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Research
Cite this article: Drotlef DM, Appel E, Peisker
H, Dening K, del Campo A, Gorb SN, Barnes
WJP. 2015 Morphological studies of the toe
pads of the rock frog, Staurois parvus (family:
Ranidae) and their relevance to the
development of new biomimetically inspired
reversible adhesives. Interface Focus 5:
20140036.
http://dx.doi.org/10.1098/rsfs.2014.0036
One contribution of 15 to a theme issue
‘Biological adhesives: from biology to
biomimetics’.
Subject Areas:
biomaterials, biomimetics
Keywords:
rock frog, adhesive toe pad, functional
morphology, cell shape, electron microscopy,
biomimetics
Author for correspondence:
W. Jon. P. Barnes
e-mail: [email protected]
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rsfs.2014.0036 or
via http://rsfs.royalsocietypublishing.org.
Morphological studies of the toe pads of
the rock frog, Staurois parvus (family:
Ranidae) and their relevance to the
development of new biomimetically
inspired reversible adhesives
Dirk M. Drotlef1, Esther Appel2, Henrik Peisker2, Kirstin Dening2,
Ara´nzazu del Campo1, Stanislav N. Gorb2 and W. Jon. P. Barnes3
1
Max Planck Institut fu¨r Polymerforschung, Mainz, Germany
Functional Morphology and Biomechanics, University of Kiel, Kiel, Germany
3
Centre for Cell Engineering, University of Glasgow, Scotland, UK
2
The morphology of the toe epithelium of the rock frog, Staurois parvus (Family
Ranidae), was investigated using a variety of microscopical techniques. The
toe pad epithelium is stratified (four to five cell layers), the apical parts of
the cells of the outermost layer being separated by fluid-filled channels. The
surface of these cells is covered by a dense array of nanopillars, which also
cover the surface of subarticular tubercles and unspecialized ventral epithelium of the toes, but not the dorsal epithelium. The apical portions of the
outer two layers contain fibrils that originate from the nanopillars and are
oriented approximately normal to the surface. This structure is similar to the
pad structure of tree frogs of the families Hylidae and Rhacophoridae, indicating evolutionary convergence and a common evolutionary design for
reversible attachment in climbing frogs. The main adaptation to the torrent
habitat seems to be the straightness of the channels crossing the toe pad,
which will assist in drainage of excess water. The presence of nanopillar
arrays on all ventral surfaces of the toes resembles that on clingfish suckers
and may be a specific adaptation for underwater adhesion and friction. The relevance of these findings to the development of new biomimetically inspired
reversible adhesives is discussed.
1. Introduction
In addition to tree frogs that are largely arboreal (though smaller species may be
found in the ground flora), there are also frogs that live around freshwater streams
(stream frogs) and waterfalls (rock and torrent frogs) that also possess adhesive
toe pads [1]. The purpose of this study is to investigate the morphology of the
toe pads of one such rock frog, the Lesser rock frog, Staurois parvus Inger and
Haile, found in and around waterfalls within the rainforests of Borneo. Is the
pad morphology very similar to those of tree frogs, or can we find differences
that might relate to its rather different habitat?
The foundations of our understanding of the structure and function of tree
frog toe pads dates back 40 years, but has received a significant boost in recent
years by the realization of the biomimetic significance of mechanisms of animal
adhesion (e.g. [2,3]). Many of these biomimetic studies have been aimed at
replicating the dry adhesive mechanisms of geckos (e.g. [4– 7]), but tree frog
adhesion has also been highlighted as having potential for the development
of new smart adhesives able to adhere under wet conditions [8–11].
The morphology of the adhesive toe pads of tree frogs has been extensively
investigated by both transmission and electron microscopy [12–21]. There is
also a recent study by Barnes et al. [22] that combined atomic force microscopy
(AFM) and cryo-scanning electron microscopy (including freeze-fracture).
& 2014 The Author(s) Published by the Royal Society. All rights reserved.
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2. Material and methods
2.1. Animals
The frog species of our study was the Lesser rock frog, S. parvus
(family Ranidae), a native of Borneo. The frogs (N ¼ 5) were subadult with a snout – vent length of 30 mm. They were donated by
Scho¨nbrunn Zoo (Vienna, Austria).
2.2. Sample preparation for scanning electron
microscopy
Frog toes were cut off with a scalpel and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 2 h at room temperature.
