Honeybees

For all experiments, except the field test, bees were collected from several unrelated honeybee colonies (A. mellifera ligustica) housed on the University of Queensland St. Lucia campus (Brisbane, Queensland, Australia), from April 2013 to October 2014, excluding the winter months—June to August. All colonies were freely foraging and underwent routine beekeeping inspections and honey collection during the course of the experiments. An equal number of bees from four different colonies participated. The bees were caught on sunny days in two rounds (around 0930 and 1100, h, alternatively for each colony), in a pattern ensuring that no colony was disturbed more than once every 48 h. This delay allowed the hives to fully settle down in between disturbances and indeed no increase in aggressiveness was observed over time. To select for the population of bees involved in colony defence (guards and soldiers), the bees were collected by waving a large black feather in front of the hive entrance for a few seconds. Once the feather was covered in about 30–40 attacking bees, it was quickly placed into a sealable plastic bag and in a freezer at −20 °C. The state of the bees was checked after 5 min and then every 1–2 min until they were all motionless (on average 8.25 min in the freezer). The bees showing the quickest recovery were selected and placed alone or in pairs into 50 ml syringes (Terumo) containing a wet tissue and three droplets of sugar water (50% sugar water, vol/vol). The tip of the syringe was cut and replaced with a plastic sliding door held with a paper clip. In case of pairs, one honeybee was marked with a red dot on the thorax (enamel paint), while the other was left unmarked. Similarly, half of the single honeybees were marked in the same manner, while the other half remained unmarked, to control for a possible effect of the enamel paint. The data revealed no difference in aggressive behaviour between marked and unmarked bees (χ2=1.575, df=2, P=0.455). Once this step was complete, all honeybees were allowed to recover for another 10 min and up to 80 min before being tested in the set-up investigating aggressive behaviour. If one or both bees showed signs of poor recovery when put in the set-up (difficulty to hold upside down, clumsy and/or slow walk), the whole trial was excluded from further analysis. All the materials used to contain the bees were washed with detergent, rinsed and dried after each use.

For the main aggression experiment, a large number of different odourants were tested. As it was not technically possible to test them all simultaneously, the odourants were distributed into four sets, each including IAA and the solvent TEC as reference points. As there was no statistical difference between these references across the four sets (see results), the data were pooled. As a consequence, the IAA and TEC groups include 128 pairs of bees, whereas every other group includes 32 pairs. The experiment testing the role of social interactions included 32 pairs or individuals per group. This sample size was chosen based on pilot experiments. The experiment using the full SAP included 48 pairs of bees per odour condition. The sample size for this experiment was increased, to gain the statistical power necessary to detect this smaller effect. No bee was tested more than once nor released (they were killed).

The field test was performed at the apiary of the University Paul Sabatier (Toulouse, France) at the end of summer 2015 (August–September). Three colonies of the same subspecies (A. mellifera ligustica) participated in this experiment. They were all freely foraging, treated against Varroa and underwent routine beekeeping inspections during the course of the experiment. The colonies were tested no more than once every 24 h, in the morning of sunny days.

Odourants

All odourants were pure chemicals (98–99.9% purity) from Sigma-Aldrich and kept in the freezer (−20 ° C). Before each set of experiments, a fresh batch of odourant dilutions was prepared using TEC for solvent and kept for the whole length of this experiment. These odourants were delivered at room temperature (25 ° C) and kept in the fridge (4 ° C) when not in use. Table 1 presents all the odourants used and their concentrations. The concentration of 0.075% (vol/vol) for all plant odours was chosen taking as reference the concentration of Pr, the combination of plant-derived odours used in our work (0.03% Z-3-hexen-1-ol, 0.03% E-2-hexenal and 0.015% α-pinene in TEC). This concentration was shown to reduce corticosterone, glucose and redox responses elicited by psychological stress in rats25,56, and was thus used as a starting point for our study. For the field test, the concentration of the odourants was increased to 1%, to cope with the large volume of air in which the odour had to be delivered.

Aggression assay

Assessment of the bees’ aggressiveness was done in circular arenas (Fig. 1a, 14 cm diameter, 4 cm high) made of transparent plastic. A sliding door on the side allowed introduction of the honeybees from the syringes. The various odourants used were blown into the arena through three entry points (4 mm ID) regularly spaced and at middle height along the wall. The arena lid was regularly drilled with about 40 holes (1 mm ID), to avoid building up of the odour inside the arena. A 1-cm hole was also opened in the middle of the arena floor to allow passage of the step motor axle (Aviosys DYO AK27PCB). The step motor was connected to a DC power unit set to 9 V and 0.25 A. Before each trial, the arena was wiped clean using a 70% ethanol solution and a wet filter paper was put on the floor to maintain the humidity. A dummy was placed horizontally on top of the step motor axle with blue tack. Four dummies were made, each consisting of the barrel of a 3-ml syringe (cylinder of 5–6 cm long, 1 cm in diameter) covered with a rectangular patch of black suede leather (4.5 × 7 cm) and prolonged on one end with a soft black feather. The use of the four dummies was always balanced across the different conditions. The leather patch was held with four pieces of yellow electrical tape and was changed whenever it had been stung. Stung leather patches were rinsed with clear water and left to dry outside for at least 24 h before being used again; the feather was also cleaned with 70% ethanol. To increase the jerkiness of the movement, the step motor was used at its lowest speed: as a result, the dummy rotated horizontally across the middle of the arena floor, while the black feather gently brushed the sides. The size and shape of the dummy allowed the honeybees to freely move along the sides and lid of the box without touching it. The purpose of the black feather was to disturb the bees without causing them pain. Indeed, this feather was merely touching the bees and was not strong enough to change the path of a walking honeybee.

