The pretarsus of the honeybee

Abstract

Although the honeybee (Apis mellifera L.) is a well-studied species, the functional morphology of its pretarsal structure is still not fully understood. We conducted an in-depth scanning electron microscopic study on these complex structures to contribute to the comprehension of the pretarsal structure-function relationships. As a result, this study has provided valuable information on the ultrastructure of the pretarsus, and in particular on the spines of the unguitractor surface and the small spines and scalloped surface of the claws with longitudinal grooves. Special attention was given to the adhesive contact zone of the arolium with its highly specialized fibrillary cuticle texture. Remarkably, several of the observed pretarsal structures, such as the pyramidal structures on the unguitractor and the thin hairs on both the grooved claws, and the hairs of the manubrium have not been previously described. All observed structures in this study were characterized with respect to their possible physiological and mechanical roles.

Full text

The pretarsus of the honeybee
Michel Asperges 1,*, Jan D’Haen
2, Ivo Lambrichts
3 &
Frank Van Belleghem 4
1,* Universiteit Hasselt, Faculteit Wetenschappen Campus Diepenbeek Agoralaan Gebouw D,
BE-3590 Diepenbeek, Belgium.
2 Universiteit Hasselt, Instituut voor Materiaalonderzoek Campus Diepenbeek Wetenschapspark 1,
BE-3590 Diepenbeek, Belgium.
3 Universiteit Hasselt, Faculteit Geneeskunde en Levenswetenschappen Campus Diepenbeek
Agoralaan Gebouw D, BE-3590 Diepenbeek, Belgium.
4 Open Universiteit, Faculty of Management, Science and Technology, Valkenburgerweg 177, 6419 AT
Heerlen, The Netherlands and Universiteit Hasselt, Centre for Environmental Sciences, Department
Biology, Agoralaan, Diepenbeek, Gebouw D, BE3590 Diepenbeek, Belgium.
* Corresponding author: michel.asperges@uhasselt.be
2 E-mail: jan.dhaen@uhasselt.be
3 E-mail: ivo.lambrechts@uhasselt.be
4 E-mail: frank.vanbelleghem@uhasselt.be
Abstract. Although the honeybee (Apis mellifera L.) is a well-studied species, the functional
morphology of its pretarsal structure is still not fully understood. We conducted an in-depth scanning
electron microscopic study on these complex structures to contribute to the comprehension of the
pretarsal structure-function relationships. As a result, this study has provided valuable information on
the ultrastructure of the pretarsus, and in particular on the spines of the unguitractor surface and the small
spines and scalloped surface of the claws with longitudinal grooves. Special attention was given to the
adhesive contact zone of the arolium with its highly specialized brillary cuticle texture. Remarkably,
several of the observed pretarsal structures, such as the pyramidal structures on the unguitractor and the
thin hairs on both the grooved claws, and the hairs of the manubrium have not been previously described.
All observed structures in this study were characterized with respect to their possible physiological and
mechanical roles.
Keywords. Honeybee, pretarsus, arolium, arcus, manubrium, planta, unguitractor.
Asperges M., D’Haen J., Lambrichts I. & Van Belleghem F. 2017. The pretarsus of the honeybee. Belgian Journal
of Zoology 147 (2): 87–103. https://doi.org/10.26496/bjz.2017.8
Introduction
All insects including the honeybee (Apis mellifera L.) possess three pairs of segmented legs attached
to the thorax. The honeybee’s segmented leg (Fig. 1) consists of the coxa, the trochanter, the femur, the
tibia and ve tarsomeres, comprising the basitarsus, three small tarsomeres and the pretarsus (Asperges
2011).
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This work is licensed under a Creative Commons Attribution 3.0 License. ISSN 2295-0451
Research article
https://doi.org/10.26496/bjz.2017.8

Although the honeybee is a well-studied species, the functional morphology of its pretarsal structure
is still not completely elucidated. Using light microscopy snodgrAss (1956) described all parts of the
pretarsus accurately, but did not propose any structure-function relationship. It was later suggested that
the pores on the pretarsus could possibly secrete footprint pheromones (Lensky et al. 1985; BiLLen
1986). In 2003, as part of an extensive electron microscopic study, goodmAn (2003) was the rst to
provide a general description of the pretarsal functional morphology. In addition, JArAu et al. (2004)
and WiLms & eLtz (2008) proposed the occurrence of epithelial glands around the tendon to mark food
sources. In order to contribute to the elucidation of the functional morphology of the pretarsal structures,
we conducted an in-depth scanning electron microscopic study on these complex structures. In this way,
we aimed to provide more detailed information on the ultrastructure allowing better comprehension of
the pretarsal functional morphology.