They were then rinsed three times (5 min per rinse) in 0.1 M
phosphate buffer containing 2% sucrose. Toes were post-fixed
with 1% osmium tetroxide in 0.1 M phosphate buffer for 1 h,
then washed three times in distilled water (15 min per wash),
before en bloc staining with 0.5% aqueous uranyl acetate for
1 h in the dark (as this is a light-sensitive stain). Following two
further washes in distilled water, samples were dehydrated
through an acetone series (30, 50, 70 and 90%, 15 min in each),
then four changes of absolute acetone (15 min each) and four
changes of dried absolute acetone (3A molecular sieve). Toe
samples were placed in critical point drying (CPD) specimen processing capsules (type C211, TAAB Laboratories Equipment Ltd,
Aldermaston, Berkshire, UK) under dried absolute acetone and then
critical point dried using a Polaron Critical Point Drying Apparatus
E3000 (Quorum Technologies, Lewes, East Sussex, UK). Dried
frog toes were orientated on aluminium pin scanning electron
microscopy (SEM) stubs using double sided copper conductive
tape and quick drying silver DAG paint, all from Agar Scientific
(Stansted, Essex, UK). Mounted toes were gold/palladium coated
(approx. 20 nm thickness) using a Polaron SC515 SEM coating
system (Quorum Technologies) and then viewed on a JEOL6400
SEM (JEOL, Tokyo, Japan) running at 6–10 kV. The above describes
procedures used in Glasgow. Procedures carried out in Kiel were
similar, with specimens being viewed on a Hitachi S-4800 SEM
(Hitachi High-Technologies Corp., Tokyo, Japan).
2.3. Sample preparation for transmission electron
microscopy
Frog toes were cut off as described earlier, with the initial sample
preparation procedures following those described above for SEM.
Following fixation in glutaraldehyde, post-fixation in osmium
2
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pad and the periphery. Experimental evidence is lacking, but
an obvious function for these shorter pathways from centre to
edge of pad would be to promote rapid drainage of excess
fluid from underneath the pad. Such elongated channels were
also found in the torrent frog, Odorana hosii, but only around
the edges of the toe pads in the rock frog, Staurois guttatus [34].
A biomechanical study has been carried out on the Trinidadian
stream frog, Mannophryne trinitatis, by Barnes et al. [35], while
Endlein et al. [34] made a detailed comparison between the
adhesive abilities of the rock frog S. guttatus and a rhacophorid
tree frog of comparable size. In both studies, the stream/rock
frog was good at adhering to rough wet surfaces, but very
poor at sticking to dry rough ones. They also slipped on
smooth wet surfaces. These data suggest that torrent/stream
frogs can cope with running water so long as the surfaces are
rough. This study searched for morphological features that
could underlie these abilities and discusses the implications of
these findings for the development of new smart adhesives.
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These investigations show that the cells that make up the
stratified columnar epithelium of the toe pads differ from
those of the skin on the dorsal surface of the toes or indeed
any other epidermal cell type [1], being specialized for their
adhesive function. Indeed, circumferal and, in many species,
proximal margin grooves separate the toe pad epithelium
from neighbouring, less specialized skin. Accessory adhesive
tubercles, found more proximally on the digits as well as on
the main body of the feet, may be similarly specialized, but
their precise role in adhesion and/or friction remains to be
confirmed. The outer layer of cells, largely hexagonal in surface view, appears flat-topped at the microscale, the apical
portions of individual cells being separated from each other
by deep, fluid-filled channels, giving an aspect ratio of
ca 0.5 for the outer parts of the epithelial cells. The fluid
within the channels is secreted by mucous glands that have
ducts ending in these channels, the channels serving to
spread the mucus over the surface of the pad. Additionally,
on surfaces wetted by rain, the channels may act to disperse
excess fluid to the pad edge. Finally, the epithelial cells are
covered by a dense array of nanopillars [23] that originate
from desmosomes [12]. Within the epithelial cells, there are
large numbers of keratin fibrils (tonofilaments), arising from
the nanopillars, that are oriented approximately normal to
the surface [12,20]. They are the main cytoskeletal elements
of the toe pad epithelium and will confer a degree of rigidity
to the cells [15]. Additionally, by analogy with comparable research on insects [24–26], it is probable that they are
important for both toe pad adhesion and detachment.
Arboreal frogs (tree frogs) are found in at least seven different families including the Hylidae and Rhacophoridae,
whose members are almost exclusively arboreal in the New
and Old Worlds, respectively. The morphological features
described above show little variation between the different
families of tree frog and, as common ancestors with toe pads
are lacking, clearly demonstrate convergent evolution [1].
