrows: termination of the experiments, due to the death of bee MI and to technical difficulties with the video recording in bee M2. The figures to the right state the total number of hours of resting behaviour accumulated during each 24-h cycle. All clock times: MEST. Both animals from outdoor hives. Bee M1 July, bee M2 August 1984

different nights with two bees have been analysed in great detail - with the aid of the time display on the monitor, it was possible to determine the duration of single episodes of a behavioural category with an accuracy of __+ 1 s (Figs. 3-5). Analysis of the data from the remaining tapes was undertaken with lower time resolution.

Single forager bees which have been isolated from the colony and are no longer able to pursue their normal activities still show a daily, rhythmical alternation between activity and rest. For most, often all, of the day, isolated honeybees engage in locomotion, feeding and grooming. During the night, bees remain in one and the same location for extensive periods of time. Most of the bees observed to date in the lucite chamber finally came to rest on a side wall with their heads directed upwards. The majority of resting bees in the hive also displays the 'head-up ' constellation. Thus, resting bees clearly prefer this position. Just as was the case in the observation hive, bees which rested on a horizontal surface (the chamber floor) provided the most definitive evidence of a loss of muscle tone in the legs. Figure I shows such a bee. The reduction in leg-muscle tone is so pronounced that, following a gradual lowering of the body (Fig. I a-c), the bee finally tilts over and comes to lie on its right side (Fig. I d). The antennal con-

stellation in Fig. I a typically appears during profound rest.

Figure 2 presents data from the video tapes analysed with low time resolution. With one exception (bee M1, day 2), locomotion first ceased after the start of the D-phase. The bees continued to rest beyond the start of the L-phase - this is much more pronounced in bee MI than bee M2. Unin- terrupted activity always occurred between 15:00 and 19:00 h. Resting behaviour which occurred after the main nightly bout was fragmented and absent after day 3 (bee M1). The amount of activity thus increased with increasing experimental duration.

Video tapes from cycle 5 (bee M1) and cycle 2 (bee M2) were subjected to a detailed motility analysis with high time resolution, since, during these nights, both bees showed the minimum number of changes of location during the resting phase. Both bees also did not display any resting behaviour during the days immediately before and after these nights.

The results of this detailed analysis are presented in Fig. 3. The resting behaviour of stationary bees could be described in terms of the following categories: (1) total absence of head and body movements ( ' immobility'); (2) head and body movements which were always associated with


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Fig. 3A-D. Categories and cumulated amounts of behaviour shown by two single, unrestrained bees on two different nights. Durations of individual episodes were determined from video tapes. B, D Data obtained by scoring both tapes a second time just for categories of antennal motility. The double-headed arrows under A and C represent the intervals during which the bees remained completely stationary. During this time, bee M1 sat on the right wall of the lucite chamber, with its head pointing upwards; bee M2 remained on the left chamber-wall, also with its head pointing upwards. All clock times: MEST. Same animals as in Fig. 2

large antennal movements; (3) antennae motionless (includes sporadic, minute movements); (4) antennal movements of small amplitude (' small antennal movements '); (5) large antennal movements; (6) grooming movements (not included under (2) and (5) above). The animals switched from one kind of behaviour to another at irregular intervals. In addition, movements of the proboscis and bursts of abdominal pumping (= respiratory) movements which occurred at fairly regular intervals (approx. 50-70 s) were also clearly visible.

The duration of single episodes of each of the various overt behaviours mentioned above was determined by visual inspection of the video tapes. For each behavioural category, the sum of all episodes per half-hour was plotted as a function of time of day. Figures 3A (bee M1) and 3C (bee M2) show the nightly time course of changes in the motility of head and body. After the cessation of locomotion, there is a gradual increase in the half-hourly amount of immobility (black areas). High values are maintained for several hours. Overall immobility subsequently declines before locomotion resumes in the morning. With very few exceptions, episodes of head and body movements and of grooming movements occurred during each 30-min interval. The most obvious difference be-

tween the two bees is the latency between light-off and the first appearance of immobility.

Since antennal movements are still present when all head and body movements have ceased, the video tapes were evaluated a second time in order to investigate the course of antennal motility during the night (Fig. 3B, D). Large antennal movements which occurred during grooming behaviour have been scored as 'grooming movements' . The black areas in B and D (antennae motionless) are much smaller than the black areas in the upper half of the diagram. When all antennal movements have ceased, the bees are virtually im- mobile. Motility is evident only in the form of respiratory movements and occasional jittery movements of the tarsi. Prominent peaks in antennal immobility occur at the same time as the peaks in immobility of head and body. In both animals, the largest amount of antennal immobility occurred between 03 : 00 and 04: 00 h. Further results of the motility analysis are presented in Table 1.

The diagrams in Fig. 3 demonstrate that, between 02:00 and 07:00 h, antennal immobility oc- cupies, on average, at least 50% of each half-hour interval (mean values for this 5-h period: 51% for bee M1 and 62% for bee M2). The corresponding values for immobility of head and body are even

W. Kaiser: Behavioural and electromyographical sleep signs in honeybees

Table 1. Quantitative analysis of the behaviour of resting bees

571

Bee M1 Bee M2

1 Total amount of immobility of head and body 8.8 h 4.9 h 2 Total amount of antennal immobility ('deep sleep') 3.5 h 3.1 h 3 Total amount of head and body movements 1.2 h 1.4 h 4 Total amount of small antennal movements ('light sleep') 4.1 h 1.8 h 5 Total amount of large antennal movements 2.4 h 1.3 h 6 Total amount of grooming movements 1.1 h 1.0 h 7 Longest, uninterrupted time-interval without change of location 9.6 h 7.2 h 8 'Deep sleep' onset latency 79.1 min 54.1 min 9 Total 'sleep' time [(2)+(4)] 7.6 h 4.9 h

