Interrelations of gas exchange cycles , body movements and heartbeats in the foragers of bumblebee Bombus terrestris ( Hymenoptera : Apidae ) at low temperatures

Rhythmic body movements, cyclic gas exchange and heart activity were studied at low temperature (mostly at 5°C) in bumblebee Bombus terrestris foragers. Simultaneous measurements by means of an electrolytic respirometer combined with an optical system by infrared radiation, revealed a co-ordination between discontinuous gas exchange cycles and body rhythmic move­ ments. No distinct correlation was found between intermittent heart activity and body movements. The carbon dioxide bursts were actively ventilated by abdominal vigorous pumping movements. These bursts followed each other with intervals of 25-30 minutes. The periods of heart activity regularly alternated with heart pauses. The frequency of the heartbeat was similar to that of the weak abdominal pulsations (0.2-0.25 Hz) which were imperceptible with the naked eye. All bumblebees displayed abdominal pulsations which were independent of vigorous pumping movements. In bumblebee B. terrestris foragers haemolymph oscillation was assumed at 5°C, as is known in the honeybee and some other Bombus species.


INTRODUCTION
Bumblebee workers often have to survive in tempera tures near zero.So far little is known about the breathing patterns of bumblebees at low temperatures close to their cold stupor points.
In many insects respiration occurs by discontinuous gas exchange cycles or DGCs, which means that the carbon dioxide is released discontinuously by bursts.Commonly the DGC is devided into three phases according to the action of the spiracles: the "C phase" of closed spiracles, the flutter or "F phase" when only a little carbon dioxide is released but oxygen is consumed, and finally, there is the phase of opened spiracles or "O phase" when carbon dioxide is released in bursts (Kestler, 1984;Hadley, 1994;Lighton, 1994;Snyderetal., 1995).
The bumblebee workers are very active at room tem perature and only the lowering of temperature allowed for examination of their respiratory patterns.The aim of the present paper was to study the possible coordination between gas exchange, abdominal movements and heart beats in the foragers ofbumblebee B. terrestris at 5°C.

MATERIAL AND METHODS
The bumblebee B. terrestris colonies (Natupol hives) were purchased from Koppert Biological Systems and were hold in an orchard in Tartu, Estonia.Thirty individuals from three colonies were tested.The bumblebee colonies and individuals used in this study were not parasitized, nor infected by diseases.
For the experiments foragers weighing 220-260 mg were selected from the colony and adapted to 5°C for one day before laboratory measurements.
Metabolic rate, discrete CO2 releases and body movements were recorded by means of an electrolytic differential microres pirometer, where the consumed oxygen was replaced continu ously by adjusting the level according to pressure changes in the insect chamber (for more details see Kuusik et al., 1991Kuusik et al., , 1996;;Tartes et al., 1999Tartes et al., , 2000)).In the present modification of this res pirometer, standard plastic tubes (Eppendorf, volume of 1.5 ml) were used as insect chambers, and were connected to the respirometric system by rubber tubing (inner diameter 0.5 mm).
The units on the graphs "VO2 (ml h-1)" represent the rate of the production of oxygen by electrolysis.Using a personal com puter the mean metabolic rate was calculated by averaging data over a longer period involving at least 3 DGCs (see Tartes et al., 2000).
The respirometer was combined with an infrared (IR) optical device.Two IR-emitting diodes were placed on the side of the chamber near to the ventral side of an insect.Two IR-sensitive sensors were placed on the opposite side of the chamber.Light from IR-diodes was modulated by the contractions of the heart and skeletal muscles.The output voltage level reflected the vigour of the muscular contractions of the insect.
We used 30 bumblebee individuals in our measurements.On the graphs the patterns of single individuals are represented, but these general patterns were also characteristic of all of the indi viduals studied.

Cyclic gas exchange
The bumblebee workers exhibited clear cycles of dis continuous gas exchange while restrained and motionless.The immobility of bumblebees was achieved by exposing them to 5°C; i.e., above the cold stupor point and below the threshold of activity.The intervals between CO2 bursts were 20-30 minutes after the measurements had lasted 3-4 hours and the bumblebees had fully recovered from the handling stress (Fig. 1).After every CO2 burst a characteristic peak followed, obviously due to abdominal contraction (Fig. 2).At 0°C the gas exchange was con tinuous.