Frog pads employ wet adhesion [27,28] that functions
optimally when the fluid layer under the pad is very thin
( 1 mm) and there are no air pockets [29,30]. Given the presence of this fluid joint, the fact that toe pads also generate
significant friction forces is surprising. Federle et al. [29]
have proposed that this is achieved through direct contact
between the tops of the nanopillars and the substrate in
adhering frogs, and their experimental data demonstrate
static as well as dynamic friction. Toe pads detach by peeling
from the rear (i.e. in a proximal to distal direction) [28]. This is
a natural consequence of the forward and upward movement
of the distal part of the limb at the beginning of the locomotor
swing phase, but does mean that, on slopes, downwardfacing pads would have a tendency to peel spontaneously.
As a consequence, tree frogs on slopes usually place their
pads so that they face uphill [30]. Frictional forces prevent
sliding on sloping surfaces, produce the close contact upon
which good adhesion depends and, on overhanging surfaces
where the limbs are spread out sideways [31], prevent peeling
of the pads from the surface [32].
Much less is known about adhesion and toe pad morphology in rock or torrent frogs. Ohler [33] carried out a
preliminary study of toe pad morphology of torrent-living
ranid frogs, demonstrating that the toe pads of Amolops spp.
had distinct anatomical differences from the typical pattern
seen in tree frogs. The toe pad epithelial cells were elongated,
providing much straighter channels between the centre of the
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Frog toes were tied with a synthetic fibre to prevent blood loss, cut
off with a scalpel and plunge-frozen in liquid propane cooled by
liquid nitrogen. This procedure avoided formation of ice crystals
and prevented damage of pad structure. The specimens were
stored in liquid nitrogen. Cryo-SEM analysis was performed with
a Hitachi S-4800 SEM (Hitachi High-Technologies Corp.) using a
cryo-preparation chamber Gatan ALTO-2500 (Gatan Inc., Abingdon, UK). The frozen frog toes were fixed with Tissue-Tek O.C.T
(Sakura Finetek Europe B.V., Zoeterwoude, The Netherlands) to
the specimen holder and immediately plunged in liquid nitrogen
to avoid thawing. They were then placed in the cooled (21408C)
cryo-preparation chamber (using liquid nitrogen). The frozen specimens were sputter-coated with a gold–palladium layer (1 : 9, layer
thickness 10–20 nm) and transferred to the microscope. The
samples were examined at 21208C at an accelerating voltage of
2–5 kV.
2.5. Sample preparation for atomic force microscopy
The AFM studies were performed with a NanoWizard I AFM (JPK
Instruments AG, Berlin, Germany). The frog toe was tied with a
synthetic fibre and cut off with a scalpel. The toe was fixed with
superglue to a glass slide in an AFM BioCell chamber (JPK
Instruments AG, Berlin, Germany) and submerged in frog Ringer
solution. The measurements were conducted with a pyrex-nitrideprobe cantilever (silicon nitride SPM-Sensor, NanoWorld AG,
Neuchaˆtel, Switzerland) in intermittent (tapping) mode. The resolution of the scans was 1024 1024 pixels. The images were
processed with the SPIP software (Scanning probe image processing
software, v. 5.1.2, Image Metrology A/S, Hørsholm, Denmark).
3. Results
3.1. Toe morphology (SEM and cryo-SEM)
3.1.1. Toe pads
The toe pads are located on the expanded tips of each digit, of
which there are four in each forelimb (figure 1) and five in each
hindlimb. Additionally, there are accessory adhesive structures
1 mm
Figure 1. SEM image of forelimb of S. parvus; ventral view showing toe pads
and subarticular tubercles (arrows). The ‘thumb’ (digit 1) is the small pad on
the right hand side.
called subarticular tubercles located more proximally on
the digits (figure 1, arrows). The toe pads are surrounded by
circumferal and proximal grooves (figures 1 and 2). The proximal groove, which delineates the proximal margin of the
pad, is shallow as seen in the longitudinal section of the pad
(figure 2b, small arrow), while the circumferal groove that
delineates the distal and lateral pad margins is a large deep
channel, especially at the distal tip of the pad (figure 2b, large
arrow). Interestingly, as this is not a common feature of tree
frogs, the larger pads extend distally around the front of the
toe (figure 2a,b). The following features were visible.
