Morphological studies of the toe pads of the rock frog, …rsfs.royalsocietypublishing.org/content/royfocus/5/1/... · Morphological studies of the toe pads of the rock frog, Staurois

on May 21, 2018http://rsfs.royalsocietypublishing.org/Downloaded from

rsfs.royalsocietypublishing.org

ResearchCite 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]

& 2014 The Author(s) Published by the Royal Society. All rights reserved.

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 ofthe rock frog, Staurois parvus ( family:Ranidae) and their relevance to thedevelopment of new biomimeticallyinspired reversible adhesives

Dirk M. Drotlef1, Esther Appel2, Henrik Peisker2, Kirstin Dening2,Aranzazu del Campo1, Stanislav N. Gorb2 and W. Jon. P. Barnes3

1Max Planck Institut fur Polymerforschung, Mainz, Germany2Functional Morphology and Biomechanics, University of Kiel, Kiel, Germany3Centre for Cell Engineering, University of Glasgow, Scotland, UK

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 epi-

thelium 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, indi-

cating 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 rel-

evance of these findings to the development of new biomimetically inspired

reversible adhesives is discussed.

1. IntroductionIn 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).

rsfs.royalsocietypublishing.orgInterface

Focus5:20140036

2

on May 21, 2018http://rsfs.royalsocietypublishing.org/Downloaded from

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 sur-

face 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 compara-

ble 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 dif-

ferent 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 pres-

ence 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, downward-

facing 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 mor-

phology 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

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.

2. Material and methods2.1. AnimalsThe frog species of our study was the Lesser rock frog, S. parvus(family Ranidae), a native of Borneo. The frogs (N ¼ 5) were sub-

adult with a snout–vent length of 30 mm. They were donated by

Schonbrunn Zoo (Vienna, Austria).

2.2. Sample preparation for scanning electronmicroscopy

Frog toes were cut off with a scalpel and fixed in 2.5% glutaral-

dehyde 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 pro-

cessing 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 electronmicroscopy

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

1 mm

Figure 1. SEM image of forelimb of S. parvus; ventral view showing toe padsand subarticular tubercles (arrows). The ‘thumb’ (digit 1) is the small pad onthe right hand side.

rsfs.royalsocietypublishing.orgInterface

Focus5:20140036

3

on May 21, 2018http://rsfs.royalsocietypublishing.org/Downloaded from

tetroxide, en bloc staining with uranyl acetate and washing in dis-

tilled 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 formvar-

coated 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 micro-

scope (Carl Zeiss Ltd, Cambridge, UK), running at 120 kV. Images

were captured using an Olympus iTEM soft imaging system.

2.4. Sample preparation for cryo-SEMFrog 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., Abing-

don, 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 speci-

mens 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 microscopyThe 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-nitride-

probe cantilever (silicon nitride SPM-Sensor, NanoWorld AG,

Neuchatel, Switzerland) in intermittent (tapping) mode. The resol-

ution 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. Results3.1. Toe morphology (SEM and cryo-SEM)3.1.1. Toe padsThe 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

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 prox-

imal 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 cellsAs 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 prob-

ably 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 hexago-

nal 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

approximatelyanterior–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 signed-

rank 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 epithe-

lial cells meet. The channel walls can be seen to be ridged, the

200 mm 100 mm

ventral

(a) (b)

dorsal

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)

100 mm

1 mm 500 nm

10 mm

(b)

(c) (d )

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 (mainlyhexagonal in shape) surrounded by deep channels with a single mucous pore (centre). (c) Edge of a pad epithelial cell showing dense array of nanopillars coveringthe pad surface. (d ) High-power view of nanopillars.

rsfs.royalsocietypublishing.orgInterface

Focus5:20140036

4

on May 21, 2018http://rsfs.royalsocietypublishing.org/Downloaded from

ridges probably reflecting the presence of bundles of keratin

tonofilaments beneath the surface.