Abdominal pumping movements
We regarded vigorous abdominal contractions as abdominal pumping movements.These visually well observable movements in bumblebee workers were always associated with discontinuous gas exchange cycles and the tracheae were seemingly actively venti lated by these movements.Usually the pumping move ments started with the beginning of a CO2 cyclic release  (Fig. 1 and 2), but sometimes the pumping movements began at the end of the CO2 burst.The period of pumping lasted 2-4 minutes.About 20% of the bursts of an indi vidual were not associated with abdominal pumping.There were also bumblebee individuals whose DGCs never concurred with bouts of vigorous abdominal pump ing.The common frequency of the abdominal pumping movements was 0.5-0.7 Hz.
The metabolic rate during the vigorous pumping move ments was 2-3 times higher than the metabolic rate during the pumping pause (0.6-1.2 ml g-1 h-1 and 0.2-0.5 ml g-1h-1, respectively).

Abdominal pulsations
The clear rhythmic signals with relative low amplitudes we regarded as "abdominal pulsations", as they were externally imperceptible even under a stereomicroscope.
Abdominal pulsations were easily seen on the respirometric recording after the ending of the vigorous pumping movements (Fig. 2).These pulsations caused rhythmic air pressure pulses inside the respirometer.The patterns of abdominal pulsations were often mixed with other signals.

Heartbeats
On the recordings of the respirometer and IR device, several signals were mixed with those of heartbeats.There were periods of very regular signals of low ampli tudes, seen well only on the recordings of IR device (Fig. 1, 3).The simultaneous IR irradiation through the abdominal second and fifth segments showed the clear peristaltic nature of these movements, which were recog nized as forward directed heartbeats (Fig. 4).We con cluded that these weak heartbeat signals were recorded  during the "heart pause", because after the pause these signals transformed into peaks of the same frequency, but of much higher amplitudes (Fig. 3).By our methods we could not determine the time when the "heart pause" ended.Abdominal pumping movements occurring during the CO2 cyclic release (Fig. 1) suppressed the weaker sig nals, including possible heartbeats which were mixed with the pumpings.The usual frequency of the supposed heartbeats was 0.25-0.3Hz at 5°C.
The heartbeat frequency of some bumblebee workers was similar to that of the abdominal pulsations, and some times a strict synchronization occurred between these two events.The amplitude of ventilation strokes was com monly 4-5 times higher than that of heartbeats.Thus it was evident that our cardiographic method allowed us to record the heartpulses only during the periods when other body movements were not mixed with heartbeats.

Shivering
By means of the IR optical device we recorded move ments in the thorax with the frequency of 1.3-1.6Hz at 5°C, which we interpreted as fibrillar weak "shivering" of wing muscles (Fig. 5).The signals of the supposed shiv ering were sometimes superimposed on the signals of more vigorous muscular contractions with lower fre quency.We recorded a clear shivering with a frequency of 5-7 Hz in the stressed bumblebee foragers at 10°C (Fig. 6).