Comparison of data obtained from a detailed analysis of the behaviour of bee MI and bee M2. Data were collected at night during time-intervals in which locomotion was absent. 'Deep sleep' onset latency is the time interval between the end of the last bout of locomotion and the start of the first episode of antennal immobility

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Fig. 4. Mean durations of episodes of immobility of head and body (A, C) and of antennal immobility (B, D). The curves were derived from a moving averages analysis of the raw data of Fig. 3, i.e., same bees and same nights. The y-coordinate value of each point represents the mean duration of 10 consecutive episodes of immobility. The individual episodes were separated from one another by various intervals of time. The corresponding x-coordinate value represents the midpoint between the start of the first episode and the end of the tenth. 372 and 327 values contributed to the upper and lower curves, respectively, for bee M1; the corresponding numbers for bee M2 are 114 and 165. Points which lay too close to one another to allow clear graphic representation have been eliminated without altering the courses of the curves. The intercepts of the dotted lines with the abscissae indicate the estimated times of occurrence of zero episode duration - these points cannot be measured. Note different scales on the ordinates. Double-headed arrows as in Fig. 3

higher (82% for bee M1 and 80% for bee M2). The parameter of immobility thus warranted closer investigation.

Since the method employed to present the data in Fig. 3 results in at least a partial elimination

of information about the internal structure of bees' resting behaviour a moving averages analysis (Har- tung et al. 1982) was performed on the raw data which contributed to the black areas in Fig. 3. It is evident from Fig. 4 that the mean episode dura-


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tions of both categories of immobility fluctuate throughout the night. The times of occurrence of the prominent peaks in Fig. 4A, C correlate with the large amounts of head and body immobility which occur between 03:00 and 04:00 h (Fig. 3A, C). These large values are obviously due to increased mean episode duration. Later in the night, large amounts of immobility are related to an increase in the number, rather than duration, of episodes.

Prominent peaks between 03 : 00 and 04: 00 h are not evident in Fig. 4 B, D (immobility of antennae). The mean episode duration of antennal immobility is maintained above at least 20 s for 5-6 h during the night. In Fig. 4 D there is even a marked increase in mean episode duration as the night pro- gresses.

The means of the durations of all episodes of the two behavioural categories in Fig. 4 are: bee MI - 84.9 s for immobility of head and body, 38.6 s for antennal immobility; bee M2 - 152.5 s for immobility of head and body, 68.1 s for antennal immobility. These considerable differences were further investigated with the help of histograms showing the relative frequencies of occurrence of individual episode durations. This analy-

sis, which is presented in Fig. 5, revealed that the higher mean values for bee M2 were due to the presence of episodes of very long duration (Fig. 5A) and of relatively more episodes in the upper half of the duration scale (Fig. 5 B). Bee M2 has 26 episodes of antennal immobility which last longer than 120 s as compared to only 9 for bee M1.

Discussion

The behaviour of resting bees at night can be ap- propriately described in terms of changes in motility. Bees do not enter a state of prolonged 'sleep immobilization' (Hoffmann 1937). Following the cessation of locomotion, the amount of overt activity per unit time decreases gradually, i.e., immobility increases (Fig. 3). Pronounced immobility is subsequently maintained over long periods of time at night. The end of the resting phase is characterized by a gradual decrease in immobility.

Since antennal movements continue to occur when head and body movements have ceased, their presence or absence probably represents a very sensitive measure of the state of activation of stationary, resting bees. Episodes of small antennal movements usually preceded and succeeded episodes of antennal immobility.

In humans, a correlation exists between the amount of motility and the depth of sleep (Wilde- Frenz and Schulz 1983). Thus, by analogy, it is tempting to describe the phenomenon which pre- vails during the state of antennal immobility in bees as 'deep sleep' and that prevailing during small antennal movements as 'light sleep'. This interpretation is supported by measurements of reaction thresholds and muscle tone (see below).

Total 'sleep' time in the bee was therefore estimated as the total of these two states [Table I (9)]. Tobler (1983) has used the presence of complete behavioural immobility as a measure of 'sleep' time in cockroaches. In these nocturnal insects, antennal movements also occur in the absence of head and body movements. The cockroach was found to have a total 'sleep' time of 14 h, which is similar to the amount of resting behaviour displayed by bee MI on days 1-3 (Fig. 2). However, Tobler's resolution in time for scoring her video tapes was only 2.5 min.

Bees M1 and M2 differed clearly both in the amount o f ' s l eep ' which they accumulated and in ' deep sleep' onset latency, although both displayed very similar amounts of antennal immobility ( 'deep sleep') [Table 1 (2), (8), (9)]. Bee M2 thus had relatively more 'deep sleep' than bee M1


which 'slept' longer. These results provide the foundations for a speculative comparison with human long and short sleepers. Amongst humans, habitual short sleepers have relatively more deep (stage 3 +4) sleep than normal or habitual long sleepers (Home 1983) and relatively short sleep onset latencies (Benoit et al. 1983). The fact that the mean episode duration of antennal immobility for bee M2 was longer than for bee M1 supports this speculation, since longer episode durations may reflect a greater depth of ' s leep ' : in rats, it has been demonstrated that, when the mean duration of non-REM episodes is long, a relatively large amount of deep (delta) sleep is present (Borb61y and Neuhaus 1979).