3.1.2. Toe pad surface micropattern—epithelial cells
As shown in figure 3a,b, the toe pad surface is made up of
flat-topped epithelial cells of ca 13 mm diameter separated by
channels with a width of ca 2 mm in fixed material, but probably narrower in living cells where channel width varies with
the amount of fluid under the pad. Mucus glands are rare in
toe pads (less than five per pad), less common than in other
skin areas. The duct of one such mucus gland is shown in
figure 3b, opening into an intercellular channel. The majority
of epithelial cells are irregular hexagons, but other shapes are
also seen. In a total number of 122 epithelial cells, the hexagonal shape was predominant (56%), followed by pentagonal
(33%) and heptagonal (11%). A single octagonal epithelial
cell was also recorded. The white lines on figure 3a show the
existence of relatively straight channels crossing the pad in an
approximately anterior–posterior direction. We have quantified
the lengths of such channels in two pads (10 channels in each
pad), including those in figure 3a. Relative to a straight line
(length ¼ 1), such channels have a length of 1.13 + 0.04
(mean + s.d., N ¼ 20). This is significantly better than a pattern
of regular hexagons, which have a relative channel length
of 1.33. Interestingly, channels going across the pad also have
a relative channel length of less than 1.33, but are not as straight
as the anterior–posterior channels ( p , 0.001, Wilcoxan signedrank test). Such channels should promote good drainage of
excess water in the rock frogs’ stream habitat, those running in
an anterior–posterior direction being of greater significance as
pads normally face uphill. For full details of this analysis, see
electronic supplementary material, table S1 and figure S1.
Figure 3c shows such a channel at a point where three epithelial cells meet. The channel walls can be seen to be ridged, the
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2.4. Sample preparation for cryo-SEM
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tetroxide, en bloc staining with uranyl acetate and washing in distilled water, the toes were processed through a dehydration series
of ethanol starting with 30%, then 50, 70 and 90% (15 min in each
solution), before four changes in absolute ethanol (for 15 min each)
and drying in absolute ethanol (3A molecular sieve; four changes
of 15 min each). The absolute ethanol was then replaced with 100%
propylene oxide for three 15 min changes, and then into a 50 : 50
propylene oxide/Araldite 502/812 resin mix (TAAB Laboratories
Equipment Ltd) minus accelerator overnight. The toes were
given several changes of pure resin minus accelerator over the
next day; then placed in pure resin plus accelerator overnight
and fresh resin embedded in flat-bed moulds the next morning.
They were then left to polymerize at 608C for 48 h. Semi-thin
0.35 mm light microscope sections were cut using a Leica Ultracut
UTC Ultratome (Leica Microsystems, Milton Keynes, UK) and a
Drukker histomicrotome knife with trough (Drukker International,
Cuijk, The Netherlands). Sections were dried on a glass slide on a
hotplate (808C) and then stained with 1% toluidine blue/1% borax
in distilled water. Ultrathin sections (60–70 nm) were also cut using
the Leica Ultracut UTC Ultratome and a Drukker Ultramicrotome
knife. Sections were picked up on slot or 100 mesh formvarcoated copper grids and contrast-stained with 2% methanolic
uranyl acetate for 5 min and Reynolds lead citrate for 5 min. Toe
samples were viewed on a LEO912AB transmission electron microscope (Carl Zeiss Ltd, Cambridge, UK), running at 120 kV. Images
were captured using an Olympus iTEM soft imaging system.
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(a)
4
(b)
200 mm
100 mm
Figure 2. (a) SEM image of digit 2 of forelimb of S. parvus, showing toe pad epithelium, circumferal and proximal grooves. (b) Longitudinal section of toe pad
(light microscope image) showing the same features. Note relatively large size of circumferal groove (large arrow) compared with proximal groove (small arrow).
(a)
(b)
100 mm
(c)
10 mm
(d)
1 mm
500 nm
Figure 3. SEM images of toe pad epithelium of S. parvus. (a) White lines show relatively straight channels crossing the pad. (b) Polygonal epithelial cells (mainly
hexagonal in shape) surrounded by deep channels with a single mucous pore (centre). (c) Edge of a pad epithelial cell showing dense array of nanopillars covering
the pad surface. (d ) High-power view of nanopillars.
ridges probably reflecting the presence of bundles of keratin
tonofilaments beneath the surface.
3.1.3. Toe pad surface nanopattern—nanopillars
As in tree frogs, the surface of the epithelial cells is covered in a
dense array of nanopillars that form a surface nanopattern consisting of regular polygons 200–300 nm in diameter (figure 3d).
Their height is not measurable from this image, partly because
they are so densely packed and partly because they merge with
the ridges that characterize the channel walls. However, transmission electron microscopy (TEM) images show their height
to be 400–500 nm, giving them an aspect ratio of 1.4–2.0
(figure 10). Bundles of filaments, presumed to be keratin tonofilaments from immunohistochemical studies on other frog
species (e.g. [36]), are visible through the walls of the nanopillars in some of our cryo-SEM images (figure 4b). Such images
(figure 4a,b) also revealed that the nanopillars have a slightly
concave terminal and in, unspecialized ventral toe epithelium
at least, can have aspect ratios of up to 2.5. One of the
advantages of using cryo-SEM is that it allows one to view
the mucus covering of the pad. In figure 4a, the frozen mucus
can be seen surrounding but not covering the nanopillars and
also filling their dimpled tops.
