morphological analysis of the primary center receiving spatial information transferred by the waggle...

Morphological Analysis of the Primary CenterReceiving Spatial Information Transferred by theWaggle Dance of Honeybees

Hiroyuki Ai* and Hiromi Hagio

Division of Biology, Department of Earth System Science, Fukuoka University, Fukuoka 814-0180, Japan

ABSTRACTThe waggle dancers of honeybees encodes roughly the

distance and direction to the food source as the dura-

tion of the waggle phase and the body angle during the

waggle phase. It is believed that hive-mates detect air-

borne vibrations produced during the waggle phase to

acquire distance information and simultaneously detect

the body axis during the waggle phase to acquire direc-

tion information. It has been further proposed that the

orientation of the body axis on the vertical comb is

detected by neck hairs (NHs) on the prosternal organ.

The afferents of the NHs project into the prothoracic

and mesothoracic ganglia and the dorsal subesophageal

ganglion (dSEG). This study demonstrates somatotopic

organization within the dSEG of the central projections

of the mechanosensory neurons of the NHs. The termi-

nals of the NH afferents in dSEG are in close apposition

to those of Johnston’s organ (JO) afferents. The sensory

axons of both terminate in a region posterior to the

crossing of the ventral intermediate tract (VIT) and the

maxillary dorsal commissures I and III (MxDCI, III) in

the subesophageal ganglion. These features of the ter-

minal areas of the NH and JO afferents are common to

the worker, drone, and queen castes of honeybees.

Analysis of the spatial relationship between the NH

neurons and the morphologically and physiologically

characterized vibration-sensitive interneurons DL-Int-1

and DL-Int-2 demonstrated that several branches of DL-

Int-1 are in close proximity to the central projection of

the mechanosensory neurons of the NHs in the dSEG.

J. Comp. Neurol. 521:2570–2584, 2013.

VC 2013 Wiley Periodicals, Inc.

INDEXING TERMS: vibration; body angle; Johnston’s organ; neck hairs; subesophageal ganglion honeybee standard brain

Honeybees share information concerning profitable

flowers by a species-specific behavior, the waggle dance

(von Frisch, 1967). During waggle-dance communication,

hive-mates of the dancer are able to decipher the vector

information contained within the dance, and subse-

quently, they arrive at the indicated flower patch. Riley

et al. (2005) confirmed this phenomenon by recording

the actual flight paths of recruited bees.

It was suggested that Johnston’s organ (JO) on the ped-

icel of each antenna detects the vibration for decoding

the distance information (von Frisch, 1976) and that the

neck hairs (NHs) detect the body angle for decoding the

direction information (Fig. 1; Lindauer and Nedel, 1959).

More recently, the central projections of these two sen-

sory organs that receive this vector information have

been clarified. The afferents originating from the JO pro-

ject into the dorsal lobe (DL) through antennal nerve tract

6 (T6; Suzuki, 1975) and then bifurcate within the DL into

T6I and T6II. The JO afferents in T6I terminate in the

medial posterior protocerebral lobe (PPL) close to the

esophagus, whereas those in T6II terminate in the dorsal

subesophageal ganglion (dSEG; Ai et al., 2007). In con-

trast, the afferents of NH neurons project into the pro-

thoracic ganglion (pro-TG) through lateral nerve tract 1

and terminate ipsilaterally in a region close to the midline

of the pro-TG. From there, the NH afferents extend axons

into the posterior and anterior regions of the pro-TG.

These afferents extend axons through the ventral nerve

cord (VNC) posteriorly, terminating close to the midline of

the mesothoracic ganglion (meso-TG; Sandeman et al,

1997). Anteriorly, the axons extend through the VNC to

terminate at the dSEG (Brockmann and Robinson, 2007).

Thus, the JO and NH sensory afferents terminate in

Grant sponsor: Ministry of Education, Culture, Sports, Science andTechnology of Japan; Grant number: 22570079.

*CORRESPONDENCE TO: Hiroyuki Ai, Division of Biology, Department ofEarth System Science, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku,Fukuoka 814-0180, Japan. E-mail: [email protected]

VC 2013 Wiley Periodicals, Inc.

Received November 15, 2012; Revised December 8, 2012; AcceptedDecember 27, 2012

DOI 10.1002/cne.23299

Published online January 8, 2013 in Wiley Online Library(wileyonlinelibrary.com)

2570 The Journal of Comparative Neurology | Research in Systems Neuroscience 521:2570–2584 (2013)

RESEARCH ARTICLE

diverse and different regions of the central nervous sys-

tem. However, it is noteworthy that terminations within

the dSEG are common to both JO and NH afferents, sug-

gesting a putative role for the dSEG as an integration cen-

ter for direction and distance information (Ai et al., 2007).