3.1.3. Toe pad surface nanopattern—nanopillarsAs in tree frogs, the surface of the epithelial cells is covered in a

dense array of nanopillars that form a surface nanopattern con-

sisting 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, trans-

mission 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 tono-

filaments from immunohistochemical studies on other frog

species (e.g. [36]), are visible through the walls of the nanopil-

lars 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 tuberclesProximal 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

(a)

2 mm

1 mm

(b)

Figure 4. High-power images of nanopillars in S. parvus. Cryo-SEM images ofunspecialized ventral epithelium showing (a) the tops of the nanopillars sur-rounded by frozen mucus and (b) fibrils (bundles of tonofilaments) visiblethrough the walls of the nanopillars (arrows).

rsfs.royalsocietypublishing.orgInterface

Focus5:20140036

5

on May 21, 2018http://rsfs.royalsocietypublishing.org/Downloaded from

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 shal-

lower 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 epi-

thelium. 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 epitheliumExamination 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 sur-

face is covered by nanopillars, but at lower density than in the

more specialized epithelia of the toe pads and subarticular tuber-

cles. 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 ear-

lier (figure 4a,b) were taken from this area, their lower density

making structural features easier to see and measure.

3.1.6. Dorsal toe epitheliumIn contrast to the ventral epithelium, the epithelium of the

dorsal surface of the toes has neither nanopillars nor intercellu-

lar 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].

3.2. Toe pad anatomy (light microscopy and TEM)3.2.1. Epithelial cell layersExamination 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, consist-

ing 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. Per-

ipheral 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 intercellu-

lar channels when the second layer becomes the outer layer,

through the shedding of the outer layer. By contrast, unspecia-

lized ventral epithelium and the dorsal epithelium of the toe

are only 15–20 mm thick (figures 2b and 8b). The dorsal epi-

thelium has no channels, while the channels separating the

outer portions of the outer layer of cells in unspecialized ven-

tral toe epithelium are only about 1 mm deep. The extent of

the keratinization is much reduced. The epithelium of the sub-

articular tubercles is broadly similar to that of the toe pads,

though its thickness is reduced (data not shown).

3.2.2. Transmission electron microscopyThe electron microscope reveals a lot more detail of the ultra-

structure 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 differ-

ent 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 dark-

staining 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,

(a)

100 mm 500 nm

(b)

(c)

10 mm 10 mm

(d )

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, intercellularchannel (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 oftubercle 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)

5 mm 500 nm

(b)

Figure 6. SEM images of nanopillar array and intercellular channels of unspecialized ventral epithelium of S. parvus digits. (a) Low power; arrow indicates openingof mucous gland. (b) High power.

rsfs.royalsocietypublishing.orgInterface

Focus5:20140036

6

on May 21, 2018http://rsfs.royalsocietypublishing.org/Downloaded from

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 invagi-

nated, 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 surfaceof the toe pads

3.3.1. Cell surface probed by the atomic force microscopyThe 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, there-

fore, the depth of the channels could not be estimated.

Figure 11b shows the nanostructures on the epithelial cell sur-

faces. 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

(a)

10 mm 2 mm

(b)

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 ofa ( presumed) mucous gland.

(a)

10 mm 10 mm

(b)

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 channelsbetween the outer portions of the outer cell layer (arrows). (b) Unspecialized ventral epithelium (near proximal groove) showing small shallow grooves (arrows) betweenthe outer portions of the outer cell layer.

rsfs.royalsocietypublishing.orgInterface

Focus5:20140036

7

on May 21, 2018http://rsfs.royalsocietypublishing.org/Downloaded from

centre of the nanopillar. Our best data give a figure for the

depth of the dimple in the range 6–8 nm.

4. DiscussionThe 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 cur-

rent 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 pat-

tern 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 sur-

rounded 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 epi-

thelial 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 interrup-

tions of the hexagonal pattern to account for curvature,

confined boundary conditions and transition zones from smal-

ler 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 heptago-

nal 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 tor-

rent frogs such as Amolops spp. [33], in rock frogs of the genus

Staurois ([34] and this study) and also, somewhat surpris-

ingly, 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 epi-

thelium, 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

(a)

1 mm

1 mm 2 mm*

*

1 mm

(b)

(c) (d )

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 theepithelial cells when the outer layer is shed.

