Dynamic Range Compression in the Honey Bee Auditory System toward Waggle Dance Sounds

Honey bee foragers use a “waggle dance” to inform nestmates about direction and distance to locations of attractive food. The sound and air flows generated by dancer's wing and abdominal vibrations have been implicated as important cues, but the decoding mechanisms for these dance messages are poorly understood. To understand the neural mechanisms of honey bee dance communication, we analyzed the anatomy of antenna and Johnston's organ (JO) in the pedicel of the antenna, as well as the mechanical and neural response characteristics of antenna and JO to acoustic stimuli, respectively. The honey bee JO consists of about 300–320 scolopidia connected with about 48 cuticular “knobs” around the circumference of the pedicel. Each scolopidium contains bipolar sensory neurons with both type I and II cilia. The mechanical sensitivities of the antennal flagellum are specifically high in response to low but not high intensity stimuli of 265–350 Hz frequencies. The structural characteristics of antenna but not JO neurons seem to be responsible for the non-linear responses of the flagellum in contrast to mosquito and fruit fly. The honey bee flagellum is a sensitive movement detector responding to 20 nm tip displacement, which is comparable to female mosquito. Furthermore, the JO neurons have the ability to preserve both frequency and temporal information of acoustic stimuli including the “waggle dance” sound. Intriguingly, the response of JO neurons was found to be age-dependent, demonstrating that the dance communication is only possible between aged foragers. These results suggest that the matured honey bee antennae and JO neurons are best tuned to detect 250–300 Hz sound generated during “waggle dance” from the distance in a dark hive, and that sufficient responses of the JO neurons are obtained by reducing the mechanical sensitivity of the flagellum in a near-field of dancer. This nonlinear effect brings about dynamic range compression in the honey bee auditory system

Figure 6

<p>Intensity characteristics of SEPs in response to acoustic stimuli. (A) Schematic representation of the preparation is shown. Honey bee heads are fixed and exposed from the end of a micropipette tip, and the recording electrode is inserted at the joint between the scape and pedicel (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000234#pone-0000234-g001" target="_blank">Fig. 1C</a>) and the reference electrode penetrates the head cuticle. Sound stimuli are delivered through a Tygon tube ending in a 7 mm opening close to the honey bee. The dashed line indicates the hemispherical zone where full near-field acoustic conditions are maintained. The SEP magnitudes in response to 750 (B and D) and 265 (C and E) Hz tone stimuli are plotted against the flagellum (B and C) and air particle (D and E) displacements. The SEP magnitudes linearly increase with the flagellar tip displacement up to 100 nm. The response of JO neurons can be detected by 20 nm displacement of flagellar tip in response to 750 Hz stimuli. The background SEP magnitude is <0.02. The response of JO neurons saturates by the flagellar tip displacement above 200nm in response to 265 Hz stimuli. (F) Bar graph shows the mean SEP magnitudes in response to 265 Hz acoustic stimuli at 7 mm/s air particle velocity. SEPs were measured before (Control) and after (Lesioned) lesions in the pedicel. Error bars indicate standard error (N = 5 antennae per treatment, where the SEP of each antenna is the averaged response to 10 trials). (G) SEPs were measured before (Control) and after cutting off the distal one-third (1/3 severed) and two-thirds (2/3 severed) segment of the flagellum. Bar graph of the mean SEP magnitudes as above is shown. Error bars indicate standard error.</p

Figure 2

<p>Organization of the honey bee JO. (A) TEM picture of the honey bee JO is shown. The morphology and positions of the cuticular knob (1), epithelial cell (2), quasi-longitudinal section of long sensory processes (3), quasi-transverse section of scolopidia (4), soft chitin (5), pedicel (P), and flagellum (F) are indicated. (B) The summary of the ultrastructural analysis of the honey bee JO is shown. At the joint of the pedicel (P) and flagellum (F), the cuticle is organized in a complex pattern of radial fibrils (horizontal lines), with circular fibrils surrounding the cuticular “knobs” (K) to which the scolopidia are attached. There are approximately 48 knobs evenly distributed around the circumference of the flagellum at its joint with the pedicel. Epithelial cells (EC; blue) exhibit extensive apical microvilli, likely for the copious secretion of cuticle proteins. The epithelial cell cytoplasm is filled with spongiform membranous organelles, likely also reflecting high secretion levels. Each cuticular knob is the attachment site of 3–10 scolopidia. Each scolopidium forms an independent dendritic cap (red), and these are surrounded by cap cells (CC) which enclose electron dense rods (green) that are thick apically but divide more basally into multiple finer rods. The scolopale cell (SC) of each scolopidium forms a spindle-shaped cage of scolopale rods (organe) and encloses an extracellular scolopale space (ss), through which the ciliary outer dendritic segments of three neurons (N) extend. Morphologically, honey bee JO scolopidia are amphinematic, containing cilia of both Types I and II (see classification described in ref. 30). The two Type I cilia are of uniform diameter, contain an axoneme along their entire length, and attach to the basal end of the dendritic cap; the single Type II cilium contains an axonemal segment up to the dendritic cap, then a wider non-axonemal segment with loose microtubules that continues throughout the length of the dendritic cap. Basally, there are accessory cells (AC) of uncertain classification. The structure of the honey bee JO is consistent with a sensory function for flagellar vibration.</p