Coding of the topology of the receptors of the insect

auditory system is commonly achieved by somatotopic

organization of the projections of their sensory neurons in

the central nervous system (Ignell et al., 2005; Kamikou-

chi et al., 2006). In honeybees, somatotopic organization

of the JO is present in the PPL (Ai et al., 2007). Likewise,

to decode the orientation of the food source, honeybees

must recognize which of the NHs are compressed by the

posterior capsule of the head (Fig. 1). At least three sub-

regions of NHs have been described: the dorsal, central,

and ventral NHs (Sandeman et al., 1997). The dorsal and

ventral NHs are longer (>100 lm) than the central NHs

(<100 lm), so they can be conveniently identified from

both their position and their length. In this study, we

investigated whether there are differences among these

three NH groups with respect to the central projection

patterns of their afferents within the pro-TG and meso-TG

and the dSEG, i.e., whether there is a somatotopic organi-

zation of the dSEG indicative of a role in integration of

waggle dance sensory information.

The spatial relationship between the termination

regions of these two sensory organs in honeybees is

unknown. Here we identify a putative integration area of

these sensory organs by two methods: by double staining

of the mechanosensory neurons of NHs and the JO and

by registering the positions of the NH and JO afferents

within an averaged three-dimensional honeybee brain

image (the ‘‘honeybee standard brain’’ [HSB]). For analy-

sis of the spatial relationships among the different neu-

rons using the HSB, laser scanning confocal microscopy

(LSM) images were transformed through two processes:

affine transformation and elastic registration within the

virtual space of the HSB (Rohlfing, et al., 2004). In addi-

tion, we investigated whether workers, drones, and

queens have similar projection areas of the JO and NH

afferents in the dSEG.

Previously, we have morphologically and physiologi-

cally characterized several types of vibration-sensitive

interneurons, which arborize in the dSEG, whose vibration

response patterns exhibit on-off phasic excitation, tonic

excitation, or tonic inhibition, suggesting a possible role

in coding of the duration of airborne vibrations (Ai et al.,

2009). These interneurons have dendritic arborization

close to the JO axon terminals (Ai, 2010). Here, we inves-

tigated the spatial relationship between morphologically

and physiologically characterized vibration-sensitive inter-

neurons and the NH terminals.

MATERIALS AND METHODS

Honeybees (Apis mellifera L.) were captured at the en-

trance of hives kept in Fukuoka University between April

and September, 2010, 2011, and 2012. The experiments

used more than 100 worker honeybees, two queens, and

six drones.

Anterograde staining of subgroups of NHsThere are three subgroups of NHs on each side of the

neck: the central, dorsal, and ventral subgroups (Fig.

1A,C). To stain subgroups of NHs specifically, a small

patch of the NHs was shaved off with a razor blade. Then,

a small incision was made to expose the cell bodies of

the NH neurons, onto which a single crystal of dextran

tetramethylrhodamine (D3308; Invitrogen, Carlsbad, CA)

was placed. The preparations were kept for 48 hours at

4�C. The success rate in obtaining the complete staining

of just a few sensory afferents of interest was about 10%.

Double-fluorescence dye injection using crystals of both

dextran-fluorescein (D3306; Invitrogen, Carlsbad, CA),

and dextran-tetramethylrhodamine was also applied to

different subgroups of NHs. The success rate for these

experiments was quite low, because the subgroups of the

NHs represented a relatively small area of the prosternal

organ.

Anterograde staining of the antennal nerveand the NH sensory nerve

It has been shown that axons within the antennal nerve

project into the antennal lobe, the DL, the subesophageal

ganglion (SEG), and the PPL (Pareto, 1972; Suzuki, 1975;

Mobbs, 1982; Arnold et al., 1985; Flanagan and Mercer,

1989; Maronde, 1991; Kloppenburg, 1995; Abel et al.,

2001; Kelber et al., 2006; Kirschner et al., 2006). The

Abbreviations

AL antennal lobeAN antennal nerveCA calyx of mushroom bodyCB central bodyDL-Int-1 dorsal lobe interneuron 1DL-Int-2 dorsal lobe interneuron 2dSEG dorsal subesophageal ganglionHSB honeybee standard brainEs esophagusJO Johnston’s organLo loburaMe medullameso-TG mesothoracic ganglionMxDCI, III maxillary dorsal commissure I and IIINHs neck hairsPC protocerebrumPPL posterior protocerebral lobepro-TG prothoracic ganglionSEG subesophageal ganglionT5 tract 5T6I, II tracts 6-1 and 2VIT ventral intermediate tract