Odour delivery in the arena

Medical grade air (BOC, North Ryde, Australia) was delivered from a 680-l tank and fed into a custom-designed olfactory stimulus controller. This olfactometer delivered a constant clean air flow of 1 l min−1. Polytetrafluoroethylene (PTFE) Teflon tubing (3 mm ID) led this air flow to the base of a 15-ml Falcon tube, inside which filter papers carrying the odourants were placed. Further up the sides of the Falcon, three more Teflon pipes were connected, which terminated on the other end into truncated pipette tips. The resulting device could be easily plugged in and out of the three odourant entry points of the arena (Fig. 1). To avoid contamination, eight of these devices were made and each one was used for the delivery of a single odourant (or combination of odourants) during the course of an experiment. In between experiments, they were thoroughly washed with 70% ethanol and left to dry for at least 24 h before being used for another set of odours.

During each trial, two pieces of filter papers were put in the Falcon tube dedicated to the odour delivery. Depending on the odour combination tested, they were either both blank (none, no odour control), one soaked with 10 μl of an odourant and the other with 10 μl of solvent (odourant alone) or one soaked with 10 μl of an odourant and the other with 10 μl of the alarm pheromone (IAA+odourant or SAP+odourant). For example, for the TEC control both papers were soaked with TEC, for testing Lim alone the combination was Lim+TEC and for testing the interaction between Lim and the alarm pheromone one filter paper carried IAA and the other Lim. To ensure homogeneity of the data, presentation of the different odourants was balanced over colonies and time of the day.

Trials and scoring of the aggression assay

All trials were recorded with an high definition camera positioned above the arena. Each trial went as follows: first, the camera and the step motor moving the dummy were switched on. The tip of the syringe containing the honeybees was then inserted inside the arena. The olfactometer was always switched on just before introduction of the honeybees in the arena, while the arena door was already open but not yet the syringe door. As a result, the bees received a quick puff of odour just before facing the dummy, thus mimicking the successive steps of colony defence usually occurring in nature. The syringe door was then opened and, if necessary, the bees were gently pushed inside the arena with the plunger. The odourant air flow was left running during the whole length of a trial (3 min). During the trial, the rotating direction of the dummy was manually and randomly changed multiple times.

The stinging response of a bee was scored visually and defined as the bee holding onto the dummy for at least 3 s, with the tip of the abdomen pressed against it in the characteristic stinging position; the vast majority (90.2%) of the attacks recorded were further confirmed by the presence of the stinger apparatus still embedded in the dummy leather. Another 5.7% of the aggressive bees stayed exclusively on the feather, which was considered the reason why their stinger was not pulled away. Finally, the remaining 4.1% of attacks scored correspond to bees either choosing to bite the dummy, or (in very few cases) to bees clearly attempting to sting the dummy, although the reason why the stinger could not be recovered was unknown. Fewer than 1% of the trials were considered borderline (for example, when an agitated bee contacted the dummy multiple times but did not exhibit any of the other criteria) and were excluded. For each trial, the aggressive response was scored as 1 if at least one of the bees attacked the dummy and 0 if all bees remained calm.

Field test

Before the beginning of the experiment, the size of the landing board was standardized (5.5 × 53 cm) for all the colonies and an open box was created around the hive entrance by placing two vertical wooden walls (10 cm high) on each end of the landing board and a transparent plastic roof on top (Fig. 2c). This box was closed at the beginning of each test by the addition of a front door, thus creating a stable atmosphere of about 2.5 l at the hive entrance in which an odour could be delivered. To this end, two 15 ml Falcon tubes containing a filter paper carrying 10 μl of the odour were inserted into holes in the lateral walls (Fig. 2c). Four small holes were drilled at the bottom and the lid was modified so that the tubes could be easily connected to the output of an aquarium air pump (Rena 300, delivering a total air flow of about 3.3 l min−1). To avoid contaminations, a pair of tubes was made for each odour tested. To measure aggressiveness at the colony level, a black leather patch (4.5 × 7 cm) on a wooden pole was placed in front of the hive entrance, 1–2 cm away from the landing board, and jiggled via a small motor29,30 (Lego Power Functions XL-Motor). A square marker on the ground ensured that the flag positioning was the same across days. The tests started by closing the front door of the box and switching on the air pump to deliver the odour, while the flag remained motionless just outside the box. The order of presentation of the odourants was randomized. After 2 min of odourant exposure, the flag motor was switched on and the door was removed, thus allowing the bees to confront the moving flag (with the odour delivery still on; Fig. 2c). This step lasted for another minute (hence a total of 3 min for the whole test), after which the motor was stopped and the flag quickly sealed in a plastic box so that no additional bees could access it. The number of stingers embedded in the leather was then counted and used as a measure for aggressiveness at the colony level. The flags were discarded after they were stung and all the material was washed with 70% ethanol between trials. All trials were recorded with a camera placed above the landing board. Each of the three colonies was tested six times with each odour (n=18 per group) and data were normalized per colony (see below) to account for different inter-colony aggressiveness before being pooled.