In humans and mammals, longest episodes of non-REM sleep occur at the beginning of the sleep phase and decline thereafter (Borb61y 1984). In rooks and some other diurnal birds, the episode durations of non-REM sleep show a plateau-like distribution across the night (Szymczak 1987). In the bee, the nightly time courses of both the amount of antennal immobility and mean episode duration of antennal immobility also show a plateau-like distribution.

A comparison of the present results, obtained in the laboratory, with data collected under natural/seminatural conditions, reveals the absence of major, laboratory-induced artefacts. Grziwa (1961) monitored the noise level in a normal hive over a 24-h cycle and used this as an index of general activity. The minimum noise level occurred between 01:20 and 04:40 h. The times of sunrise and sunset did not have any obvious immediate influence on the activity (noise level) in the hive. Kronenberg and Heller (1982) also found that the lowest levels of locomotor activity (qualitatively estimated by direct observation) of bees kept in an observation hive within a metabolism chamber (LL, 25 ~ occurred during the subjective night. There was a gradual rise toward and fall away from the daytime peak values.

Part 2." Observations on restrained, intact bees

a) Antennal positions during the day and the night

Methods

A small tab was glued onto the dorsal surface of the thorax of individual foragers which had been immobilized by cooling them in a refrigerator. The bees were then returned to the hold- ing cage and allowed to recover. For the experiments, the tab

was clamped into a holder and the bee was mounted horizontally and at a fixed distance above a lightweight, pivot-mounted treadwheel. The animal's legs could rotate the wheel. The bee's behaviour was monitored continuously under DD-conditions with the video equipment described above. Movements of the treadwheel were readily visible on the monitor. The IR illuminated the frontal, dorsal region of both compound eyes and the ocelli as well. In order to obtain comprehensive information about the exact momentary position of the antennae, surface mirrors were placed above and beside the bee. The picture on the video monitor thus consisted of 3 segments - two which originated from the mirrors and one direct frontal view.

Results

A few details about the anatomy of the bee's antenna are necessary for a better understanding of the results to be presented below. The antenna consists of two elements which can be moved actively. (1) An elongated basal segment (scape) which con- nects to the head capsule via a complex ball-joint structure. The scape can be moved by 4 muscles which originate in the head capsule. (2) A short distal segment (pedicel) to which the long flagellum is attached. There is a hinge-joint between the scape and the pedicel. Two antagonistic muscles in the scape can move the pedicel-flagellum up and down. The antennae can thus occupy almost any position in the space located in front of the bee's head.

Results from this experiment are shown in Fig. 6. Figure 6 A depicts a situation which occurs during locomotor activity during the subjective day; Fig. 6B shows antennal positions that could be observed when the bees remained very still at night. During the resting state at night, the head tilts downwards. The scapes lie almost horizontally and are close to the surface of the head, the pedicels and their flagella hang almost vertically (Fig. 6 B, a-c). Figure 6 B, d shows the frontal view at another point in time during the night: the tips of the flagella have approached each other and the head capsule as well. This postural constellation of the antennae was always correlated with particularly high reaction thresholds (see below). Symmetrical positions of the antennae were very common at night.

The postural changes which occurred when bees made the transition from activity to rest only appeared gradually. The final state was reached via a series of sub-stages which were also characterized by differences in antennal motility. One of these substages, which belongs in fact to the resting state, is what I shall call antennal swaying: both antennae make simultaneous, parallel small swaying movements. These movements are performed by the scapes which, by this time, have already


B

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Fig. 6A, B. Typical positions of the head and antennae of a bee which was fixed above a treadwheel. The drawings were traced from the screen of the video monitor. A During an episode of activity in the daytime; B during profound rest at night. The video camera could simultaneously register three views of the bee: side view (a) and top view (e) reached the camera via surface mirrors, the frontal view (b) was recorded directly. A(d) and B(d) are frontal views that were recorded at other times during the day and night, respectively. Animal from an outdoor hive. August 1983

approached the horizontal plane. The pedicels and the flagella point downward. One cycle is com- pleted in about 3-4 s.

Discussion

The bees in the lucite chamber displayed, during their nightly times of rest, certain antennal positions (Fig. 1) which had previously also been seen in the observation hive. Figure 6B demonstrates that the same antennal positions are also displayed at night by bees resting on a treadwheel. Thus, they are not artefacts. Schulze (1924) and Ander- sen (1968) have reported that resting insects show specific antennal postures. The postural constellations of bees' antennae which are observed during rest at night (Fig. 6B, a-c) are probably due to

reduced tone in the antennal muscles. This assumption is supported by Rathmayer's (1962) findings.

It is unlikely that the position of the antennae per se is a sufficiently accurate behavioural indi- cator for the internal state of activation of the animal. Antennal motility is also an important factor (see above). The behavioural state of a bee at any particular time can thus only be adequately as- sessed when both parameters (motility and position) are measured simultaneously. We are pres- ently engaged in developing such a measuring sys- tem.

b) Changes in thoracic temperature The body temperature of resting bees in the observation hive was equal to the ambient temperature at the edges of the combs and lower than that of active hive bees (see above). In order to investigate the relationship between activity, resting behaviour and body temperature in detail, long- term experiments were performed on individual forager bees.

Methods Animals were mounted above a treadwheel as described above. Motion of the wheel was registered photoelectrically and the resulting impulses were summated every minute by a digital counter. The corresponding analogue values were recorded continuously with a pen recorder (Servogor). Since both backward and forward movement of the treadwheel produced identical impulses, the difference between on-the-spot movements and true locomotion could only be determined by making simultaneous video observations. These demonstrated unequivocally that high treadwheel-activity values were always associated with true locomotion.

