The effects of changes in the magnetic azimuth on bee resting behaviour

Only honey bee foragers were used in this research and this experiment was conducted in a dark room. The bees were captured in the afternoon and maintained in a plastic cup (diameter 9 cm, height 6 cm) with water and food. The cups with the bees were placed in a refrigerator (~4 °C) and the cold-anaesthetised bees were transferred individually into Petri dishes (diameter 9 cm, height 1.5 cm), which were placed on a wooden board in the coil at 20:00. On each experimental day, 18 bees were tested simultaneously and each dish was surrounded by a strip of paper to prevent the bees from seeing each other.

The magnetic stimulus was generated by square coils. The square coils were implemented using a scheme similar to the Merritt four coil system57. Four sets of thin enamelled wire (Ø = 0.5 mm, ρ = 1.7 × 10−8 Ω·m) were wound into four coils that consisted of 26, 11, 11 and 26 turns, sequentially. The side length of each square coil was one metre and the spacing of the neighbouring coils was consequently 37.74, 25.62 and 37.74 cm. The four coils were serially connected to a DC power supply to generate a uniform magnetic field at the central area of the coils. The coil was placed vertical to the geomagnetic field (approximately 40 μT) and generated a 65 μT field, which induced the horizontal component to rotate 60° clockwise.

The experiment took place between 0:00 and 4:30 and images of the bee behaviour were recorded every five minutes by an infrared camera, which was placed 50 cm above the wooden board. Except for the lens, the camera was wrapped in aluminium foil grounded with wire to minimise the potential influence of the magnetic field. The square coils were turned on/off to change the magnetic azimuth every 5 minutes during the stimulus period (from 1:30 to 3:00). The data of the ‘before stimulus’ (from 0:00 to 1:30) and ‘after stimulus’ (from 3:00 to 4:30) served as the control group. The images were visually checked on the computer and we counted the body turns when the body axis slewed more than 10°.

PER training

To examine the magnetic sensing capability of the honey bee, a magnetic field generator was built to deliver short pulses of magnetic fields to the body of the honey bee. Using ferritic stainless steel with low magnetic susceptibility (Ø = 60 mm, μ = 875 × 10−6 H/m, χ m = 700), we constructed a semicircular magneto core with an air gap. A thin enamelled wire coil (Ø = 0.5 mm, ρ = 1.7 × 10−8 Ω·m) consisting of 1200 turns was wound onto the magneto core to induce a magnetic field in the air. The coil wire received an electric current whose amplitude and frequency were regulated by an external controller, which allowed the direction of the magnetic field to be modulated or the core to be demagnetised.

Only honey bee foragers were used in this research. After undergoing cold anaesthesia, the honey bees were fixed at the tip of a 1000 μL pipette with a drop of beeswax-resin mixture melted by a heated soldering iron. The compound eyes were painted over with black acrylic fabric rubber to reduce visual interference. Each honey bee was then placed at the centre of the coils overnight. The orientation of the body axis was parallel to the geomagnetic field and the magnetic stimulus generated by the magnetic field generator was vertical to the geomagnetic field. The intensity of the stimulus was 200 μT, which is about five times the geomagnetism (approximately 40 μT) and the stimulus was 5 Hz from an alternating field.

The bees were trained to have a PER. The CS (5 Hz of alternating field for 10 s) was a magnetic stimulus and the unconditioned stimulus (US) was a 50% sucrose reward. Before training, each honey bee was first offered sucrose to make sure that she had a normal PER response to the US. Figure 4 shows how the training and test process were conducted.

Figure 4 The training protocol for the proboscis extension reflex (PER) of a honey bee being conditioned by magnetic stimulus (The images of the bees were drawn by C.H.L.). (a) The conditioned stimulus (CS; 5 Hz of an alternating field for 10 s) was a magnetic stimulus and the unconditioned stimulus (US) was a sucrose reward. Before training, each honey bee was first offered sucrose to verify a normal PER response to the US. Each honey bee was then conditioned by the CS, immediately followed by the US. After resting for 10 s, each honey bee was again tested by the CS to complete the training and test cycle. If a bee extended its proboscis during the test period, this suggested that it had learned the association in this trial. (b) The process of training and test cycles (shown in green). The interval between the two training and test cycles was 5 minutes and we conducted 20 cycles each day. Full size image

The effects of cutting the VNC on magnetoreception

To confirm the transmission of the magnetic signals, microsurgery was used to cut the nerve connection between the iron granules in the abdomen and the brain. The successfully trained bees (those that had previously responded twice in a row to the magnetic stimulus) in PER training were used for the experiment. The surgical process was carried out as follows. (1) Bees were fixed on Styrofoam using pins that were not inserted into the honey bee’s body. (2) A small incision was made on the underside of the first abdominal sternum. The ventral nerve cord and a globe, known as the first abdominal ganglion, were visible. (3) The VNC was cut above the first abdominal ganglion to ensure that no signals from the abdomen could pass to the brain. (4) The wound was covered with petroleum jelly to reduce transpiration. After microsurgery, each bee was subjected to the magnetic stimulus again and the response was recorded.

To eliminate the possibility that it was the surgery that resulted in the bees being unable to have a PER, the odour PER was applied as a control. The odour PER training process was conducted as follows. (1) The odour stimulus was applied for 6 seconds as the CS. (2) The sucrose reward was given immediately after the CS. (3) Then, there was a 10 second rest to allow each bee to retract its proboscis. (4) The stimulus was then reapplied to test those bees that did not follow the CS. The successfully trained bees (those that responded twice in a row to the magnetic stimulus) had their VNCs cut as described above. After surgery, the bees were again subjected to the odour stimulus and their responses were recorded.

Neural signals in response to the magnetic field

The magnetic field generator used in this experiment was the same as the one used to test the effects of the bee rest behaviour in response to magnetic azimuth changes. In this experiment, the glass microelectrodes were used to record the neural response. The microelectrode was made from a glass capillary tube (AF100-64-10, Sutter Instrument Co., USA), which was manufactured with a micropipette puller (Model P-87, Sutter Instrument Co., USA). The well-prepared microelectrode was sharp at the tip and tough enough to penetrate the nerve cord. A head stage was used to receive the signals from the electrode. The ‘probe+’ of the head stage was attached to a silver wire (782500, A-M Systems, Inc.), which was coated with AgCl on the surface and then the wire was placed in the microelectrode as the recording electrode. The ‘probe-’ and the ‘probe ground’ of the head stage were tied together and attached to another silver wire as an indifferent electrode. The electrical signals could then be amplified by an AC/DC Differential Amplifier (Model 3000, A-M System, Inc., Sequim, WA, USA), transmitted to the computer and recorded by the program DataWave SciWorks (Version 7.2, DataWave Technologies Co., Loveland, CO, USA). The sampling rate was 5 kHz and we used a digital filter set as 70 Hz high pass. Finally, the response was drawn by using the program Origin (Version 7, OriginLab Co., Northampton, MA, USA).

Only foragers were used in this experiment. The bees were fixed on the pipette by using the same method used in the PER training and all legs were removed to reduce interference. We recorded the signals from the ventral nerve cord between the thorax and the brain, which we located after removing the membrane on the ‘neck’. The indifferent electrode was placed in the mesothorax. Each bee was placed parallel to the geomagnetic field and the magnetic field generator was placed vertical to the geomagnetic field and generated a 65 μT field, which induced the horizontal component to rotate 60° clockwise.