200 nm

Figure 10. High-power TEM image of nanopillars in S. parvus. Section of toepad epithelium showing nanopillars filled with tonofilaments lying at rightangles to the pad surface.

rsfs.royalsocietypublishing.orgInterface

Focus5:20140036

8

on May 21, 2018http://rsfs.royalsocietypublishing.org/Downloaded from

[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 con-

clusion 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 impor-

tant in frogs that live in wet environments, such as rock and

torrent frogs. Indeed, Ohler [33] observed elongated epi-

thelial 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 chan-

nels 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 drai-

nage 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

Z range: 1.591 mm

(a) (b)

(c)

25

2

1

0

15

20

25

30

35

0

0.4 1.2 2.0position (mm)

heig

ht (

nm)

2.8 3.6 4.4

2.5(mm)

(mm

)

5.0

1900

2000

2100

2200

2300

0 25

1.2

0.8

0.4

(mm)

(mm

)

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 surfacenanostructures. The horizontal line indicates the position of the height profile shown in (c). (Online version in colour.)

rsfs.royalsocietypublishing.orgInterface

Focus5:20140036

9

on May 21, 2018http://rsfs.royalsocietypublishing.org/Downloaded from

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 sub-

articular 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 pro-

file), 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, con-

cave) 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 repor-

ted 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 fric-

tion. This paper does not address this matter directly, but the

TEM results indicate that the distal regions of the pads are ker-

atinized 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 compo-

site 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 particu-

lar 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

rsfs.royalsocietypublishing.orgInterface

Focus5:20140036

10

on May 21, 2018http://rsfs.royalsocietypublishing.org/Downloaded from

[12,14,20,21]. For example, in all groups, it appears that the

outer cell layer consists of dying or dead cells and is highly

keratinized, that nanopillars originate from desmosomes,

and that the keratin filaments are linked to desmosomes.

Our images also indicate that the specialized outer layer

develops in a similar way to the tree frogs. The degree of

evolutionary convergence is thus remarkable, though it is

probable that most of the underlying mechanisms (e.g. mech-

anisms of keratinization) will have been present in common

ancestors of these groups. Thus, in our view, it is the detailed

structure of the toe pad that arises by convergence in the

different frog families and that it does so predominantly

using pre-existing cellular mechanisms.

The adhesive mechanisms of climbing animals such as

geckos, anoles and skinks among reptiles, tree, torrent and rock

frogs among amphibians, and a wide variety of insects have

many features that are of interest to materials scientists. These

include the combination of good adhesion with easy detachment,

the fact that the adhesive can be used repeatedly without dete-

riorating, the ability to self-clean (e.g. [48]) and thus quickly

and efficiently remove attached dirt particles, and the property

of only sticking when required [2,4]. The amphibians additio-

nally have the ability to adhere under wet conditions. Thus,

understanding the adhesion principles behind the structural fea-

tures described in this paper will enhance the development of

novel adhesion strategies for reversible attachment in artificial

systems. The initial work on the tree/rock/torrent frog system

has already begun with theoretical studies of capillary adhesion

of soft, deformable surfaces [49,50] and nanofabrication and

testing of hexagonal patterns of cells surrounded by channels

(Varenberg & Gorb [46,51], whose work was insect-inspired,

and Drotlef et al. [11], who systematically examined the adhesive

and frictional properties of tree frog toe pad analogues under a

variety of conditions). More generally, studies on bioinspired

adhesive materials are increasingly combining materials with

very different properties in the development of new hybrid

adhesive surfaces (e.g. [52,53]). In respect of the frog adhesion

research, possible applications include improved design in wet

weather tyres [35,54,55], non-slip footwear, plasters for surgery

able to adhere to tissue, holding devices for surgery and the

assembly of functional elements in miniature electronic devices

(microelectromechanical systems). As both Barnes et al. [22] and

this paper make clear, both nanopillars and fibrils will also

need to be considered if the full biomimetic potential of the

rock frog adhesive system is to be realized.

Ethics statement. The frogs were sacrificed for these studies by submer-sion in water containing a lethal dose of benzocaine, in accordancewith current laws on animal experimentation in the UK.

Acknowledgements. We thank Margaret Mullin of Glasgow University’sLife Sciences Electron Microscopy Facility for her expert assistancewith both the scanning and transmission electron microscopy.

Funding statement. This work was supported by grants from theDeutsche Forschungsgemeinschaft (SPP1420 ‘Biomimetic materialsresearch: functionality by hierarchical structuring of materials’) toA.d.C. and W.J.P.B. and by an STMS grant from COST ActionTD0906 on ‘Biological adhesives: from biology to biomimetics’ toD.M.D.