Thoracic temperature was measured continuously with a thermocouple which was glued directly onto the depilated cuti- cle of the thorax with a drop of wax rosin. This drop also insulated the soldered tip of the thermocouple from the surrounding air. The copper/constantan thermocouple (diameter of each wire: 0.076 mm) was connected to a battery-driven digital thermometer (BAT 12, Sensortek Inc.) with an analogue output. The digital display was correct to _+ 0.1 ~ The analogue signal was registered continuously with a pen recorder. Our measurements confirmed Esch's (1960) finding that, when fine thermocouples are used, the thoracic temperature thus measured is maximally 0.1 ~ lower than the temperature of the thoracic muscle mass.

Experiments were performed in DD. Animals could be observed with video equipment, as described above.

Results

A total of fifteen 24-h cycles from long-term experiments (minimal duration: 96 h) on 3 bees yielded data about the relationship between activity/inactivity and thoracic temperature. The period- length (z) of the rest/activity rhythm was determined from twelve 24-h cycles.


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During the subjective day, the animals were active, albeit with pauses, whilst during the subjective night they showed little or no locomotor activity (Fig. 7). Figure 7 also shows that, throughout the subjective night, the thoracic temperature remains at the prevailing ambient level (25-26 ~ This value, however, is also attained during the subjective day, when the bee does not engage in locomotor activity for at least 6 rain (Fig. 8). This figure shows an excerpt from day 5 of the same experiment as Fig. 7, on an expanded time scale. The bee's activity episode began here during the objec- tive night because r is less than 24 h (see below).

Figure 8 clearly illustrates the close correlation between locomotor activity and thoracic temperature.

Observations of bees' behaviour via the video camera demonstrated that, prior to resuming locomotion following longer pauses, the animals first exhibited antennal and body movements as well as grooming movements�9 These movements were also accompanied by leg movements which led to small forward and backward movements of the treadwheel. During this preparatory behaviour, the bees also performed pumping respiratory movements with their abdomens and the thoracic tern-


perature began to rise. Only after this initial increase in thoracic temperature did the bees begin performing locomotor movements. These were followed by a further temperature increase.

All records showed a clear circadian alternation between activity and rest. The amount of locomotor activity increased, however, with increasing experimental duration. The time at which activity started in each cycle was the reference point for determining z. In two of the three bees, the second cycle started later than the first; all other cycles, however, began earlier than the preceding one. r was therefore less than 24 h and ranged between 22 h 18 min and 22 h 53 min, with a mean of 22 h 38 rain.

Discussion

The flight muscles of bees can generate heat in the absence of visible movements of the wings (Esch 1960, 1964). Thus, the pattern of thoracic temperature changes in the presence or absence of overt activity is of major interest. The data presented here (Fig. 7) demonstrate that, at an ambient temperature of 25 ~ rest in bees at night is associated with a prolonged depression of thoracic temperature. There is no activity-independent, en- dogenous temperature rhythm.

Temperatures around 25 ~ are commonly encountered within the hive, outside of the brood area (Hess 1926, and own unpublished observations). Bees only enter a state of 'cold-immobilization' well below this temperature (Heinrich 1981; Rothe 1983). Thus, the prevailing ambient temperature at night could not have induced the reduction of motility. The observation that the start of locomotion in bees which had been resting was always preceded by an increase in thoracic temperature (Fig. 8) confirms results obtained by Rothe (1983).

The experiments presented here were performed in DD in order to see whether the rest/ activity patterns of isolated forager bees under constant conditions differed significantly from those evident in the presence of a Zeitgeber. In constant darkness, major bouts of activity continue to alternate with prolonged resting phases. The mean period-length of the rest/activity rhythm in DD (22 h 38 min) agrees well with results obtained previously in this laboratory under similar conditions (Kaiser and Steiner-Kaiser 1983, Fig. 2). It is also in agreement with the results of Moore and Rankin (1985), who studied the locomotor rhythms of unrestrained, individual bees maintained in small plexiglas chambers (DD, 25 ~ Locomotor activity was measured opto-

electronically, without simultaneous behavioural observations.

c) Reaction thresholds for the elicitation of grooming movements

In order to measure behavioural reaction thresholds both at night and during the day, we needed a clearly-defined behavioural act which could also be reliably elicited in resting animals. My student, W. Eusemann, discovered that bees perform grooming movements (the forelegs sweep over the head and antennae) when pulses of infrared radiation of sufficient intensity are directed at the frontal surface of the head. To the best of my know- ledge, this is the first description of this behavioural reaction in honeybees.

Methods

Individual bees were mounted on a treadwheel and their locomotor activity and thoracic temperature were recorded continuously (see above). During threshold measurements, the animals' behaviour was monitored with video equipment (see above). These experiments were performed under LD conditions. The intensity of the 'day light ' was 1.6 W ' m-2 .

Thresholds were determined only when locomotor activity was absent, since locomotion apparently has an inhibiting influence on thermally-induced grooming (see also Pflumm 1969). Thus, measurements could be made throughout most of the D-phase. During the L-phase, reaction thresholds could be measured reliably only during locomotion-free intervals lasting at least l0 min. During such pauses, the thoracic temperature sinks to the environmental level (see Fig. 8). This was also a necessary precondition for the comparison of daytime and nighttime threshold values, since thresholds which are obtained by stimulating temperature receptors can only be compared when the temperatures of the surrounding tissues are identical.