3.1.4. Epithelium of toe tubercles
Proximal to each toe pad are located subarticular tubercles
(figures 1 and 5a). Their epithelial surface possesses similar
features to those of the toe pads (figure 5c,d). Polygonal
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ventral
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dorsal
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3.1.6. Dorsal toe epithelium
(a)
3.2. Toe pad anatomy (light microscopy and TEM)
3.2.1. Epithelial cell layers
1 mm
Figure 4. High-power images of nanopillars in S. parvus. Cryo-SEM images of
unspecialized ventral epithelium showing (a) the tops of the nanopillars surrounded by frozen mucus and (b) fibrils (bundles of tonofilaments) visible
through the walls of the nanopillars (arrows).
epithelial cells (mostly 5 or 6-sided) are separated by channels
(figure 5c,d), but the channel depth in tubercular epithelium
is significantly shallower (1 –3 mm, figure 8b) than in the
pad epithelium (10 mm, figure 8a), and significantly shallower and narrower in the peripheral region of the tubercles
(figure 5d) than in the centre (figure 5c). Epithelial cells in
the tubercle are also covered by an array of nanopillars
with similar dimensions and arrangement as in toe pads,
but at a significantly lower density (figure 5b). By contrast,
the density of mucous pores (several of which are visible in
both figure 5c,d) appears higher than in the toe pad epithelium. These images also show marks on their surface
(actually gaps in the nanopillar array as figure 5b makes
clear) that clearly indicate the position of the layer of cells
that previously had lain above them (figure 5d, arrows).
Such marks are also present on the toe pad epithelium
(obvious in figure 11a, less so in figure 3b). The different
cell layers are thus displaced with respect to each other in
both x and y axes, as previously noted for tree frogs [22].
3.1.5. Ventral unspecialized toe epithelium
Examination of the ventral epithelium of the toes, excluding toe
pads and subarticular tubercles, shows a degree of adaptation for
adhesion and friction. As figure 6 shows, the epithelial cell surface is covered by nanopillars, but at lower density than in the
more specialized epithelia of the toe pads and subarticular tubercles. Such nanopillars are absent at the cell boundaries, giving
rise to narrow, shallow, intercellular channels. Mucous pores
are common. The cryo-SEM images of nanopillars discussed earlier (figure 4a,b) were taken from this area, their lower density
making structural features easier to see and measure.
Examination of longitudinal sections of the toes under the light
microscope reveals that the epithelium of the toes of S. parvus is
stratified. In the toe pads, it has a thickness of ca 50 mm, consisting of four to five cell layers, with deep channels separating the
outer portions of the outer cell layer (figure 8a). This layer has
dark pyknotic nuclei and consists of dying or dead cells. Peripheral portions of the cytoplasm of the two outmost layers
are stained darkly, owing to the presence of keratin. The
large vesicles in the second layer will give rise to the intercellular channels when the second layer becomes the outer layer,
through the shedding of the outer layer. By contrast, unspecialized ventral epithelium and the dorsal epithelium of the toe
are only 15–20 mm thick (figures 2b and 8b). The dorsal epithelium has no channels, while the channels separating the
outer portions of the outer layer of cells in unspecialized ventral toe epithelium are only about 1 mm deep. The extent of
the keratinization is much reduced. The epithelium of the subarticular tubercles is broadly similar to that of the toe pads,
though its thickness is reduced (data not shown).
3.2.2. Transmission electron microscopy
The electron microscope reveals a lot more detail of the ultrastructure of the different layers. As, with each sloughing of
the outer layer, layer IV will progressively become layer III,
then II and finally I (the outer layer), examination of the different layers also provides insights into how the specialized outer
layer with its deep channels, nanopillar arrays and keratin
fibrils develops from unspecialized beginnings. The outer
layer, seen in figure 9a, shows the cells to be filled with darkstaining keratin tonofilaments that have their origin in the
nanopillars, three of which are shown in enlarged view in
figure 10. Completely filling the nanopillars (figure 10), they
initially run normal to the cell surface (figure 9a), but gradually
bend proximally before stopping about halfway across the cell.