References

1. Duellman W, Trueb L. 1997 Biology of amphibians,2nd edn. Baltimore, MA: Johns Hopkins UniversityPress.

2. Barnes WJP. 2007 Biomimetic solutions to stickyproblems. Science 318, 203 – 204. (doi:10.1126/science.1149994)

3. Creton C, Gorb S. 1997 Sticky feet: from animals tomaterials. MRS Bull. 32, 466 – 468. (doi:10.1557/mrs2007.79)

4. Autumn K. 2007 Gecko adhesion: structure, functionand applications. MRS Bull. 32, 473 – 478. (doi:10.1557/mrs2007.80)

5. Bhushan B. 2007 Adhesion of multi-levelhierarchical attachment systems in gecko feet.J. Adhes. Sci. Technol. 21, 1213 – 1258. (doi:10.1163/156856107782328353)

6. del Campo A, Greiner C, Arzt E. 2007 Contactshape controls adhesion of bioinspired fibrillarsurfaces. Langmuir 23, 10235 – 10243. (doi:10.1021/la7010502)

7. Zhao B, Pesika N, Zeng H, Wei Z, Chen Y, Autumn K,Turner K, Israelachvili J. 2009 Role of tilted adhesionfibrils (setae) in the adhesion and locomotion ofgecko-like systems. J. Phys. Chem. B 113,3615 – 3621. (doi:10.1021/jp806079d)

8. Barnes WJP. 2007 Functional morphologyand design constraints of smooth adhesivepads. MRS Bull. 32, 479 – 485. (doi:10.1557/mrs2007.81)

9. Barnes WJP. 2012 Adhesion in wet environments—frogs. In Encyclopedia of nanotechnology, part 2 (ed.B Bhushan), pp. 70 – 83. Berlin, Germany: Springer.

10. Gupta R, Frechette J. 2012 Measurement andscaling of hydrodynamic interactions in the presenceof draining channels. Langmuir 28, 14 703 – 14 712.(doi:10.1021/la303508x)

11. Drotlef D-M, Stepien L, Kappl M, Barnes WJP, ButtH-J, del Campo A. 2013 Insights into the adhesivemechanisms of tree-frogs using artificial mimics.Adv. Funct. Mater. 23, 1137 – 1146. (doi:10.1002/adfm.201202024)

12. Ernst V. 1973 The digital pads of the tree frog, Hylacinerea. I. The epidermis. Tissue Cell 5, 83 – 96.(doi:10.1016/S0040-8166(73)80007-2)

13. Ernst V. 1973b The digital pads of the tree frog,Hyla cinerea. II. The mucous glands. Tissue Cell 5,97 – 104. (doi:10.1016/S0040-8166(73)80008-4)

14. Welsch U, Storch V, Fuchs W. 1974 The finestructure of the digital pads of rhacophorid treefrogs. Cell Tiss. Res. 148, 407 – 416. (doi:10.1007/BF00224267)

15. Green DM. 1979 Treefrog toe pads: comparativesurface morphology using scanning electronmicroscopy. Can. J. Zool. 57, 2033 – 2046. (doi:10.1139/z79-268)

16. McAllister A, Channing C. 1983 Comparisons oftoepads of some Southern African climbing frogs.S. Afr. Zool. 18, 110 – 114.

17. Green DM, Simon P. 1986 Digital microstructure inecologically diverse microhylid frogs generaCophixalus and Sphenophryne (Amphibia: Anura)from Papua New Guinea. Aust. J. Zool. 34,135 – 145. (doi:10.1071/ZO9860135)

18. Hertwig I, Sinsch U. 1995 Comparative toe padmorphology in marsupial frogs (genus Gastrotheca):arboreal versus ground-dwelling species. Copeia 1,38 – 47. (doi:10.2307/1446798)

19. Ba-Omar TA, Downie JR, Barnes WJP. 2000Development of adhesive toe-pads in the tree frog(Phyllomedusa trinitatis). J. Zool. Lond. 250,267 – 282. (doi:10.1111/j.1469-7998.2000.tb01077.x)

20. Mizuhira V. 2004 The digital pads of rhacophoridtree-frogs. J. Electron Microsc. 53, 63 – 78. (doi:10.1093/jmicro/53.1.63)