The source of infrared radiation for the stimuli was a low- voltage 100 W incandescent halogen light bulb which was run from a constant-current DC-source. The radiation first passed through an edge filter (RG 830, 3 mm thick, cutoff at 830 nm, Schott) and was then collected by an ordinary condensor lens so that it formed a circle, which was somewhat larger than the activity range of the antennae, just anterior to the head. By changing the current passing through the light bulb, it was possible to continuously vary the intensity of the radiation stimulus. This procedure meant, however, that the spectral energy distribution varied according to intensity - this minor source of error has not been compensated for in the present experiments.

The threshold for the grooming reaction at any particular time was determined by successively increasing the stimulus intensity. Stimuli of 3 s duration were delivered at 100-s intervals. The intensity of successive stimuli increased by a factor of 1.15 when the intensity was near threshold. Stimuli which were very close to threshold induced the bees to only raise their heads without making accompanying grooming movements. After the first grooming movement had been elicited, the intensity of the next stimulus was increased by a factor of 1.06 - this usually elicited a second grooming movement. The intensity was then reduced to 94% of the value which

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Fig. 9A-D. Time courses of the threshold intensity of infrared radiation which elicits grooming of the head and antennae. Individual bees were mounted on a treadwheel. A-C Three curves from 3 different bees. With the exception of two values in B (A), all measurements were made during one 24-h cycle. The two exceptions were obtained during the next cycle. D Results from one bee whose reaction thresholds were measured during 5 consecutive cycles. Numbers 1-5 denote the cycle number. All animals from a flight room, January and February 1984

had elicited the first grooming movement. If this intensity was not sufficient to elicit a reaction, the original value was re- tested. When it again elicited a grooming movement, it was considered to represent the threshold intensity. Determination of one threshold value required a test series of 7-14 measurements, lasting 15-25 rain.

R e s u l t s

Reaction thresholds were measured in 4 bees. The results in Fig. 9A-C were, with the exception of two points in B (filled triangles), obtained over one 24-h cycle. The experiment shown in D lasted for 5 days. The good agreement between the curves A-C is evident. The highest threshold values occur towards the end of the D-phase and are approx. 2-3 times higher than the lowest reaction thresholds measured during the day. Analyses of the video tapes made during the threshold determinations revealed that higher thresholds were associated

with reduced antennal motility and more pronounced downward tilting of the head (see Fig. 6B). Maximum threshold values were obtained when the flagella of the antennae approached the head capsule (Fig. 6 B, d).

In Fig. 9D, the differences between threshold values on nights I and 2 may reflect incomplete 'acclimatization' to the experimental conditions on night 1. Video recordings from this bee showed more antennal movements and less downward tilting of the head on the first than on the second night. The thresholds measured during the L-phase on days 1-5 of this long-term experiment show very little variation.

Determination of reaction thresholds during longer pauses in locomotion in the L-phase was, on occasion, immediately followed by a bout of intense locomotor activity, lasting up to I h. There was also a concomitant rise in thoracic tempera-


ture. This kind of phenomenon was never encountered at night. During the D-phase, the bee's head and antennae would sink downwards again within a few seconds following the presentation of a threshold stimulus. Neither locomotor activity nor an induced increase in thoracic temperature were registered.

Discussion

Readiness to react to external stimuli is an essential criterion when assessing the momentary state of activation of an animal. A bee's readiness to perform grooming movements in response to stimulation with pulses of IR was found to be an appropriate measure of activation state in our test situation.

In contrast to humans (Michelson 1897; Wil- liams et al. 1964), bees display highest reaction thresholds towards the end of their nightly resting phase. The course of the reaction threshold during the night (Fig. 9) is similar to the time-course of the half-hourly amount of antennal immobility at night (Fig. 3 B, D). This indicates that a correlation exists between the amount of antennal immobility and reaction threshold. The presence of such a correlation was confirmed by video recordings made simultaneously with threshold measurements.

The procedure employed to determine reaction threshold could have favoured the expression of habituation-like processes. Two facts indicate that such processes did not exert a major influence on the daily course of the bee's reaction threshold. Firstly, threshold values within a test series (see Methods) remained essentially constant. Secondly, there is little variation in the daytime threshold values across days 3-5 in Fig. 9 D.

High thresholds for responses to sensory stimulation in resting insects have been described qualitatively (Fiebrig 1912; von Frisch 1918; Haufe 1963; Andersen 1968). Relatively mild thermal stimuli elicit grooming movements in many insect species (' W/irmeputzen ', Herter 1953). The results reported here demonstrate that this reaction also occurs in honeybees. Heran (1952) demonstrated that the antennae play a key r61e in thermorecep- tion in bees. It is thus very likely that, in the present experiments, the pulses of IR stimulated the antennae and thereby elicited the grooming reaction.

a) B e h a v i o u r a l e x p e r i m e n t s

Methods

Individual bees were mounted above the lightweight treadwheel described above. Instead of being fixed at a set distance above the wheel, the animals were attached via the thorax to one end of an oil-damped balance arm. The arm was counter-bal- anced so that an active bee just carried its own weight. This arrangement enabled the observer to assess both the inclination of the head and the amount of tone present in the leg muscles. Experiments were performed under LD conditions, with continuous video monitoring. The intensity of the light during the L-phase was 0.5 W . m -z. The angle of inclination of the head, ~, was defined as the angle between the direction of gravity (vertical line on the video-monitor screen) and a line joining the base of the right antenna with the anterior edge of the right mandible. (The optical axis of the camera was aligned perpendicular to the median plane of the bee which was seen in silhouette.) With the help of a template, this head angle was measured once every 10 min directly on the screen of the TV monitor. Clock time was read off the video tape.