Figure 1

<p>External morphology of the honey bee antenna. The honey bee antenna was examined by scanning electron microscopy with different magnifications (A; x40, B; x150, C; x 300). Two proximal antennal segments (scape and pedicel) and the ten segments of the flagellum are indicated by arrows in A. Arrowhead in C indicates the position of electrode insertion for SEP recordings. The scale of each panel is shown by a white bar.</p

Figure 8

<p>The age-dependent response of honey bee JO neurons to acoustic stimuli. (A) Representative time traces of SEPs of 0, 3, 6, 11 and 21 day old bees (the 21 day old bees are foragers (21F)) in response to 500 Hz pulse and “waggle dance” sound stimuli are shown. Each SEP is the averaged response of 10 trials in a single antenna. Note the vertical scale is 1 mV. (B) Bar graph of 10-trial SEP magnitudes obtained with 265 Hz continuous sound stimuli at 7 mm/s air particle velocity for N = 5 antennae per treatment. Error bars indicate standard error. (C) The time traces of flagellar vibrations of 0 day old bees (Day 0) and 21 day old foragers (21F) in response to 500 Hz pulse and “waggle dance” sound stimuli are shown. Each vibration velocity shown is the averaged response of 10 trials in a single antenna. The vertical scales of the velocity to 500 Hz pulse and “waggle dance” sound stimuli are 0.25 and 1 mm/s, respectively. (D) Bar graph shows the mean vibration magnitudes in response to 265 Hz continuous sound stimuli at 7 mm/s air particle velocity. Error bars indicate standard error.</p

Figure 3

<p>Simultaneous measurements of the flagellar vibration velocity and the particle velocity of the surrounding air. Experimental arrangement in top and lateral views is shown. The laser doppler vibrometer (LDV), the honey bee antenna, and the sound tube were linearly aligned along with the optical axis of the LDV as shown. The vibrometer was positioned at 31 cm away from the flagellum. Sound stimuli are delivered through a Tygon tube ending in a 7 mm opening close to the honey bee. The dashed line indicates the hemispherical zone where full near-field acoustic conditions are maintained. The scanning probe to measure the particle velocity of the surrounding air was positioned at 2 mm away from the flagellum. The probe head was aligned perpendicular to the direction of sound propagation. The joints between head and scape as well as scape and pedicel were fixed with glue to prevent the antennal movements by muscle. The spot position of laser beam was fixed at the tip of the flagellum. All items are not in scale.</p

Figure 4

<p>Intensity and frequency characteristics of honey bee flagellar vibrations in response to acoustic stimuli. The vibration (response) magnitudes, the ratio of the flagellar vibration velocity to the air particle velocity, of 4 live (solid square) and 2 dead (open triangle) bee flagellar tips to pure-tone stimulation at different frequencies (50–1000 Hz) are plotted against the particle velocity (A-G). The data points are parallel to the intensity axis at 50 (A), 160 (B), 500 (E), 750 (F), and 1000 (G) Hz, demonstrating that the flagellar vibration velocity linearly increases as a function of the air particle velocity. The response magnitudes to 265 (C) and 350 (D) Hz stimuli are non-linear, they show the maximum only at intensities between 0.3–4 mm/s, and then decrease at intensities above 4 mm/s. (H) Bar graph shows the mean response magnitudes to stimulation at different frequencies obtained with 0.3 mm/s air particle velocity. Error bars indicate standard error. The resonance frequency of the honey bee flagellum would be around 350 Hz.</p