Primary Center of Vector Information in Bees

The Journal of Comparative Neurology | Research in Systems Neuroscience 2571

central projection in the antennal lobe has been revealed

in more detail (Suzuki, 1975; Mobbs, 1982; Abel et al.,

2001; Kelber et al., 2006; Kirschner et al., 2006). In our

previous study, the central projections of the JO were

investigated by using three-dimensional reconstruction (Ai

et al., 2007). The sensory neurons originating from the

NHs have also been investigated, in this case by dye injec-

tion into the first lateral nerve of the pro-TG, a nerve that

innervates only the NHs (Lindauer and Nedel, 1959; San-

deman et al, 1997; Brockmann and Robinson, 2007). To

investigate the spatial relationship between the central

projections of JO afferents and those of NH afferents, dou-

ble-fluorescence dye injection was applied to the proximal

cut stump of the antennal nerve at the base of the pedicel

and to the first lateral nerve of the pro-TG. JO is located in

the pedicel. If the dye is injected proximal to the pedicel,

the sensory fibers running from both the pedicel and the

flagella will be stained in the brain. However, JO afferents

are easily distinguished from other afferents because of

their characteristic morphologies (Ai et al., 2007).

Figure 1. Neck hairs (NHs) and their correspondence with the body orientation on the vertical comb. A: The NHs on the left side. There

are three subregions, dorsal, central, and ventral NHs, recognized by the position and their length. B: Detection of the body angle on the

vertical comb. The bee detects the inclination of the head capsule against the thorax (gray arrows). C: Drawing of detection of body

angle during the waggle phase. The three subgroups of NHs are differentially subjected to pressure by the posterior head capsule. For

example, when the bees are oriented in the opposite direction from gravity (head upward, 0�), the ventral NHs contact the posterior head

capsule. When the bees are oriented to the right (90�), the central hairs on the right side make contact with the posterior head capsule.

Scale bar ¼ 500 lm.

Ai and Hagio

2572 The Journal of Comparative Neurology |Research in Systems Neuroscience

After anesthetizing the bees for 5–10 minutes, all wings

and legs were removed and the sensory nerves supplying

the NHs were exposed and cut at the base of the NHs.

The antenna was cut in the distal region of the scape. The

brain, pro-TG, and meso-TG complex, with the antennal

and NH nerves, was exposed and excised from the body.

The stumps of the antennal nerve and NH nerve were

carefully placed into the tips (internal diameter �70 lm)

of tapered separate glass electrodes filled, respectively,

with 0.8% dextran-fluorescein and with 0.8% dextran-tetra-

methylrhodamine. Alternatively, crystals of these fluores-

cence dyes were placed directly onto the stump of the

antennal nerve and the NHs. The dye was then distributed

by axonal transport into the brain. After incubation at 4�C

for 24–48 hours, the brain, pro-TG and meso-TG com-

plexes were dissected out, rinsed in PBS (pH 6.7) several

times, fixed in 4% paraformaldehyde in 0.1 M PBS for 12

hours, dehydrated, and cleared in methyl salicylate.

Confocal microscopy and three-dimensionalreconstruction

The posterior aspects of stained and cleared specimens

of the central projections of antennal and NH afferents

were viewed with a confocal laser scanning microscope

(LSM 510; Carl Zeiss, Jena, Germany) using a Zeiss Plan-

Apochromat �10/0.45 dry lens objective for low-magnifi-

cation images and a Zeiss Plan-APO LD �25/0.8 Imm

Korr DIC or a Zeiss Plan-Neofluar �40/1.30 oil lens objec-

tives at high magnifications. Optical sections were made

at 3 lm (low magnification) or 1 lm (high magnification)

throughout the entire depth of each specimen. Dextran-

tetramethylrhodamine was excited by the 543-nm line of a

HeNe laser, whereas dextran-fluorescein was excited by

488-nm line of an argon laser. The stained sensory fibers

were reconstructed three-dimensionally from optical sec-

tions by using the label field editor in Amira 4.1 software

(Mercury Computer Systems Inc., San Diego, CA). The out-

lines of the neuropil and the brain/SEG complex were also

traced with the label field editor to identify the location of

the stained sensory fibers in the brain/SEG complex.