Results

Quantitative data were obtained from 2 bees which were observed continuously for 24 and 48 h respectively. Figure 10 shows the daily course of the head angle, ~. During the night, the head displays varying degrees of downward tilt (negative c~-values). Particularly low values occur late at night. In contrast, during the day, almost all c~-values are posi- tive: the head is held raised (see also Fig. 6A). The transitions between activity and rest are characterized by gradual changes in c~. Light-on is associated with a transient raising of the head (most pronounced in Fig. 10 A). There after, c~ reassumes negative values, or remains negative, before gradually returning to its daytime level. Video recordings showed that, during the night, there was also a reduction in the distance between the bee's body and the treadwheel (see also Fig. 1). Increasingly negative a-values were commonly associated with progressive decreases in the body-treadwheel sepa- ration.

b) E l e c t r o m y o g r a p h i c r e c o r d i n g s

The tone of neck muscle 42 was measured experi- mentally during long-term electromyographic recordings. This is one of the neck muscles which raises the bee's head; it also participates in turning the head (Snodgrass 1956; Markl 1966).

III. Daily changes in neck-muscle tone

Activity-related changes in the angle of inclination of the bee's head (see Fig. 6) and the electrical activity of one neck muscle were investigated.

Methods Individual bees which had been immobilized by cooling were mounted firmly on a small holder with wax rosin. The longitu- dinal body axis was almost horizontal. The legs were fixed


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but the abdomen could move freely. In order to keep the elec- trodes in place, the head was fixed to the thorax, in a natural position, by a wax-rosin bridge.

The active electrode was a piece of insulated steel wire, 30 tam in diameter, whose end was cut off just prior to insertion into the muscle. Only the cut surface of the electrode made electrical contact with muscle tissue. The electrode pierced the left episternum and was introduced laterally into the underlying muscle. After reaching its final position, about 0.2 mm below

the surface of muscle 42, the electrode was fixed firmly into place with drops of wax rosin on the episternum and thorax. The position of the electrode was verified at the end of the experiment by dissection and measurement under microscopic control. The indifferent electrode was a platinum wire, 100 gm in diameter, whose tip was located in the haemolymph of the neck approx. 0.5 mm beneath the neck membrane. This electrode was introduced dorsally, very close to the anterior edge of the prothorax and somewhat lateral to the midline of the neck; it thus did not pierce the head artery. The indifferent electrode was not insulated but carefully earthed, and it pre- vented the large potentials which originate in the thoracic musculature from reaching the active electrode in muscle 42.

The potential changes recorded by the active electrode were led off to a preamplifier (Tektronix, Type 122, low cutoff frequency 80 Hz, high cutoff frequency 1000 Hz) connected to an FM tape recorder (Tandberg, TIR 100). The tape recorder had a bandwidth of 0-2500 Hz at the recording speed (7 1/2 ips) used during the experiments.

The electromyogram was recorded intermittently: an external timing device switched on the taperecorder for 20 s at 5-min intervals. In the graphic presentation of the data (Fig. 12), con- tinuity on the time-axis (binwidth 5 min) was obtained by as- signing the amplitude of the 20-s EMG recording to the entire 5-min interval. When more than one amplitude was present in the trace, that amplitude which was dominant for at least 10 s was chosen to represent the entire interval. The method of recording EMGs discontinuously may have led to a slight overestimation of muscle activity which lasted only a little longer than 20 s.

Experiments were performed under LD conditions. The intensity of the 'daylight' was 0.5 W.m 2 Bees were fed with honey only during the L-phase. The ambient temperature ranged between 24 and 27 ~

Results

D a t a a r e p r e s e n t e d f r o m one l o n g - t e r m e x p e r i m e n t in w h i c h the bee s u r v i v e d fo r 4 d a y s . A su rvey o f i n d i v i d u a l E M G s s h o w e d t h a t al l r e c o r d i n g s c o u l d be a s s i g n e d to o n e o f f o u r c lasses o n the bas i s o f the a m p l i t u d e o f the l a r g e s t p o t e n t i a l s p r e s e n t in e a c h r e c o r d i n g . T h e s e c lasses t hus r e p r e - sen t d i f f e r en t levels o f m u s c l e ac t iv i ty . W h e n the E M G s were e x a m i n e d w i t h ve ry h i g h t e m p o r a l res- o l u t i o n , i t was a p p a r e n t t h a t e a c h level a l so con - t a i n e d al l o f the p o t e n t i a l f o r m s a n d a m p l i t u d e s p r e s e n t a t al l l o w e r m y o g r a m levels.

F i g u r e 11 s h o w s f o u r o r i g i n a l E M G - r e c o r d - ings, e a c h o f w h i c h is r e p r e s e n t a t i v e fo r one level o f m u s c l e a c t i v i t y ( n u m e r a l s to the r igh t ) . T h e m o s t c o n s p i c u o u s f e a t u r e o f the m y o g r a m s is the m a r k e d r e d u c t i o n o f a m p l i t u d e a t n igh t . Leve l 4 was c h a r a c t e r i z e d b y a m a r k e d v a r i a b i l i t y in a m p l i - tude . T h e a m p l i t u d e a t the l o w e s t level (1) is o n l y a b o u t twice as l a rge as t h a t o f the no i se level o f the r e c o r d i n g . T h e no i se level was e s t a b l i s h e d b y c o n t i n u i n g to r e c o r d f r o m the bee p a s t the p o i n t o f t ime a t w h i c h i t d ied .