Even the peripheral cytoplasm appears dark, owing to keratin
deposition. Figure 9b shows the cell border between layers I
and II. Here, the outer cell layer is devoid of both tonofilaments
and keratin, though both are present in the peripheral part of
layer II. The outermost part of layer II also shows how the
nanopillars arise, as bundles of tonofilaments are becoming
separated from each other by clear cytoplasm and there are
some invaginations of the cell boundary into these clear areas
(figure 9b, arrows). Figure 9d shown the same layer I/layer II
boundary, but now including one of the large vesicles (seen
in figure 8a) that will give rise to the deep channels between
the outer parts of the epithelial cells in layer I. Figure 9c,
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2 mm
In contrast to the ventral epithelium, the epithelium of the
dorsal surface of the toes has neither nanopillars nor intercellular channels; i.e. it shows no adaptations for either adhesion or
friction. At low power (figure 7a), the surface appears relatively
smooth, though large Y-shaped pores, possibly of mucous
glands (though the pore structure is very different than that
of the ventrally located mucous pores), are common. At
higher power (figure 7b), the dorsal epithelium is seen to be
covered by nanostructures (150 nm across) that are joined
end to end to form a network that covers the cell surface.
Their appearance is similar to that of vermicular (worm-like)
structures seen on lizard scales [37].
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(a)
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(b)
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100 mm
(d)
10 mm
10 mm
Figure 5. SEM images of subarticular tubercle epithelium of S. parvus. (a) Basal tubercle of digit 4 of forelimb. (b) High-power view of nanopillars, intercellular
channel (black arrow) and gap in the nanopillar array at the position of a cell boundary of the previous outer cell layer (white arrow). (c) Epithelium of centre of
tubercle with larger intercellular channels. (d ) Epithelium near edge of tubercle with smaller intercellular channels (black arrows indicate channels; white arrows,
gaps in the nanopillar array as described in b).
(a)
(b)
5 mm
500 nm
Figure 6. SEM images of nanopillar array and intercellular channels of unspecialized ventral epithelium of S. parvus digits. (a) Low power; arrow indicates opening
of mucous gland. (b) High power.
which illustrates the boundary between layers II and III, shows
an even earlier stage in the development of the structure of the
outer layer. Both cell layers are clearly living tissue, with typical
nuclei visible in the cells shown. The cell boundary is invaginated, but to a lesser extent than the layer I/II boundary. In
some cases, short tonofilaments can be seen to be going both
inwards and outwards from desmosomes joining the two
surfaces together (figure 9c, arrows). Electron-dense particles,
probably corneous (horny) material containing keratin [38],
are visible in both layers.
3.3. Atomic force microscopic studies of the surface
of the toe pads
3.3.1. Cell surface probed by the atomic force microscopy
The AFM studies provide further insight into the structure
and components of toe pad epithelium. Unlike SEM, TEM
and light microscopy, the tissue does not require to be
fixed, so problems of shrinkage and distortion are avoided.
However, as an AFM cantilever is not able to penetrate
narrow clefts, it cannot provide data on the depths of the
channels between either epithelial cells or nanopillars. AFM
scans showed the polygonal epithelial cells separated by
channels that did not exceed 1 mm in width (figure 11a),
considerably narrower than they appear in SEM images
(figure 3b). They also show the gaps in the nanopillar array
that reflect the position of the previous cell layer, a feature
not obvious in figure 3b. The narrowness of the channels
did not allow the cantilever tip to penetrate them and, therefore, the depth of the channels could not be estimated.
Figure 11b shows the nanostructures on the epithelial cell surfaces. The height profile (figure 11c) quantifies the extent to
which the tops of the nanopillars are not flat-terminated
but slightly concave. Precise quantification is difficult because
it is necessary for the profile to run precisely through the
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(c)
500 nm
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(a)
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(b)
(a)
(b)
10 mm
10 mm
Figure 8. Light microscope sections of ventral epithelium of toes of S. parvus (100 objective). (a) Toe pad epithelium showing four cell layers with deep channels
between the outer portions of the outer cell layer (arrows). (b) Unspecialized ventral epithelium (near proximal groove) showing small shallow grooves (arrows) between
the outer portions of the outer cell layer.
centre of the nanopillar. Our best data give a figure for the
depth of the dimple in the range 6–8 nm.
4. Discussion
The surface of toe pads of tree, rock and torrent frogs shows a
particular topography with distinct features at the micro- and
nanoscales [20 –23,29,33,34,39 –41]. The role that this surface
structure plays in reversible attachment and locomotion of
the animals in wet and even flooded environments is a current focus of research for zoologists and material scientists
eager to transfer new ‘wet’ adhesive principles from natural
to artificial systems.
The outermost cell layer of the toe pad consists of epithelial
cells forming polygonal pillars of 10–15 mm width. As in hylid
and rhacophorid tree frogs [22,39], the majority are hexagonal
but pentagonal and heptagonal geometries also occur. The
occurrence of such geometries in a predominantly hexagon pattern has recently been studied in the corneal nipple arrays that
cover the ommatidia (facets) of the compound eyes of the
butterfly, Nymphalis antiopa [42]. Nipples are occasionally surrounded by either seven or five neighbours and five/seven
pairs are relatively common, a feature shared by the toe pads
of the tree frog, Rhacophorus prominanus, where pentagonal epithelial cells are surrounded by more heptagons than would be
expected by chance and vice versa [22]. Space filling due to the
domed surface of each ommatidium is considered by Lee and
Erb to be one reason for the existence of such irregularities.