Material and methods
Light microscopy
Amputated whole legs from fresh honeybees were immersed in 10% KOH in water (25°C). After a few
days the solution turned brown. The solution was refreshed (every two days) until it remained colorless
and the legs had become transparent. This clearing operation could take up to two weeks and more.
Following the clearing operation the legs were rinsed in water followed by dehydration in a graded
ethanol series (30%, 50%, 70%, 95%). Whole legs or parts of legs were mounted on glass slides within
a drop of water for further observation using an Olympus CH2 equipped with a Nikon Coolpix 950
camera, and a Zeiss “Primo Star” microscope.
Scanning electron microscopy
Fresh amputated legs were xed for 24 hours in 2% glutaraldehyde buffered in 0.05 M sodium cacodylate
(pH 7.3) and 0.15 M saccharose. The xed legs were rinsed for 2 × 10 minutes in 0.05 M sodium
cacodylate and 0.15 M saccharose (pH 7.3). Subsequently, the legs were dehydrated in a graded ethanol
series of 30%, 50%, 70% and 95% for 30 minutes each step. The legs were critical-point dried with a
Polaron critical-point dryer. To enable observation by SEM the samples were Au/Pt sputter coated using
a Leica E:ACE600 Sputter coater. The SEM images were rst recorded using a FEI Quanta 200FEG-
SEM scanning electron microscope.
The images presented in this report are only SE (secondary electron) images. These images were mostly
used to reveal the surface morphology of the specimen. SE image contrast is mostly generated by
differences in SE emission efciency with topography.
Figure 1 – The hind-most leg of a honeybee with pollen-loaded corbiculum attached to the tibia.
Belg. J. Zool. 147 (2): 87–103 (2017)
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Results
Tarsus
Each leg has ve tarsomeres (Fig. 2) of which the rst or most proximal one is the basitarsus. The
basitarsus of a worker bee has specialized functionalities, such as collecting pollen. The next three more
distal tarsomeres are rather small. The foot ends in the pretarsus. There are no muscles in the segments
behind the tibia, however a long unguitractor tendon runs through all the segments from the femur down
to the pretarsus.
The basitarsus and the small subsegments of the tarsus move freely with respect to one another by
monocondylic articulations. The articulations shown in Fig. 3A–B consist of exible membranes arising
from a large ‘elbow’- or ‘knee’-like joint. The same type of connection exists between the basitarsus and
the tibia. The tendon of the musculus retractor unguis of the femur and tibia, called “retrotractor of the
claws”, ends in the pretarsus on the unguitractor and arcus.
Pretarsus
The pretarsus is a complex structure (Fig. 4A–B) consisting of two segments: the complex foot and the
fth most distal tarsomere. On the foot two pairs of claws can be distinguished. Between the claws a
soft arolium can be seen, including the arcus, a dark ‘U-shaped’ band on the upper part of the arolium.
Figure 2 – Detailed image of the tarsomeres. The basitarsus in the left upper corner is more distally
followed by three tarsomeres and the pretarsus. The long unguitractor tendon runs through all the leg
segments.
ASPERGES M. et al., The pretarsus of the honeybee
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The arolium, the ‘sole’ of the foot, is connected to the planta, a small sclerotized plate, attached to the
unguitractor. The manubrium is located on top of the planta. The unguitractor is connected to the long
unguitractor tendon (Figs 5–7) coming from the tibia and femur musculus retractor unguis through the
different tarsomeres.
Figure 3 – A. Micrograph of the leg joints between the basitarsus and the second tarsomere. B. Second
and third tarsomere with ‘knee-like’ joint and in the middle the unguitractor tendon (t) (arrow) clearly
visible.
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Figure 4 – A–B. SEM of the pretarsus.
Figure 5 – Pretarsus with the fth tarsomere and the foot.
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Figure 6 – Scheme of the pretarsus.
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Claws
There are two pairs of hollow, strongly sclerotized claws (Fig. 8A–B), each claw tapering to a point.
Each claw exists of two lobes of unequal length with long stout spines on the outer surface. The lobes of
the claws are outgrowths of the membranous lateral walls of the base of the tarsus. They arise between
the articular condyle above and the auxiliary sclerite below, just at the top of the unguitractor plate. They
are freely exible but not musculated. The claws are unable to grip on hard or smooth surfaces. When
the claw touches a substrate, it exes as the points of the claws spread sideways. This causes a chain
reaction on the unguitractor, the planta plate and further on the arcus, pulling down the arolium, which
spreads out on the smooth surface to be gripped.
SEM micrographs (Fig. 9A–B) show that the claw surface is not smooth but scalloped with longitudinal
grooves on both outer and inner surfaces. The surface is covered (Fig. 10) with very small, short spines.
These seem to have a tactile or sensory role.
Arolium
The arolium (Fig. 11A) is a soft cuticular sac located between the two pairs of claws at the front of the
foot. The upper surface of the arolium is covered with very short hairs while the undersurface is almost
Figure 7 – Foot of the honeybee.