Registration of the morphologies of sensoryafferents and interneurons into thehoneybee standard brain

In addition to double-staining analyses, we used the

honeybee standard brain (HSB) to analyze the spatial rela-

tionships among JO afferents, NH afferents, and

morphologically and physiologically characterized vibra-

tion-sensitive interneurons. First, the neuronal profiles of

stained JO afferents, NH afferents, and interneurons

DL-Int-1 and DL-Int-2, obtained from different prepara-

tions, were reconstructed in Amira 4.1 (Evers et al.,

2005). Subsequently, the neuropilar outlines were traced

manually and segmented with the Amira 4.1 label field edi-

tor. These neuropilar label fields were used to register the

segmented neuron of each preparation into the HSB fol-

lowing the method described by Brandt et al. (2005). This

registration process involves both affine transformations

and elastic geometric deformations. The affine transforma-

tion matrixes and the deformation vector fields were

applied to the segmented neurons, and these were fitted

into the HSB. To identify a possible overlap region

between the NH afferents and each morphologically and

physiologically characterized interneuron, Amira’s ‘‘surface

distance’’ was used to calculate the distance between

neurons. As landmarks for identifying the common projec-

tion areas of the sensory afferents of JO and NHs, the ven-

tral intermediate tract (VIT) and the maxillary dorsal com-

missures I and III (MxDCI and -III) were also registered

into the HSB. Adobe Photoshop 5.0 was used to enhance

the contrast of images and to store all photographs.

RESULTS

Central projection of three neuralsubgroups of NHs

Dye injection specifically into the central (n ¼ 7), dor-

sal (n ¼ 7), and ventral (n ¼ 6) NHs revealed projection

patterns corresponding to the three neural subgroups of

the NHs (Fig. 2). In general, the projection patterns of the

three subgroups were similar; afferents from each of the

neural subgroups supplied fine collaterals in the pro-TG

and sent axons both through the anterior VNC toward the

SEG and through the posterior VNC toward the meso-TG

(Fig. 2). In the dSEG, all afferents from different sub-

groups bifurcated into the dorsal and ventral branches

and then ramified to form fine collaterals with blebby ter-

minals (Fig. 2A–C). In the pro-TG, the NH afferents bifur-

cated into two main tracts close to the proximal region of

nerve N1. In the thicker tract, the axons extended close

to the midline of the pro-TG toward the posterior ipsilat-

eral VNC. The axons running in the VNC terminated along

the midline of the meso-TG (Fig. 2G–I). The other thinner

tract also sent axons to the midline of the pro-TG and

then bifurcated into two subtracts. One tract sent axons

toward the anterior ipsilateral VNC, and the other was

elongated posteriorly along the midline of the pro-TG,

with fine ramifications with blebby terminals (Fig. 2D–F).

To compare spatial projection patterns of the different

NH subgroups, pairs of the three subgroups were stained

differentially with fluorescent dyes. No dye coupling was

detected between the subgroups (Figs. 3C,D). However,

because the central NHs were very close to both the dor-

sal and the ventral NHs, dye mixing always occurred

between this group and the other groups during the



incubation following dye injection. Therefore, double

staining was carried out on the dorsal and ventral NH

groups. There were no clear differences between the pro-

jection areas of these two subgroups in the pro-TG and in

the meso-TG (data not shown). In the pro-TG and the

meso-TG, the afferents of the dorsal NHs and the ventral

NHs were in close apposition to each other. However, in

the dSEG, the bifurcating points of the dorsal and ventral

branches were clearly different (arrows in Fig. 3A,B; n ¼5). The bifurcating point of the afferents originating from

the dorsal NHs was more anterior to that of the ventral

NHs (arrows in Fig. 3B). The three-dimensional represen-

tation of axon terminals showed that their termination

fields were spatially well segregated in the dSEG: axon

Figure 2. Central projections of the afferents originating from the right-side NHs of the three subregions (dorsal NHs, central NHs and

ventral NHs). A,D,G show those of the dorsal NHs; B,E,H those of the central NHs; and C,F,H those of ventral NHs. The NHs project into

the subesophageal (SEG; A–C), prothoracic (pro-TG; D–F), and mesothoracic (meso-TG; G–I) ganglia. There were no clear differences in the

branching patterns of the afferents originating from NHs of the three subregions in these ganglia. The dSEG are outlined by dashed lines,

which is the terminal regions of the NH afferents in SEG. In F, the ventral NHs of the left side were also weakly stained. See Results for

details. Scale bars ¼ 50 lm in C (applies to A–C); 100 lm in F (applies to D–F); 100 lm in I (applies to G–I).

Ai and Hagio


terminals of the dorsal NHs were located anteriorly to

those of the ventral NHs (Fig. 3A,B). In the anterior region

of the dSEG, there were blebby terminals of the dorsal

NH afferents but not of the ventral NH afferents (arrow-

heads in Fig. 3C). In the posterior region of the dSEG, the

blebby terminals of both dorsal and ventral NH afferents

were intermingled (arrowheads in Fig. 3D). To visualize

the spatial maps of the terminal regions of the NH sub-

groups, we registered these afferents into the HSB

(Fig. 4A–C). The central projections of the NH subgroups

segregated in the dSEG along the anterior to posterior

axis (arrowheads in Fig. 4E,F). The JO afferents send

axons in each terminal region of the NH subgroups;

however, they do not cover the whole terminal regions of

the NHs (Fig. 4D–F).

