Antenna movements as a function of odorants' biological value in honeybees (Apis mellifera L.)

Abstract

In honeybees, the antennae are highly mobile sensory organs that express scanning movements in various behavioral contexts and toward many stimuli, especially odorants. The rules underlying these movements are still unclear. Using a motion-capture system, we analyzed bees' antennal responses to a panel of pheromonal and other biologically relevant odorants. We observed clear differences in bees' antennal responses, with opposite movements to stimuli related to opposite contexts: slow backward movements were expressed in response to alarm pheromones, while fast forward movements were elicited by food related cues as well as brood and queen related pheromones. These responses are reproducible, as a similar pattern of odor-specific responses was observed in bees from different colonies, on different years. We then tested whether odorants' attractiveness for bees, measured using an original olfactory orientation setup, may predict antenna movements. This simple measure of odorants' valence did however not correlate with either antennal position or velocity measures, showing that more complex rules than simple hedonics underlie bees' antennal responses to odorants. Lastly, we show that newly-emerged bees express only limited antennal responses compared to older bees, suggesting that a significant part of the observed responses are acquired during bees' behavioral development.

Figure 1

Antennal movements recording. (A) Schematic representation of the apparatus for recording bees’ antennal movements. The bee is placed under a camera which detects the colored dots previously applied on bees’ antenna tips. The recording is made in a dark room and the camera is surrounded by a ring light source to control lighting and allow best detection of the color dots. The bee is placed in front of an odor stimulation device. (B) Representation of the calculated antennal movement variables: the distance to antenna base (r), the angular position (θ) defined as the angle between the line connecting the antenna tips to their base (r) and an antero-posterior line passing through the corresponding antenna base. The angular velocity (Vθ) is calculated as the angle θ traveled by each antenna during a frame (1/90 s), expressed in degrees per second. (C,D) Average recordings of (C) antennal angular position (θ) and (D) angular velocity (Vθ) in response to the air control (gray line) and to two odorants which induced marked changes: the royal jelly component octanoic acid (blue) and the alarm pheromone component 2-heptanone (red). Curves show average values every 200 ms from the data acquired in the first experiment (N = 24, Fig. 2A,B). Octanoic acid induced a forward motion of the antennae with an acceleration, whereas 2-heptanone induced a backward motion of the antennae with a deceleration. The changes in angular position (Δθ) or angular velocity (ΔVθ) were calculated as the difference between these values during odor presentation (5 s) and before (15 s).

Figure 2

Screening of antennal response to a large panel of odorant. (A–D) Histograms showing the change in antennal movements in response to odor presentation (during–before odor) in terms of (A,C) angular position (Δθ) and (B,D) angular velocity (ΔVθ). (A,B) show antennal responses on the first year (N = 24), and (B,C) show the replication of the experiment on the following year (N = 25). Color code: air control (white), alarm pheromones (red), aggregation pheromones (green), brood pheromones (light purple), queen pheromone (dark purple), floral odors (grey), repulsive odor (orange), fecal odor (brown), and the royal jelly component (blue). Asterisks in the square next to the graph indicate a significant heterogeneity in antennal movements between odorants (RM-ANOVA, ***: p < 0.001). Asterisks on the histograms indicate significant differences in Dunnett post-hoc tests comparing each value to the air control (•: p < 0.1; * = p < 0.05). (E,F) Regressions comparing the results of the two experimental years in terms of (E) antennal angular position (θ) and (F) angular velocity (ΔVθ). Asterisks in the square next to the graph indicate significance in a Pearson correlation test (***: p < 0.001).

Figure 3

Relationship between changes in antennal angular position (Δθ) and velocity (ΔVθ) in response to odorants. (A,B) Histograms showing the change in antennal movements in response to odor presentation (during–before odor) in terms of (A) angular position (Δθ) and (B) angular velocity (ΔVθ) when pooling data from both experimental years (N = 49). Asterisks in the square next to the graph indicate significant a significant heterogeneity in antennal movements between odorants (RM-ANOVA, ***: p < 0.001). Asterisks on the histograms indicate significant differences in Dunnett post-hoc tests comparing each value to the air control (*: p < 0.05). (C) Regressions comparing bees’ responses to the stimuli in terms of antennal angular position (Δθ) and velocity (ΔVθ). Asterisks in the square next to the graph indicate significance in a Pearson correlation test (**: p < 0.01).

Figure 4

Attractiveness of the odorants and relationship with antennal movements. (A) Apparatus for measuring the orientation of 16 bees simultaneously, each confronted to a different odorant. It is made of 16 45 cm glass lines with each 3 equally interspaced infra-red portals (inset). At the start of the experiment, a box containing a worker is placed on one side of each line. A box containing a filter paper soaked with 5 µl odorant solution is placed on the other. The recordings start when opening the doors of the boxes containing the bees and lasts 10 min. From the numbers of passages through the monitors for each odorant and the air control, an attractiveness index is calculated (see text). (B) Histograms showing the relative attractiveness index of the odorants. Each bee was used to record the response to only one stimulus: air control N = 46, 2-heptanone N = 40, octyl acetate N = 44, isopentyl acetate N = 42, benzyl acetate N = 43, citral N = 40, geraniol N = 41, β-ocimene N = 43, methyl linoleate N = 40, ethyl oleate N = 42, QMP N = 37, octanal N = 39, linalool N = 40, citronellal N = 42, 3-methyl indole N = 43, octanoic acid N = 39. Asterisks in the square next to the graph indicate significant a significant heterogeneity in the attractiveness index of the different odorants (RM-ANOVA, ***: p < 0.001). (C,D) Regressions showing (C) the change in antennal angular position (Δθ) or (D) angular velocity (ΔVθ) as a function of each odorant’s attractiveness index. NS in the square next to the graph indicates the lack of statistical significance (p = 0.124 and p = 0.126 respectively).

Figure 5

Influence of odorant concentration on antennal movements. (A,B) Curves showing the change in (A) antennal angular position (Δθ) and (B) velocity (ΔVθ) in response to increasing concentrations (from 10^–7 to 10⁰) of three odorants diluted in mineral oil (N = 43). Ctrl: average response to 4 control stimuli. Asterisks in the square next to the graph indicate significant interactions between stimulus and concentration (RM-ANOVA, *: p < 0.05; ***: p < 0.001). Asterisks on the graph indicate significant differences in Dunnett post hoc tests comparing each concentration to the control (*: p < 0.05, red: 2-heptanone, green: geraniol).

Figure 6

Odor-induced antennal responses in newly emerged bees. (A,B) Histograms showing the change in antennal movements in response to odor presentation (during–before odor) in terms of (A,C) angular position (Δθ) and (B,D) angular velocity (ΔVθ). Stimuli include an air control (white), two alarm pheromone components (red), one brood pheromone component (light purple), the queen mandibular pheromones (dark purple) and a major component of the royal jelly odor (blue). The asterisk in the square next to the graph indicates a significant heterogeneity among odorants (RM-ANOVA, *p < 0.05). NS: non-significant. Asterisks on the graphs indicate significant differences in Dunnett post-hoc tests comparing each value to the air control (*p < 0.05).