Indeed, this mechanism of introducing curvature into a close-
packed structure may be quite common in nature. For example,
the honeycombs of bees and social wasps use similar interruptions of the hexagonal pattern to account for curvature,
confined boundary conditions and transition zones from smaller to larger cells [43,44]. Tree and rock frog epithelial cells are
much less regular in shape than either corneal nipple arrays
or honeycombs and the presence of relatively straight channels
crossing the pad (see below) also necessarily affects cell shape,
changing regular hexagons into rather squarer cells. However,
as in butterfly corneal nipple arrays, the coexistence of heptagonal and pentagonal shapes with the predominantly hexagonal
one is probably required to fill the pad space and accommodate
irregularities caused by the pad curvature and the mucus pores.
The polygonal epithelial cells are separated at their tips by
narrow (ca 1 mm width) channels of around 10 mm depth.
This is a characteristic feature of the toe pad epithelia of
tree frogs, suggesting an important functional role for the
channels. In ranid frogs, this feature has been observed in torrent frogs such as Amolops spp. [33], in rock frogs of the genus
Staurois ([34] and this study) and also, somewhat surprisingly, in the unexpanded toe pads of the amphibious
leopard frog, Rana pipiens [45]. In S. parvus, the channels
are restricted to the ventral epithelium of the toes; i.e. pads
(deep channels), subarticular tubercles (shallower channels)
and unspecialized ventral epithelium (channels are hardly
more than gaps in the nanopillar array) as illustrated in
figures 3b, 5, 6 and 8. They are absent from dorsal toe epithelium, where nanopillars are also absent (figure 7). The
role of the channels in reversible ‘wet’ adhesion has been
recently investigated in artificial tree frog toe pad replicas
Interface Focus 5: 20140036
Figure 7. SEM images of dorsal epithelium of S. parvus digits. (a) Low-power image of several epithelial cells. (b) High-power image of cell surface and the pore of
a ( presumed) mucous gland.
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10 mm
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(a)
8
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rsfs.royalsocietypublishing.org
(c)
(d)
*
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*
2 mm
Figure 9. TEM images of toe pad epithelium of S. parvus digits. (a) Outer cell layer showing nanopillars and dense bundles of keratin tonofilaments (arrows).
(b) Border between outer cell layer on left and the second layer of cells; arrows show invaginations of cell membrane. (c) Border between cell layers II (left) and III;
arrows indicate desmosomes. (d) Lower-power image of border between layers I and II showing large vacuoles (stars) that will form the channels between the
epithelial cells when the outer layer is shed.
200 nm
Figure 10. High-power TEM image of nanopillars in S. parvus. Section of toe
pad epithelium showing nanopillars filled with tonofilaments lying at right
angles to the pad surface.
[11]. The channel structure allows draining of a wetting fluid
out of the contact surface and establishment of direct (dry)
contact in the presence of shear forces (i.e. friction). This conclusion is supported by data from live frogs studied using
interference reflection microscopy, which allows direct
measurement of the fluid layer thickness under the pad
[29]. This draining role would be expected to be more important in frogs that live in wet environments, such as rock and
torrent frogs. Indeed, Ohler [33] observed elongated epithelial cells in the toe pads of torrent frogs (Amolops spp.)
which, she hypothesized, provided shorter and straighter
channels for the removal of excess fluid from under the
pad. Elongated epithelial cells do not appear to be a
common feature of rock frog pads in spite of their habitat
in the vicinity of waterfalls. However, as shown by Endlein
et al. [34] in S. guttatus and here in S. parvus, there are channels crossing the pad ( particularly in a distal/proximal
direction as would be appropriate for water drainage) that,
while not straight, are significantly shorter than would be
found in an array of regular hexagons (figure 3a). A role in drainage of excess fluid thus seems probable. Although the
presence of a deep circumferal groove (figure 2b) is not
unique to rock frogs (WJP Barnes 2012, unpublished data on
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1 mm
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9
2
Z range: 1.591 mm
2300
(mm)
2200
1
2100
1.2
2000
1900
(mm)
0
2.5
(mm)
0
0.8
5.0
0.4
0
height (nm)
35
30
25
20
25
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15
0.4
1.2
2.8
2.0
position (mm)
3.6
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Figure 11. AFM images of toe pad epithelium cells of S. parvus. (a) Topographical image at the microscale. (b) Topographical image of the toe pad surface
nanostructures. The horizontal line indicates the position of the height profile shown in (c). (Online version in colour.)
rhacophorid tree frogs), this groove, which surrounds the distal
and lateral parts of the pads, is ideally placed to channel fluid
around rather than under the pad.