Spatial relationship between the centralprojections of the antennal afferents andthe NH afferents in the dSEG

Both JO and NH afferents extend their axons into the

dSEG (Ai et al., 2007; Brockmann and Robinson, 2007).

The spatial relationship between these afferents was

examined in double-stained preparations. The axons of

the NH afferents pass through the ipsilateral VNC and ter-

minate in the ipsilateral dSEG (Fig. 2). They then bifurcate

into dorsal and ventral branches, which produce fine

arborizations in the dSEG terminating in close proximity to

the terminals of the antennal mechanosensory afferents

(magenta in Fig. 5A,B; n ¼ 11). Afferents from the hair

plates on the pedicel and from JO pass through T5 and

T6, respectively (Ai et al., 2007). The pedicel-hair-plate

Figure 3. Central projection of the dorsal (magenta) and ventral (green) NHs in the dSEG. A,B: Different locations of the bifurcation points

of the dorsal and ventral branches (A, frontal view; B, lateral view). The bifurcation point of the afferents originating from the dorsal

NHs was anterior to that of the ventral NHs (arrows). C,D: Frontal images in the optical planes indicated in B. C: In the anterior region of

the dSEG (left blue line in B), there were blebby terminals of the dorsal NH afferents, but there were no terminals of the ventral NH

afferents (arrowheads). D: In the posterior region of the dSEG (right blue line in B), blebby terminals of both dorsal and ventral NH affer-

ents are intermingled (arrowheads). Scale bars ¼ 20 lm.



afferents terminate in an anterior arborization within the

dSEG, whereas JO afferents terminate in a posterior arbo-

rization within the dSEG (Fig. 5D; Ai et al., 2007). The LSM

and three-dimensional images indicated that the sensory

afferents of the NHs overlapped with the posterior arbori-

zation of the antennal mechanosensory afferents T6II in

the dSEG, strongly suggesting that they were in close

proximity to the terminals of JO afferents (Fig. 5B–E).

We also compared the central projections of NH affer-

ents in worker honeybees with those in queens and

drones (Fig. 6; queens, n ¼ 2; drones n ¼ 6). Those in

queen and drone also projected into the dSEG. Overlap-

ping of JO and NH afferents was observed in all three hon-

eybee castes, indicating that the central projection of the

NHs was not unique to workers.

Spatial relationships between centralprojections of JO and NH afferents

To identify the common projection area of JO and NH

afferents in the dSEG, we referred to the HSB (Brandt

et al., 2005). The morphologies of JO afferents were ana-

lyzed in our previous studies (Ai et al., 2007) and were reg-

istered into the HSB (Ai, 2010). The confocal serial images

of the NH afferents and JO afferents obtained in the pres-

ent study were segmented and skeletonized and then reg-

istered into the HSB. This process demonstrated that the

axons of the NH afferents terminate close to axon termi-

nals T6II of the JO afferents in a location posterior to the

crossing of ventral intermediate tract (VIT) and maxillary

dorsal commissure I and III (MxDCI and -III; Fig. 7).

Spatial relationships between centralprojections of the NH afferents andmorphologically and physiologicallycharacterized vibration-sensitiveinterneurons

Previously, we morphologically and physiologically char-

acterized two vibration-sensitive interneurons (DL-Int-1

and DL-Int-2), which had dendritic arborizations in the

vibration primary center in the DL and the dSEG (Ai et al.,

2009). These interneurons had arborizations close to JO

terminals in the DL (Ai, 2010; Ai and Itoh, 2012), and both

Figure 4. Spatial relationships of the central projections in the dSEG of the NHs in the three subregions. Light blue, dorsal NHs; magenta,

central NHs; blue, ventral NHs; green, JO. A–C: Each of these central projections of the afferents was registered in the HSB (transparent

gray images). A, frontal view; B, lateral view; C, dorsal view. D–F: Magnified images of the boxed areas in A–C, respectively. These topolog-

ical projections reflect the peripheral NH subregions. E,F: The dorsal NHs (light blue arrow) terminated anterior to those of the central

NHs (magenta arrow), and the ventral NHs (blue arrow) terminated posterior to those of the central NHs. The transparent green-shaded

area shows the terminal region of JO.