21. Chakraborti S, Das D, De SK, Nag TC. 2014 Structuralorganization of the toe pads in the amphibianPhilautus annadalii (Boulenger, 1906). Acta Zool.(Stockholm) 95, 63 – 72. (doi:10.1111/azo.12008)

22. Barnes WJP, Baum M, Peisker H, Gorb SN. 2013Comparative cryo-SEM and AFM studies of hylid andrhacophorid tree frog toe pads. J. Morphol. 274,1384 – 1396. (doi:10.1002/jmor.20186)

23. Scholz I, Barnes WJP, Smith JM, Baumgartner W.2009 Ultrastructure and physical properties of anadhesive surface, the toe pad epithelium of the treefrog, Litoria caerulea White. J. Exp. Biol. 212,155 – 162. (doi:10.1242/jeb.019232)

rsfs.royalsocietypublishing.orgInterface

Focus5:20140036

11

on May 21, 2018http://rsfs.royalsocietypublishing.org/Downloaded from

24. Gorb SN, Jiao Y, Scherge M. 2000 Ultrastructuralarchitecture and mechanical properties ofattachment pads in Tettigonia viridissima(Orthoptera Tettigoniidae). J. Comp. Physiol. A 186,821 – 831. (doi:10.1007/s003590000135)

25. Perez-Goodwyn PJ, Peressadko A, Schwarz H,Kastner V, Gorb SN. 2006 Material structure,stiffness, and adhesion: why attachment pads of thegrasshopper (Tettigonia viridissima) adhere morestrongly than those of the locust (Locustamigratoria) (Insecta: Orthoptera). J. Comp. Physiol. A192, 1233 – 1243. (doi:10.1007/s00359-006-0156-z)

26. Dirks J-H, Li M, Kabla A, Federle W. 2012 In vivodynamics of the internal fibrous structure in smoothadhesive pads of insects. Acta Biomater. 8,2730 – 2736. (doi:10.1016/j.actbio.2012.04.008)

27. Emerson SB, Diehl D. 1980 Toe pad morphology andmechanisms of sticking in frogs. Biol. J. Linn. Soc. 13,199 – 216. (doi:10.1111/j.1095-8312.1980.tb00082.x)

28. Hanna G, Barnes WJP. 1991 Adhesion anddetachment of the toe pads of tree frogs. J. Exp.Biol. 155, 103 – 125.

29. Federle W, Barnes WJP, Baumgartner W, DreschlerP, Smith JM. 2006 Wet but not slippery: boundaryfriction in tree frog adhesive toe pads. J. R. Soc.Interface 3, 589 – 601. (doi:10.1098/rsif.2006.0135)

30. Barnes WJP, Oines C, Smith JM. 2006 Whole animalmeasurements of shear and adhesive forces in adulttree frogs: insights into underlying mechanisms ofadhesion obtained from studying the effects of sizeand scale. J. Comp. Physiol. A 192, 1179 – 1191.(doi:10.1007/s00359-006-0146-1)

31. Barnes WJP, Pearman J, Platter J. 2008 Applicationof peeling theory to tree frog adhesion, a biologicalsystem with biomimetic implications. Eur. Acad. Sci.E-Newsletter Sci. Technol. 1, 1 – 2.

32. Endlein T, Ji A, Samuel D, Yao N, Wang Z, Barnes WJP,Federle W, Kappl M, Dai Z. 2013 Sticking like stickytape: tree frogs utilise friction forces to enhanceattachment to overhanging surfaces. J. R. Soc.Interface 10, 20120838. (doi:10.1098/rsif.2012.0838)

33. Ohler A. 1995 Digital pad morphology in torrent-living Ranid frogs. Asiatic Herpetol. Res. 6, 85 – 96.

34. Endlein T, Barnes WJP, Samuel D, Crawford N, BiawAE, Grafe U. 2013 Sticking under wet conditions: theremarkable attachment abilities of the torrent frog,Staurois guttatus. PLoS ONE 8, e73810. (doi:10.1371/journal.pone.0073810)

35. Barnes WJP, Smith J, Oines C, Mundl R. 2002 Bionicsand wet grip. In Tire Technol. Int. December ‘02, pp.56 – 60. Dorking, UK: UK & International Press.