DISCUSSION
In bumblebees rhythmic movements of different origin may occur: e.g., heartbeats, abdominal pumping move ments and contractions of ventral diaphragm (Heinrich, 1976(Heinrich, , 1994)).In the present work some of the ordinary Fig. 6.An IR-sensor recording of the "shivering" of wing muscles of a bumblebee forager at 10°C.The signals of muscular vibra tion are modulated by four strokes of abdominal movements.rhythmic movements were identified in the foragers of B. terrestris.
In some Lepidoptera and Hymenoptera haemolymph oscillation has been described, which works as a mecha nism for the ventilation of tracheae and airsacs (Wasser thal, 1976(Wasser thal, , 1980(Wasser thal, , 1981(Wasser thal, , 1982)).InsleepingA.melliferathe abdominal contractions coincided with the moment of transition from a long heartbeat pause to a prolonged beating of the heart which is always forward (Wasserthal, 1996).The backward flow of haemolymph through the perineural sinus happened mainly during the heart pause.
In the adult bumblebee of Bombus vosnessenskii and B. edwardsii, the ventilation movements are highly coordi nated with the circulating actions of the dorsal vessel and ventral diaphragm, acting as a mechanism of thermoregu lation (Heinrich, 1977).In the overheated bumblebees single abdominal pumping movements were synchronized with heart pulses, from which it could be suggested that the pressure evoked by abdominal contractions assisted the pumping movements ofthe heart (Heinrich, 1994).
The methods we used allowed us to record and recog nize some rhythmic events of B. terrestris.These were first of all the discontinuous gas exchange cycles and vig orous abdominal pumping movements, which occurred during the CO2 bursts.We recorded periodically the very regular signals by IR sensors, which we interpreted as heartbeat pause with very weak heart pulsations.
In the present results B. terrestris showed a more or less regular alternation of periods of heartbeat with heart pauses, or rather with periods of very weak pulsations of the heart.No distinct coordination between the periods of heart activity and abdominal movements (pumping and pulsations) was found.However, the intermittent heart activity suggests haemolymph oscillation in B. terrestris at 5°C, similar to that of A. mellifera and other bumblebee species described above but measured at higher tempera tures.
We interpreted the very weak but regular movements caused by the abdomen as abdominal pulsations which are independent of muscular ventilation; with particular rhythms and frequencies.In our opinion the abdominal pulsations in B. terrestris are identical to the haemolymph extracardiac pressure pulses described by Slama (1976) and later found in the insects of various developmental stages and systematic groups (Slama, 1984(Slama, , 1988)).Tho racic ganglia regulate the extracardiac pressure pulses, and these pulses have a role to play in respiration and cir culation (Slama, 1994(Slama, , 1999(Slama, , 2000)).The extracardiac pressure pulses are independent of heartbeats, but these pressure pulses occurred during the CO2 bursts, as described during each burst in cecropia pupae (Slama, 1988) and in adult Periplaneta (Slama, 1999).
The vibrating of wing muscles or "shivering" was com monly recorded by wire electrodes as the action potentials of the fibrillar muscles; i.e., by an electrophysiological method (see Kammer & Heinrich, 1972, 1974).The shiv ering in Manduca sexta was studied by tracheal pressure measurements (Wasserthal, 2001).The infrared irradia tion method allowed us to measure the frequency and amplitude of the shivering in bumblebee workers in a contact-free manner.

Fig. 1 .
Fig. 1.Recording of IR-sensor (upper trace, Volts) and simultaneous respirogram (lower trace) showing cyclic gas exchange, abdominal movements and heart activity in a bumblebee Bombus terrestris forager.I -The period of abdominal pumping, together with other movements and heartbeats; II -the pattern due to abdominal pulsations mixed with heartbeats; III -heartbeats with very low amplitudes interpreted as heart pause.CO2bursts are indicated with arrows.A body rotation stroke is seen (x).

Fig
Fig. 2. A detail of Fig. 1.An optical recording (upper trace, Volts) and a simultaneous respirogram (lower trace) represent a typical example of when the onset of abdominal pumping movements (spikes) coincides with the beginning of a CO2 burst in a bum blebee.An arrow indicates a stroke of vigorous abdominal contraction, after which clear signals of abdominal pulsations followed.

Fig. 3 .
Fig.3.A detail of Fig.1where the heart pause was followed by a period of heart activity (arrow) (upper trace).During the CO2 burst, peaks are seen due to vigorous pumping movements (lower trace).

Fig. 4 .
Fig. 4. The simultaneous records of IR-sensor diodes at the second (solid line) and fifth abdominal tergites (dotted line) during "heart pause", showing the forward directed heartbeats.Systolic contraction is down and diastole is up.This optical recording was made by means of high-grade amplification.

Fig. 5 .
Fig. 5.A recording of an IR-sensor diode at the thorax side (upper trace), showing the contractions of wing muscles at 5°C.A simultaneous respirogram (lower trace) with three abdominal pulses.