Ai and Hagio


Figure 5. Double staining of antennal mechanosensory afferents and NH afferents. A: The NH afferents (magenta) terminate in close

proximity to the terminals of the antennal mechanosensory afferents (green) in dSEG. B: The sensory afferents of NHs bifurcate into dorsal

and ventral branches with fine arborizations in the dSEG (magenta). A,B: Frontal views. C–E: Three-dimensional reconstructed images of

NH afferents and antennal mechanosensory afferents. (C, frontal view; D, lateral view; E, dorsal view). The sensory afferents of NHs over-

lap the posterior arborization of the antennal mechanosensory afferents (T6II) in the dSEG, which are the terminals of JO afferents (Ai

et al., 2007). Scale bars ¼ 100 lm in A; 50 lm in B.

Figure 6. Central projections of antennal mechanosensory afferents (green) and NH afferents (magenta) in the dSEG of a drone (upper

images) and a queen honeybee (lower images). A,B: Image stack from the LSM (anterior views). C–H: Three-dimensional reconstructed

images of a drone (C, frontal view; D, lateral view; E, dorsal view) and a queen (F, frontal view; G, lateral view; H, dorsal view). In both

castes, NH afferents also projected to the terminals of T6II (arrows) in the dSEG, which is similar to that in worker honeybees. Scale bars

¼ 100 lm.

responded to vibration stimuli with tonic inhibitory, excita-

tory, or on–off phasic excitatory responses. These proper-

ties indicate that either neuron would be capable of moni-

toring the length of the vibration phase that encodes

distance information (Ai et al., 2009). In the present study,

we observed that the NH afferents terminated close to the

terminal region of JO afferents in the dSEG. To investigate

the spatial relationship between the NH afferents and

these vibration-sensitive interneurons, these interneurons

and the NH afferents were registered into the HSB (see

Figs. 8–10). This analysis revealed that the finest several

branches of the dorsal branch of DL-Int-1 are in close prox-

imity to the ventral branch of the NHs in the dSEG (Fig. 8).

It was also revealed that DL-Int-1 had more branches close

to the terminals of the dorsal NH subgroups than those

close to the other NH subgroups (Fig. 9). DL-Int-2 appears

to be an output neuron, whose axons extend from the DL-

dSEG to the lateral and posterior protocerebrum (Ai et al.,

2009). DL-Int-2 did not have dendritic arborization close to

the NH afferents in the dSEG (Fig. 10).

Figure 7. A: NH afferents and JO afferents registered into the honeybee standard brain (HSB). B–D: Three-dimensional images of the NH

afferents and JO afferents (B, frontal view; C, dorsal view; D, lateral view) in the HSB (transparent image). B is a magnified image of the

boxed area in A. The axons of the NH afferents (magenta) terminate close to the axon terminals T6II of the JO afferents (green), enclosed

by yellow dotted lines. The dSEGs are outlined by white dotted lines. The common terminal region is situated posterior to the crossing of

VIT and MxDCI and -III.

Ai and Hagio


Figure 8. A–C: Spatial relationships between the NH afferents (magenta) and a vibration-sensitive interneuron, DL-Int-1 (orange; Ai et al.,

2009), registered into the HSB (A, dorsal view; B, frontal view; C, lateral view). D,E: The minor arborization of the dorsal branch of DL-Int-1

(arrowheads in E) is in close proximity to the ventral branch of the NHs in dSEG (arrowheads in D). The distances between the NH affer-

ents and the DL-Int-1 show in pseudocolors. The dSEG are outlined by dotted lines.



DISCUSSION

This study provides a morphological analysis of the pri-

mary center concerned with the vector information trans-

ferred by the waggle dance of honeybees. We demon-

strate that the topological organization of sensory

afferents within the dSEG reflects the subregions of the

peripheral NHs. Furthermore, the terminals of the NH

afferents within the dSEG are in close apposition to those

of JO afferents and to arborizations of the morphologically

and physiologically characterized vibration-sensitive inter-

neuron DL-Int-1. These features are common to the

worker, queen, and drone castes of honeybees.

Figure 9. A: Spatial relationships among the terminal region of three NH subgroups and a DL-Int-1, registered into the HSB (lateral

view). Light blue, dorsal NHs; magenta, central NHs; blue, ventral NHs; orange, DL-Int-1. B–D: The area close to the afferents of three

NH subgroups (B, dorsal NHs; C, central NHs; D, ventral NHs) on the DL-Int-1 in the HSB (frontal views). The distances between each

NH subgroup and the DL-Int-1 show in pseudocolors. The dSEG is outlined by dotted line. The arrowheads indicate the areas close to

the afferents of three NH subgroups.