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entirely bare as observed by snodgrAss (1956). Its adhesive contact zone has a highly specialized
brillary, textured cuticle (FederLe et al. 2001). Observing the distended arolium by light microscopy
or by SEM it is clear that the arolium is covered with parallel grooves (Fig. 11B) that facilitate a grip on
smooth surfaces. It can be hypothesized that pheromones are secreted from the arolium.
The arolium is shaped by two hard pretarsal sclerites, the arcus and the manubrium. The arolium can
be observed in a retracted or distended state. In general, the claws touch the surface before the arolium.
When the foot is placed on a smooth surface the claws retract. The arolium unfolds and extends on the
surface. According to FederLe et al. (2001) the adhesion of the arolium to smooth surfaces is enabled
by a thin liquid lm between the surface and the arolium. Note that the arolium never extends without a
retraction of the claws. When the arolium unfolds, the unguitractor plate is drawn back completely into
Figure 9 – A. The claw surface possesses very small, short spines. B. The surface of the claws is scalloped
with longitudinal grooves.
Figure 8– A. Claws, each longer lobe ending in a point.B. Each claw consists of two lobes of unequal
length.
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the fth tarsomere. When the leg is lifted from the surface, the claws extend and the arolium deates
(Fig. 12A–B) until it detaches from the surface.
The arolium (Fig. 13) in the passive position, half retracted, presents dorsally a deep cavity between the
upturned lateral walls.
Figure 10 – The claw surface is covered with very small, short spines that seem to have a tactile or
sensory role.
Figure 11 – A. Distended arolium with parallel grooves. B. The adhesive contact zone has a highly
specialized brillary cuticle texture.
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Arcus
The arcus (Fig. 14) is the thick dark brown, “U”-shaped structure at the upper part of the arolium. A
contraction by the unguitractor tendon causes the arolium to atten thereby retracting or distending the
arcus.
Figure 12 – A. Retracted arolium. B. Distended arolium.
Figure 13 – In passive position (half retracted) the arolium presents dorsally a deep cavity between the
upturned lateral walls.
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Planta
The planta connects the unguitractor with the arolium. As described by goodmAn (2003) the surface
(Figs 15A–B, 16) of this planta plate is densely covered with strong distally-diverging spines. As
mentioned by steLL (2012) the traction force on the unguitractor plate is transferred to the planta plate,
which, by itself, acts on the arcus. The last one pulls down the arolium and causes it to spread out thereby
enabling grip on smooth surfaces such as ower petals or window glass.
Figure 14 – The arcus, a dark brown “U”- shaped structure.
Figure 15 – A. Micrograph of planta (p) and unguitractor (u) with spikes. B. Micrograph showing the
connection between arcus (a), planta (p) and unguitractor (u).
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goodmAn (2003) suggested that the tarsal glands, located in the fth tarsomere of each leg, produce
‘the footprint pheromones’, oily colorless secretion(s) with low volatility. The secretion product(s)
originating from a reservoir near the unguitractor in the fth tarsus spread over the surface of the planta.
In agreement with goodmAn (2003) and steLL (2012), we found no connection between the secretory
reservoir of the Arnhart gland and the foot parts. Therefore, the secretory products most likely ow
straight to the arolium.
Unguitractor
The proximal side or front of the unguitractor plate extends into the pretarsus along a membranous fold,
and the outer or distal end attaches to the planta (Fig. 17) with a thin membrane. It is attached to the base
of the claws with a tendon-like elastic structure. On its distal side, the unguitractor plate is attached to
the weakly-sclerotized hairy planta plate, which connects to the arolium by the arcus.
Based on light microscopic observations, goodmAn (2003) stated that “the unguitractor plate is covered
with a scaly surface”. However, the light microscopic micrographs presented here (Fig. 15A–B) do
not conrm this statement. When viewing under a slightly inclined angle, lots of pyramidal spines
can be seen in the transition region between the unguitractor and the planta (Fig. 15A–B). The SEM
micrographs (Figs 18, 19A–B) provide detailed images of the surface, densely covered with pyramidal-
shaped spines. When the unguitractor touches a rough surface the Van der Waal forces are strong, which,
in combination with the grip of the claws, results in a rm grip. On a smooth surface the Van der Waal
forces are too weak for a sufcient grip of the claws. In that case, the unfolded adhesive arolium clings
to the substrate thereby providing sufcient grip.
From the femur and tibia muscles a long unguitractor tendon passes (Figs 17, 20A, 21) through all
tarsomeres and connects to the unguitractor plate. In agreement with their observations in the bumblebee
(Bombus terrestris L.), JArAu et al. (2005, 2012) also reported the presence of footprint secretions
for the stingless bee (Melipona seminigra Friese). The secretion produced by the glands around the
unguitractor tendon is released at the base of the unguitractor plate. WiLms & eLtz (2008) also proposed
Figure 16 – The planta with spines.