Studies on toe pad mimics have also investigated the
importance of aspect ratio [11]. Microstructures with low
aspect ratio features are more resistant to bend and collapse
during shear and allow more effective establishment of
direct contact; i.e. higher friction forces are achieved. It is
thus possible that the lower aspect ratio subarticular tubercles
(epithelial cells are surrounded by shallow channels as
described above) have evolved to generate high friction
forces, but this remains to be tested experimentally.
Ventral surfaces are also characterized by the presence
of nanopillar arrays that cover the surface. These arrays are
particularly dense on the toe pads, but less so on the subarticular tubercles and unspecialized ventral toe epithelium
(figures 3c,d; 4; 5b and 6b). They are unlikely to play a role in
capillarity as such forces are generated by the meniscus (air–
water interface) around the edge of the pad [9], but a role in
friction and/or viscous flow between adhering surfaces
seems likely. Indeed, the thickness of the fluid layer under
the pad is such that the tops of the nanopillars are likely to
make direct dry contact with the substrate. This is shown
both by cryo-SEM images such as figure 4a and by calculations
of the thickness of the fluid layer thickness using interference
reflection microscopy [29]. Also, as noted previously in a tree
frog [23], the tops of the nanopillars are clearly concave
(figure 4b, a cryo-SEM image and figure 11c, the AFM height profile), with a dimple depth in the range 6–8 nm, similar to those
measured in tree frogs [22,23]. The reason for this concavity is
unclear, but it is noteworthy that the shape (flat, T-shaped, concave) of the tops of fibrillar microstructures can significantly
affect both adhesion and friction [11,46]. Varenberg and Gorb
propose that, under appropriate circumstances, each element
can act as a passive suction device, but this is disputed by
Drotlef et al., who favour a combination of capillary and direct
contact forces.
Interestingly, nanopillar arrays have also been reported in clingfish, where they occur around the margin of the
ventrally placed suction disc. Clingfish use their adhesive
disc to stick under water to many different kinds of surfaces
and, surprisingly as they adhere by suction, adhesion is not
hindered by surface roughness [47]. The array of nanopillars
(called microvilli by the authors), aided by secreted mucus, is
able to maintain a good seal and resist frictional forces, even
on the roughest of surfaces. As S. parvus lives in and around
fast-flowing water, it is unsurprising that the nanopillar
arrays in these frogs are present not only on the toe pads
and subarticular tubercles but also on unspecialized ventral
epithelium in the toe region. Indeed, as Endlein et al. [34]
have shown for S. guttatus, most of the ventral body surface
is used when they are clinging onto rough surfaces under
high rates of water flow.
Material properties are also critical for adhesion and friction. This paper does not address this matter directly, but the
TEM results indicate that the distal regions of the pads are keratinized and that keratin tonofilaments fill the nanopillars and
the distal parts of the two outer cell layers (figures 9 and 10).
Their dense packing resembles the structure of many composite materials and would confer higher mechanical strength
and flexibility to the epithelial cells. As frog toe pads have a
low elastic modulus [23,41], such keratinization probably
serves to increase the wear resistance of the outermost part of
the toe pads. Whether the keratin tonofilaments have a particular role in adhesion, as they do in insects [26], remains to be
investigated. However, their orientation (initially normal to
the surface they become angled to the surface in a proximal
direction as shown in figure 9a) could provide the pad with
directional adhesion, so that pads pulled towards the body
increase their contact with the substrate, while movements in
the other direction cause detachment.
The TEM images of the different cell layers within the toe
pad epithelium (figure 9a –d) are broadly similar to those of
equivalent studies on hylid and rhacophorid tree frogs
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Ethics statement. The frogs were sacrificed for these studies by submersion in water containing a lethal dose of benzocaine, in accordance
with current laws on animal experimentation in the UK.
Acknowledgements. We thank Margaret Mullin of Glasgow University’s
Life Sciences Electron Microscopy Facility for her expert assistance
with both the scanning and transmission electron microscopy.
Funding statement. This work was supported by grants from the
Deutsche Forschungsgemeinschaft (SPP1420 ‘Biomimetic materials
research: functionality by hierarchical structuring of materials’) to
A.d.C. and W.J.P.B. and by an STMS grant from COST Action
TD0906 on ‘Biological adhesives: from biology to biomimetics’ to
D.M.D.
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