Ai and Hagio


Significance of the diverse centralprojections of NH and JO afferents

Earlier authors have suggested that JO and NHs have

functions related to the waggle dance (JO: Michelsen

et al., 1992; Dreller and Kirchner, 1993; Rohrseitz and

Tautz, 1999; Michelsen, 2003; NHs: Lindauer and Nedel,

1959; Sandeman et al., 1997). These mechanosensory

organs are also believed to have roles during flight (JO:

McIver, 1985; Srinivasan and Zhang, 2004; NHs: Straus-

feld and Seyan, 1985). In fact, these sensory afferents

have diverse central projections within the central nerv-

ous system (Sandeman et al., 1997; Brockmann and Rob-

inson, 2007; Ai et al., 2007), which are consistent with

multimodal mechanoreceptive roles in different situa-

tions, e.g., during flight or walking. The JO afferents pro-

ject into two distinct regions of the brain, the PPL and the

Figure 10. A–C: Spatial relationship between the NH afferents (magenta) and a vibration-sensitive interneuron, DL-Int-2 (yellow, Ai et al.,

2009), registered into the HSB (A, dorsal view; B, frontal view; C, lateral view). D,E: DL-Int-2 (E) does not have dendritic arborization close

to the NH afferents (D) in the dSEG. The dSEG are outlined by dotted lines.



dorsal lobe-dorsal subesophageal ganglion neuromere

(DL-dSEG; Ai et al, 2007). Proposed roles for JO include

velocity detection during flight (Srinivasan and Zhang,

2004) and airborne vibration detection in the hive (Dreller

and Kirchner, 1995). The PPL is believed to be concerned

with the integration of vision with movements and veloc-

ity during flight because it is a region in which the visual

interneurons have terminal arborization. Correspondingly,

the DL-dSEG may be involved in the integration of me-

chanical (e.g., tactile and airborne vibrations) and gusta-

tory stimuli in the hive because it is a region in which the

antennal mechanosensory and gustatory neurons have

terminal arborizations (Haupt, 2007).

The NH afferents, on the other hand, project into three

distinct regions of the proto- and mesothoracic ganglia,

as well as into dSEG (Fig. 2), a pattern common to the

worker, queen, and drone castes (Fig. 6). It has been sug-

gested that the NHs monitor the angle between the head

and the thorax during flight in blowflies (Strausfeld and

Seyan, 1985) and the orientation of the body axis to the

vertical comb in honeybees (Sandeman et al., 1997). In

locusts (Braunig et al., 1983) and blowflies (Strausfeld

and Seyan, 1985), the sensory afferents of the cervical

hair plate and prosternal organ project into the pro- and

mesothoracic ganglia but not into the dSEG. These obser-

vations suggest that the termination of NH afferents in

the dSEG is unique to honeybees. One unique behavior of

honeybees is the waggle dance. The waggle dancer enco-

des the direction toward the food source as the body

angle on the vertical comb during the waggle phase of

the dance and the follower detects the body angle by

NHs (detector of direction; von Frisch, 1963). In this

study, the terminals of the NH afferents within the dSEG

are in close apposition to afferents from JO (which

detects distance). This suggests that the dSEG could

have a role in the integration of spatial information trans-

ferred by the waggle dance. However, the JO terminals

overlap only parts of the terminal regions of the NHs (Fig.

4D–F). The terminal regions of the NH subgroups might

therefore be related to other roles in addition to integrat-

ing spatial information transferred by the waggle dance.

Honeybees maintain negative geotaxis on the vertical

comb, and gravity-sensing organs, which include the NHs,

are able to detect the body’s angle (Sandeman et al.,

1997). The neuroanatomical features of the dSEG identi-

fied in this study might be related to these specific

aspects of honeybee behavior.

The present study shows that the NH afferents in

queen and drone also projected into the dSEG. Overlap-

ping of JO and NH afferents was observed in all three hon-

eybee castes, indicating that the central projection of the

NHs was not unique to workers (Fig. 6). The waggle dance

is used for transferring the vector information not only to

the profitable flowers but also to the swarming place to

the hivemates. Therefore, queen and drones might also

be able to decipher the vector information in their brains.

Significance of somatotopic organization ofthe NH afferents

Honeybees live on vertical combs within the hive.

Therefore, the direction of gravity sensed by individuals

varies depending on their orientation on the comb (Fig.

1). The inclination of the head relative to the thorax

changes with its orientation (Fig. 1B; Lindauer and Nedel,

1959). The NHs are arranged around the prosternal

appendage, forming a U shape on each side (Fig. 1C),

with long hairs in the dorsal and ventral subregions and

short hairs in the central subregion (Fig. 1A; Sandeman

et al., 1997). This distribution of hairs around the neck

suggests that honeybees may determine their orientation

with respect to gravity by sensing the inclination of the

head from the differential activation of specific subregions

of the NHs (Lindauer and Nedel, 1959). This hypothesis is

consistent with the present demonstration of a somato-

topic map in the dSEG that reflects the topological distri-

bution of the NHs. Thus, the aspects of honeybee’s spa-

tial orientation are represented in spatially organized

sensory projections in the dSEG. Although bilateral topo-

logical maps must reflect the topology of left and right

NHs in the bilateral dSEG, it is unclear whether interneur-

ons connect the two fields. In addition, the NH afferents

bifurcate into dorsal and ventral branches within the

dSEG (Fig. 2), but it remains to be established whether

these two branches are also related to the peripheral to-

pology of NHs or whether there are functional differences

between the dorsal and the ventral branches.