F i g u r e 12 s h o w s al l d a t a f r o m the s e c o n d 24-h p e r i o d o f th is l o n g - t e r m e x p e r i m e n t . T h e ve ry


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small amplitudes of level 2 predominate at night. This level is attained for the first time about 2.5 h after the start of the D-phase. Prior to this, there is a phase in which various EMG levels are present. The transition from 'waking-level' 4 to level 2 is gradual. Somewhat later in the night, level 2 is repeatedly interrupted by brief episodes of level I in which the myogram amplitude comes close to the noise level. However, level 2 also alternates with level 3. Late at night, between 01:20 and 05:10 h, the record shows an episode of particularly stable 'sleep' (levels 2 and 1 only). When the lights are switched on at 06: 34 h, there is a sudden, transient increase to level 4 in the myogram amplitude. Soon thereafter, the amplitude is back to levels 2 and 1. It takes about 1.5 h after light-on for the EMG-amplitude to reach the full 'waking' amplitude, again via intermediate stages.

Discussion

Sleep in mammals is characteristically accompanied by a loss of tone in the skeletal muscles. This phenomenon expresses itself both in body posture and in the amplitude of the EMG of the muscles (e.g., Morrison 1983). A comparison of Figs. 10 and 12 reveals that this is also true of profound rest in the bee. The results of functional-anatomi- cal investigations of the head/neck articulation in the honeybee (Lindauer and Nedel 1959) predict that a reduction of tension in the bee's neck muscles will cause the head to sink downwards.

Resting bees display reduced muscle tone not only in the neck but also in the leg muscles: video observations during the behavioural experiments showed the simultaneous occurrence of negative a-values and reduced body-treadwheel distance.

Haufe (1963) describes postures in resting mosquitoes which are obviously a consequence of reduced tone in the leg muscles. His experimental evidence indicates that mosquitoes which display these postures have relatively high reaction thresholds.

The nightly time-courses of changes in the parameters measured in Figs. 10 and 12 are not only similar to one another but also to changes in the half-hourly amount of antennal immobility at night (Fig. 3 B, D) and the values of the reaction threshold (Fig. 9).

General Discussion

The data presented in this paper demonstrate unequivocally that forager honeybees experience a state of profound rest at night. A comparison between the phenomena accompanying this state in

w. Kaiser: Behaviourat and electromyographical sleep signs in honeybees 581

bees and some phenomena which accompany sleep in humans, mammals and birds (sleep signs) will reveal whether the term sleep can justifiably be applied to the profound rest of these diurnal, social insects.

Sleep signs common to mammals (including humans), birds and bees

1. Changes in motility. Sleep in humans, mammals and birds is characterized by a relatively long-lasting reduction in overt body motility. Body movements of varying extent, ranging from slight twitches in the extremities to gross postural changes, occur during sleep. The sleep phase can also be fragmented by short bouts of wakefulness.

At night, bees remain stationary, in one and the same location, for several hours on end [Figs. 2, 3, Table 1 (7)]. During these time intervals, overt behavioural acts such as large and small movements of various parts of the body, as well as grooming, occur intermittently (Fig. 3). Motility at night, however, is greatly reduced in comparison to the daytime. This reduction is most pronounced when the antennae are motionless (Fig. 3B, D). Antennal immobility thus very probably indicates the presence of the most profound state of rest which bees can attain.

The feasibility of assessing various states of human sleep and wakefulness on the basis of detailed analysis of motility patterns has been demonstrated recently (Thoman and Glazier 1983). In humans, motility during sleep is an adequate indi- cator of the depth of sleep and the lowest levels of motility occur in stages 3 and 4 of non-REM sleep (Wilde-Frenz and Schulz 1983). A comparison of the times of occurrence of maximal amounts of immobility in humans and bees reveals that the latter - in contrast to humans - experience their deepest 'sleep' late at night. This difference is of interest; it indicates either that sleep arose indepen- dently during the evolution of these two species or that a basic, common plan has been varied. Extensive comparative investigations are required to resolve this question.

2. Changes in reaction threshold. The relationship between reaction threshold and sleep stage has been studied most intensively in humans, for whom it is well established that an increase of reaction threshold is a reliable sleep sign (see Bonnet 1982 for review). This relationship is of special signifi- cance when behavioural and/or electrophysiological criteria for the presence or absence of sleep are either unavailable or equivocal.

In humans, highest behavioural reaction thresholds occur in sleep stages 3 and 4, i.e., soon after the start of the nightly sleep phase (Williams et al. 1964). The reaction threshold for grooming in response to thermal stimuli in honeybees, in contrast, reaches maximal values late at night (Fig. 9). When honeybees are resting at night, there is a temporal correlation between the level of the reaction threshold (Fig. 9) and the amount of antennal immobility per unit time (Fig. 3). Experiments are in preparation to investigate the precise nature of this correlation. One important question is whether reaction threshold fluctuates in parallel with the duration of episodes of antennal immobility (Fig. 4).

3. Changes in muscle tone. In humans and other mammals, the onset of sleep is characterized by a decrease in the tone of head and neck muscles (Jacobson et al. 1964; Morrison 1983). The amplitude of the neck-muscle EMG attains its smallest value (virtually zero) during REM sleep. The amplitude of the EMG thus fluctuates throughout sleep. In birds, the extent of changes in neck-muscle tone varies amongst species (Dewasmes et al. 1985).

Evidence of a progressive decrease in muscle tone during rest at night in honeybees is presented in Figs. 1, 10, i 1, 12.

