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 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

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

Scolopidia lacking <i>ck</i>/MyoVIIA are not attached to the a2/a3 joint during development.

<p>A: Wild type (<i>ck¹³/CyO</i>; white labels) and mutant (<i>ck¹³/ck¹³</i>; yellow labels) cultured antennal discs labelled as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002115#pone-0002115-g002" target="_blank">Figure 2</a> support our finding that <i>ck</i>/MyoVIIA is necessary for JO organization. B: Representative EM micrographs from wild type (26 hrs APF) and mutant (28 hrs APF) antennae. Black arrowheads: dendritic caps extending beyond the a2/a3 joint boundary in controls. White arrowheads: dendritic caps at the apical levels of the scolopale space. In <i>ck</i> mutants the cap is more compact than in controls. Inset: magnification of the boxed area. C: Wild type (<i>ck¹³/CyO</i>; white labels) and mutant (<i>ck¹³/ck¹³</i>; yellow labels) cultured antennal discs labelled with anti-cadherin antibody. Green channel: GFP-NompA. Red channel: anti-cadherin. Arrows: cellular junction “tracts” in mutant discs. Open block arrows: a2/a3 boundary. Inset: enlargement of the boxed area. Arrowhead: a dendritic cap extending beyond the a2/a3 boundary. Block arrows: cell-cell junctions.</p

<i>ck</i>/MyoVIIA function is modulated by Sqh and DMBS in adult JO.

<p>A: Genetic interactions of <i>ck</i>/MyoVIIA with actin pathways in the <i>Drosophila</i> wing (top panel) and in embryonic dorsal closure (bottom panel). Boxed area indicates the portion of the pathway investigated in panel B. Based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002115#pone.0002115-Winter1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002115#pone.0002115-Mizuno2" target="_blank">[36]</a>. B: <i>ck</i>/MyoVIIA genetically interacts with Sqh and DMBS in adult JO. Responses from flies with only one functional copy of <i>Drok</i>, <i>sqh</i>, <i>zip</i>/MyoII or <i>DMBS</i> were normalized to their respective, two-copy sibling controls, all in a sensitized <i>ck</i>/MyoVIIA background. Histograms show mean +/− SD.</p

<i>ck</i>/MyoVIIA is necessary for JO organization during development.

<p>A: Comparison of control (<i>ck¹³/CyO</i>; white labels) vs. mutant (<i>ck¹³/ck¹³</i>; yellow labels) developing JO. Green channel: GFP-NompA labelling dendritic caps. Red channel: Texas red-phalloidin labelling actin filaments. Block arrows: developing scolopale rods. Open block arrows: direction of a2/a3 boundary. Box indicates disorganization of cap alignment in JO. Inset in 14 hrs control: in some cases the cap shows an elongated profile by this time. Inset in 14 hrs mutant antenna: magnification of the globular profile of the cap. Scale bar = 20 µm. B: Similar staining and labelling as “A”. Note dendritic caps juxtaposed with the perimeter of a2 (dotted line) or extending into the space between a2/a3 in controls (arrow; white labels). In mutants (yellow labels) the caps remain distanced from the future a2/a3 joint.</p

Schematic of the antenna and JO.

<p>Drawing not to scale.</p