Hypothesis of the integration in thecommon projection area of JO and NHs

We observed that JO and NH afferents both project

into the dSEG. The afferents of JO project into the PPL

and the DL-dSEG, but a somatotopic map is present only

in the PPL (Ai et al., 2007). This suggests that there is no

spatial map of airborne vibrations in the dSEG. However,

as noted, a spatial map of the NHs in the dSEG appears

to be concerned with the bee’s orientation on the vertical

comb. Based on these observations, a model of the pri-

mary processing of the waggle dance information is

hypothesized (Fig. 11). In the example shown, a forager

(waggle dancer) has just returned from a feeder at a bear-

ing 90� west of the bearing of the sun from the hive and

performs a waggle dance oriented 90� to the left. The in-

hive honeybees (followers) that follow the waggle run

from position angles between 630� of the run are

successfully recruited (Judd, 1995), suggesting that

Ai and Hagio


successfully recruited followers follow the dancer from its

tail end and align themselves parallel to the dancer to

detect the dancer’s body angle (Rohrseitz and Tautz,

1999). In this example, the left central NHs of the follower

are activated, and these afferents send signals to the cor-

responding subregion in the dSEG (in the example, ‘‘–90�’’

in the dSEG), whereas the afferents of JO also send signals

to the part of dSEG. In the dSEG, only the �90� subregion

is able to be activated by signals received from JO and the

left central NHs. We analyzed the spatial relationship

between the NH terminals and two morphologically and

physiologically characterized vibration-sensitive interneur-

ons DL-Int-1 and DL-Int-2 in the HSB (Figs. 8–10). The den-

dritic arborization of DL-Int-1 is close to the terminals of

the NHs in dSEG (Figs. 8, 9). In our previous study, the DL-

Int-1 was also close to the terminals of the JOs in the DL-

dSEG (Ai and Itoh, 2012), and DL-Int-1 has responsiveness

to the olfactory stimulus to the contralateral antenna (Ai

et al., 2009). HSB analysis does not allow visualization of

the synaptic contacts between different neurons, but it is

possible that these interneurons have synaptic inputs from

JO, NH afferents, and olfactory interneurons in the DL-

dSEG. Moreover, it has been found that there are other dif-

ferent types of vibration-sensitive interneurons arborized in

the DL-dSEG on our intracellular recording and staining

experiments and that some of them have dendritic arbori-

zation close to the terminals of the JO and NH afferents

(Ai, unpublished observations). By analyzing the morpholo-

gies and response properties of these vibration-sensitive

interneurons during stimulation of different NH subregions

and during the olfactory stimulation, it should be possible

to clarify whether these interneurons are concerned not

only with vibration processing but also with the integration

of sensory information contained in the waggle dance.

ACKNOWLEDGMENTS

I thank Prof. Dr. Randolf Menzel, Institut fur Neurobio-

logie, Freie Universit€at Berlin, and Dr. Jurgen Rybak, Max

Planck Institute for Chemical Ecology, for help with the

registration of the confocal images of neurons into the

HSB and for helpful comments on this research.

CONFLICT OF INTEREST STATEMENTThe authors declare no conflict of interest.

ROLE OF AUTHORSAll authors had full access to all the data in the study and

take responsibility for the integrity of the data and the

Figure 11. Putative model of the primary processing of the waggle dance information. In this example, the forager (waggle dancer) has

just returned from a feeder whose bearing is 90� west of the bearing of the sun to the hive. The bee performs a waggle dance oriented

90� to the left. The in-hive honeybee (follower) follows the dancer from its tail end. The left central NHs are activated, and the afferents

send signals to the corresponding subregion in the dSEG (shown as �90� in the dSEG). The afferents of JO also send signals to the part

of the �90� degree subregion in dSEG. In the dSEG, only the part of the �90� degree subregion can be activated by signals received

from JO and the left central NHs.



accuracy of the data analysis. Study concept and design:

HA. Acquisition of data:HA,HH. Analysis and interpretation

of data: HA,HH.Drafting of themanuscript: HA,HH.Critical

revision of the manuscript for important intellectual

content: HA. Obtained funding: HA. Administrative, tech-

nical, and material support: HA. Study supervision: HA.

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Ai and Hagio


morphological analysis of the primary center receiving spatial information transferred by the waggle...

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