4. Changes in body temperature. The drop in body temperature which is usually associated with the onset of sleep in humans and mammals is of relatively small amplitude, e.g., 0.5 ~ in humans, 2 ~ in rats (Borb61y 1984; Ob/tl 1984). It is, however, a reliable sleep sign in homeotherms.

Prolonged rest in bees at night is accompanied by a marked, maintained reduction in thoracic temperature (Fig. 7). Relatively short episodes of inactivity during the day (Fig. 8) were also associated with a reduction in thoracic temperature.

These four sleep signs constitute strong evidence in support of the hypothesis that bees do experience real sleep at night. Additional evidence is provided by the data obtained from visual interneurones in the bee's brain (Kaiser 1983; Kaiser and Steiner-Kaiser 1983). Not only do these neurones display properties similar to those of mammalian visual interneurones during sleeping and waking (see Introduction), they also display a circadian variation both in visual sensitivity and spontaneous activity. This circadian rhythm is of particular interest since it has been recently empha- sized that circadian processes play a central r01e in sleep regulation in humans (Daan et al. 1984).


Sleep can be described in terms of various physiological and behavioural states but this should not obscure the fact that it is, in reality, an ongoing process which is governed by active mechanisms (Galliard 1985). Figures 3, 4, 9, 10 and 12 suggest that the dynamic process (or mechanism) govern- ing sleep in bees causes a gradual increase in the amount of deep sleep in these insects at night. High values are maintained over a considerable period of time, after which the strength of the process diminishes and the animals return to the waking state. The fluctuations in the mean episode durations of immobility of head and body as well as of antennal immobility (Fig. 4) indicate that the process may contain a periodic component. Studies in humans and mammals have demonstrated that regulatory mechanisms are involved in the sleep process - sleep deprivation has been a particularly useful tool in elucidating the nature of these mechanisms (Borb61y 1982; ~kerstedt and Gillberg 1986; Dijk et al. 1987). Tobler (1983) employed this paradigm to demonstrate that a sleep-like state exists in cockroaches. The data in Figs. 3 and 4 suggest that sleep in honeybees is more ' intense' when sleep duration is short. This can be regarded as a sign of regulation. Experiments on sleep deprivation in bees are now being performed in this laboratory.

There are indications that bees do compensate for sleep loss (B6sebeck and Kaiser 1987). If this preliminary result can be substantiated, then one further criterion in Tobler's (1984) list of 'criteria for the definition of sleep' in humans and animals will have been met. Recent, long-term measurements of heart-beat frequency in forager bees show that this parameter also complies with Tobler's criteria (Jander 1987). Results from our previous electrophysiological investigations demonstrate that bees fulfill at least two of Tobler's criteria at the neuronal level as well. Both rapid state-reversibility and decreased sensitivity are displayed at night by visual interneurones (Kaiser and Steiner-Kaiser 1983, Fig. 3). (State-reversibility at the behavioural level was particularly evident during the determination of reaction thresholds.)

Sleep signs specific to bees

One of the most important sleep signs used to char- acterize sleep in mammals and birds - the EEG - is not available for the study of sleep in bees. On the basis of the experiments on reaction thresholds and muscle tone (Figs. 9, 10, 12) together with the observations of antennal motility (Fig. 3 B, D) and antennal position (Fig. 6), I believe that we

have an adequate alternative for this parameter in the bee: simultaneous determination of antennal position and motility. Experiments are in progress to determine whether the periodicities of the curves in Fig. 4 are a typical feature of sleep in the bee. Sleep in mammals and birds exhibits characteristic patterns of temporal organization (Meddis 1983).

'Sleep' behaviour in other insects

The results presented here provide no support for the existence of'sleep-immobilization' (Hoffmann 1937) in forager honeybees. This state, however, obviously does occur in a variety of other insects. It appears to be relatively common in solitary, tropical Hymenoptera. Fiebrig (1912) has pub- lished a detailed account of this bizarre behaviour which probably serves a protective function.

Fiebrig notes that there was a definite tendency for individuals of a species to congregate at a particular place, where all members of the group fell into the immobilized state. Such social aggrega- tions are not restricted to Hymenoptera: descrip- tions o f ' s l eep societies' in the Malaysian stalk- eyed fly, Cyrtodiopsis whitei, have appeared recently (Burkhardt and de la Motte 1983; de la Motte and Burkhardt 1983).

Acknowledgements. I am particularly grateful to Professors G. Fleissner, Frankfurt and W.P. Koella, Oberwil and to Dr. H. Schulz, Munich, who encouraged me to move my focus of scien- tific interest to the stimulating field of comparative sleep research. I would like to thank W. Eusemann, H. Langmaack, Y. Mainzer and A. Walter for helping to collect data and Dr. J.-P. Jander and H. B6sebeck for discussions. The instructive comments of two anonymous referees are gratefully acknowl- edged. I would also like to thank S. Rettig and U. Plank (Facul- ty of Mathematics, TH Darmstadt) for their willingness to ad- vise and assist us in matters concerning data analysis. M. Al- bert, G. Bayer, R. Frank-Bauer and R. Rohr helped to prepare the manuscript for publication. H. Gudella provided invaluable help in keeping our bees healthy. Our observation hive was a generous gift from Professor M. Lindauer and his staff. I am grateful to Gesotec GmbH, Darmstadt, for the kind loan of the thermovision camera. My wife, Jana Steiner-Kaiser, has been a source of constant support and constructive criticism throughout the course of this work and also translated the manuscript. Generous financial assistance has been provided by the Deutsche Forschungsgemeinschaft under the program SFB 45.

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Busy bees need rest, too