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Figure 17 – Overview of the pretarsus, the foot and tarsus The unguitractor is covered at one side by the
tarsus.
Figure 18 – The “scaly” appearance of the unguitractor, which is not strictly scaly, but posesses pyramidal
spines.
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the occurrence of an epithelial gland around the tendon in the honeybee. However we could not nd any
indication for the existence of one or more pores from which such secretion could be released. Studies
on honeybees (Lensky et al. 1985) and wasps (BiLLen 1986) using transmission electron microscopic
techniques (TEM) have conrmed the absence of pores, which could not be located either in the cuticle
of the arolium, or in the cuticle of the fth tarsomere. So the glandular secretion mechanism remains
unsolved. Most likely, the tendon glands rather than the tarsal glands (Arnhart’s gland) discharge
footprint secretions in honeybees, bumblebees and stingless bees. BiLLen (1984) found that this is,
however, not the case for ants.
There is a junction (Fig. 20A–B) of the unguitractor tendon to the arolium by the arcus. When the
muscle contracts, the tendon draws on the membranes of the foot and the two auxiliary sclerites on either
side of the unguitractor plate. The leverage causes the claws to ex down to provide attachment to the
surface. The arolium between the claws usually turns upwards.
Foot rupture from the fth tarsomere (Fig 21) making the long unguitractor tendon visible. This tendon
is emerging from the fth tarsomere making the long unguitractor tendon visible in the other tarsomeres.
Figure 19 – A–B. The unguitractor is covered with pyramidal-shaped spines.
Figure 20 – A. Part of the unguitractor (u) with the attached tendon (arrow, t). B. The junction of the
tendon (arrow, t) from the unguitractor to the arolium by the arcus (a).
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Manubrium
The manubrium (Fig. 22A–B) is hinged between the claws. It is a sclerotic plate (Fig. 23A–B) bearing
ve or six long bristles. These bristles are covered with small tactile or sensory hairs. The plate is
attached at one side to the arcus of the arolium.
When the manubrium is put down on the arcus, after being stimulated by the long bristles, the arcus will
stimulate the arolium to unfold.
Figure 21 – Foot ruptured from the fth tarsomere making the long unguitractor tendon visible emerging
from the fth tarsomere, and inside in the fourth and third tarsomeres.
Figure 22 – A–B. On the top of the foot the manubrium, bearing ve or six long bristles is visible.
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Discussion and conclusion
The pretarsus is a complex structure, divided into two segments: the complex foot and the tarsus. This
study conrms and elaborates on earlier studies regarding the complex honeybee foot organization. It is
clear that our in-depth study using SEM to visualize surface details has allowed us to bring forward new
information. The spiny appearance of the planta, the scaly surface at the end and the pyramidal-shaped
spines in the more proximal part of the unguitractor plate, the details of the bristles on the surface of the
manubrium, as well as the identication of the longitudinal grooves and very small sensory spines on the
surface of the claws, and nally the parallel grooves on the surface of the arolium, are clear examples
of innovative documentation. We also elucidated the junction of the tendon from the unguitractor to the
arolium by the arcus.
Based on our morphological observations, and the ndings from previous studies, we tried to comprehend
the attachment mechanism of the honeybee foot to both rough and smooth surfaces. Also important was
the fact that we did not nd any evidence for the Arnhart’s tarsal gland to be involved in the release
of the predicted footprint pheromones as suggested for the bumblebee (Bombus terrestris) (WiLms &
eLtz 2008). The fact that we did not observe secretory pores either in the cuticle of the arolium, or in
the cuticle of the fth tarsomere is in agreement with the absence of such pores in honeybees (Lensky
et al. 1985) and wasps (BiLLen 1986), although JArAu et al. (2005) did report the presence of footprint
secretions in the stingless bee (Melipona seminigra Friese). In the honeybee, the secretion produced
by the glands around the unguitractor tendon is released at the base of the unguitractor plate. Most
likely, the tendon glands rather than the tarsal glands (Arnhart’s gland) discharge footprint secretions in
honeybees, bumblebees and stingless bees although this is, however, not the case for ants (BiLLen1984).
Acknowledgments
We sincerely thank Hilde Pellaers (Instituut voor Metaalonderzoek, Hasselt University (IMO)) and Bart
Ruttens (IMO-IMOMEC) for technical aid during the preparation and observation of the SEM samples
at the IMO. We are also indebted to Natascha Steffanie and Marc Jans for their help in preparing the
SEM samples. Finally, we also wish to express our gratitude to prof. dr Roger Huybrechts (KULeuven)
and Mieke Van den Wijngaert for the review of the manuscript.
Figure 23 – A–B. Front view of the manubrium with detail of one bristle.
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