Masking experiment

In the morning, equal numbers of bees from the four colonies were caught at the hive entrance, using Falcon tubes. They were then cold anaesthetized in the freezer during 5 min and tethered in the restraining tubes used for PER conditioning57. They were fed with a droplet of sugar water (50% vol/vol) before being placed in a dark incubator (26 °C, 85% humidity) for 3 h. This is a standard procedure to homogenize the satiation level of the bees and habituate them to the restraining tube57.

The conditioning of the PER is a classical conditioning assay in which harnessed bees learn to associate odourants with the appetitive reward of sucrose solution36. When the antennae of a hungry, harnessed bee are touched with sucrose solution, the animal reflexively extends its proboscis to reach out to and suck the sucrose (PER). If an odourant is presented immediately before sucrose solution (forward pairing), an association is formed, which enables the odourant to release the PER in a following test. In our experiments, bees were exposed to an odour (CS) for 6 s followed by the presentation of sucrose solution (US, 50% vol/vol) for 3 s. The CS and US overlapped during 3 s. Bees were conditioned with four trials spaced by 13 min. Forty-five minutes after the last conditioning trial, the bees’ responses to three or four odours was tested, in a randomized order and without any sugar reward. There was a 13 min inter-trial interval between the tests. Three sets of experiments were conducted. In the first set, the bees were conditioned with IAA and tested either with IAA, PhE and IAA+PhE or with IAA, Pr and IAA+Pr. In a second set, the bees were trained with a mixture (IAA+PhE or IAA+Pr) and tested with the same mixture, IAA, the plant odour alone (PhE or Pr) and β-c (novel odour). Finally, in a third set of experiments the bees were trained with the plant odour (PhE or Pr) and tested with the same plant odour, the corresponding mixture (IAA+PhE or IAA+Pr), IAA and β-c. These six test groups include respectively 53, 54, 56, 56, 53 and 56 honeybees. These sample sizes are within the standard range used to ensure statistical power during analysis of PER experiments57.

Experiment testing the appetitive value of odours

Two populations of honeybees were tested during this experiment. Defensive bees were caught directly from colonies as described above. To test whether some odours were innately appetitive, we produced odour-naive bees by placing a capped brood frame in a dark incubator (34 °C) and collecting the newly emerged bees every day. Groups of 20 age-matched bees were then raised in meshed cages in the same incubator for 10 days. They had ad libitum access to water and an unscented sugar solution (50% vol/vol), except during the night before testing when the sugar solution was removed to increase their motivation. Fresh food and water were provided every day. All the bees were cold anaesthetized on the morning of the test day, placed in the restraining tubes used for PER testing, fed a droplet of sugar water and then left in a 26 °C dark incubator for 4 h before testing. A total of 101 naive bees and 110 aggressive bees participated in this experiment.

Each bee was presented once with the six odours tested, in a randomized order and spaced by 13 min. Importantly, no training was performed before testing and no reward was given during testing. At the end of the testing session, the PER was triggered by touching the honeybees’ antennae with sugar water and the few bees that did not respond to this stimulation were excluded from the analysis.

Statistics and calculation of theoretical data

We used χ2-tests to analyse the data produced by the experiment investigating the role of social interactions as the observations were independent and all expected cell counts were >10. To calculate the theoretical data, we considered that the frequency of aggressive trials for single bees under given conditions represent the probability p of one bee from this population to sting under these conditions. The probability of scoring an aggressive trial from two such bees was then calculated using the classical probability laws for two independent events. As a result,

Or more generally,

The expected results were then obtained by multiplying this probability by the sample size.

All the other aggression data were analysed using a GLM set-up with a logit link function appropriate for binomial data.

In the field test data set, two outliers had to be removed in each group. They all corresponded to extremely aggressive trials during which two to nine times more stingers than usual were collected. Removing them did not change the overall pattern of responses observed but allowed the data set to meet the normality assumption (Shapiro–Wilk tests) necessary to run an ANOVA with repeated measures. The data were also normalized per colony by subtracting the colony average from each data point and dividing by the colony s.d. (standard score). This was done to homogenize the data, as each colony had a different baseline aggression level (from 1.76 to 17.6 responding bees on average for the most aggressive colony). Post hoc pairwise comparisons were corrected with a Bonferroni procedure.

A potential difference between the percentages of bees exhibiting a PER response when presented with the different odourants was tested with Cochran Q test, as it is adapted to repeated measures with dichotomous responses. If this test was significant, a post hoc analysis was performed using multiple McNemar tests and a significance threshold adjusted with a Bonferroni correction. The correlation between two data sets was tested using Pearson’s r test.