36. Alibardi L. 2001 Keratinization in the epidermis ofamphibians and the lungfish: comparison withamniote keratinization. Tissue Cell 33, 439 – 449.(doi:10.1054/tice.2001.0198)

37. Spinner M, Westhoff G, Gorb SN. 2013 Subdigitaland subcaudal microornamentation inchamaeleonidae—a comparative study. J. Morphol.274, 713 – 723. (doi:10.1002/jmor.20137)

38. Alibardi L. 2010 Cornification of the beak ofRana dalmatina tadpoles suggests the presenceof basic keratin-associated proteins. Zool. Stud. 49,51 – 63.

39. Smith JM. 2003 Effects of allometric growth and toepad morphology on adhesion in hylid tree frogs. PhDthesis, University of Glasgow, Glasgow, UK.

40. Smith JM, Barnes WJP, Downie JR, Ruxton GD. 2006Structural correlates of increased adhesive efficiencywith adult size in the toe pads of hylid tree frogs.J. Comp. Physiol. A 192, 1193 – 1204. (doi:10.1007/s00359-006-0151-4)

41. Barnes WJP, Perez Goodwyn PJ, NokhbatolfoghahaiM, Gorb SN. 2011 Elastic modulus of tree frogadhesive pads. J. Comp. Physiol. A 197, 969 – 978.(doi:10.1007/s00359-011-0658-1)

42. Lee KC, Erb U. 2013 Grain boundaries andcoincidence site lattices in the corneal nanonipplestructure of the mourning cloak butterfly.Beilstein J. Nanotechnol. 4, 292 – 299. (doi:10.3762/bjnano.4.32)

43. von Frisch E. 1974 Animal architecture. New York,NY: Harcourt Brace Jovanovitch.

44. Capinera JL. 2008 Encyclopedia of entomology, 2ndedn. Heidelberg, Germany: Springer.

45. Fahrenbach WH, Knutson DD. 1975 Surfaceadaptations of the vertebrate epidermis to friction.

J. Invest. Dermatol. 65, 39 – 44. (doi:10.1111/1523-1747.ep12598036)

46. Varenberg M, Gorb SN. 2008 A beetle-inspiredsolution for underwater adhesion. J. R. Soc. Interface5, 383 – 385. (doi:10.1098/rsif.2007.1171)

47. Wainwright DK, Kleinteich T, Kleinteich A, Gorb SN,Summers AP. 2013 Stick tight: suction adhesion onirregular surfaces in the northern clingfish. Biol.Lett. 9, 20130234. (doi:10.1098/rsbl.2013.0234)

48. Crawford N, Endlein T, Barnes WJP. 2012 Self-cleaning in tree frog toe pads; a mechanism forrecovering from contamination without the need forgrooming. J. Exp. Biol. 215, 3965 – 3972. (doi:10.1242/jeb.073809)

49. Butt H-J, Barnes WJP, del Campo A, Kappl M,Schoenfeld F. 2010 Capillary forces between soft,elastic spheres. Soft Matter 6, 5930 – 5936. (doi:10.1039/c0sm00455c)

50. Zakerin M, Kappl M, Backus EHG, Butt H-J,Schonfeld F. 2013 Capillary forces between rigidspheres and elastic supports: the role of Young’smodulus and equilibrium vapour adsorption. SoftMatter 9, 4534 – 4543. (doi:10.1039/c3sm27952a)

51. Varenberg M, Gorb SN. 2009 Hexagonal surfacemicropattern for dry and wet friction. Adv. Mater.21, 483 – 486. (doi:10.1002/adma.200802734)

52. Ruffato III D, Parness A, Spenko M. 2014 Improvingcontrollable adhesion on both rough and smoothsurfaces with a hybrid electrostatic/gecko-likeadhesive. J. R. Soc. Interface 11, 20131089. (doi:10.1098/rsif.2013.1089)

53. Shahsavan H, Zhao B. 2014 Bioinspired functionallygraded adhesive materials: synergetic interplay oftop-viscous-elastic layers with base micropillars.Macromolecules 47, 353 – 364. (doi:10.1021/ma4018718)

54. Barnes WJP. 1999 Tree frogs and tire technology. InTire Technol. Int. March ‘99, pp. 42 – 47. Dorking,UK: UK & International Press.

55. Persson BNJ. 2007 Wet adhesion with application totree frog adhesive toe pads and tires. J. Phys. Cond.Matter 19, 376110. (doi:10.1088/0953-8984/19/37/376110)