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7/27/2019 Toe Pad Litoria Article
1/9
I JST (2013) 37A4: 491-499
I rani an Journal of Science & Technologyhttp://ijsts.shirazu.ac.ir
Toe-pad morphology in White's tree frog,
L itori a caeru lea(Family Hylidae)
M. Nokhbatolfoghahai
Department of Biology, School of Sciences, Shiraz University, Iran
E-mail: [email protected]
Abstract
The aim of this study was to find any structural differences between the digital pads of forelimbs and hind limbs aswell as more careful investigation of the internal and external structures of the toe-pad. In this study, pad
morphology and cytology in Litoria caeruleais described using SEM, TEM and light microscopy. At the grossanatomical level, toe-pads in hind limbs were subdivided into medial and lateral parts by two large grooves. Semi-
thin sections also showed that the toe-pad epidermis in hind limbs consisted of four layers with a cuboidaloutermost layer, while the epidermis of forelimbs consisted of 3 layers with a columnar outermost layer. SEMstudy revealed two basic shapes of epidermal cells arranged very regularly across the surface of the pad:
pentagonal and hexagonal. The pentagonal mainly occupied the most distal part of the toe. Three types of mucous-secreting pores were also seen in between the epithelial cells.
Keywords: Tree frog;Litoria caerulea; toe-pad; mucous pores; morphology
1. Introduction
The presence of enlarged toe-pads
(=adhesive/digital pads) has been reported indifferent genera and species of several frog familiesto date: Hylidae [1-3], Microhylidae [4],Leptodactylidae [5], Hyperoliidae [6-7],Rhacophoridae [8-10], Ranidae [11], Centrolenidae
[5], and Dendrobatidae [12]. These specialisedexpanded toe-pads serve to increase the surfacearea of the toes, which facilitates adhesion [13] andaid climbing ability on smooth, vertical surfaces oreven sticking overhanging surfaces [1, 12, 14-19].
Despite the diversity of taxa in which toe-pads are
found, there is a high degree of similarity in toe-padcell structure among these unrelated frogs [3-5, 10-
11, 13]. This similarity of toe-pad structure amongdifferent families of anurans has been interpreted bysome authors as being a result of convergentevolution [5, 19-20]. In contrast, the morphologicalstructure involved in toe pads like folds vary inposition and shape, can be single or double etc.[11]. These structures can bear phylogenetic signaland are distinct among clades of frogs.
As presence of toe pads is linked to life in trees,shrubs or streams, there are many anuran specieswith different adaptations that have poorly-developed toe-pads or a total lack of toe-pad.
*Corresponding authorReceived: 23 October 2012 / Accepted: 24 July 2013
The surface morphologies of the digits of non-arboreal and semi-arboreal frogs may provideevidence of the possible stages leading to the
development of the adhesive toe-pads of true treefrogs. Green [5] studied the surface structure of toe-
pads in 29 species representing 17 genera of sevenfamilies of anurans, and found the transition ofdigital pad cell specialisation. The least amount ofepidermal specialisation on the digits was found inthe terrestrial toad (Bufo americanus andGastrophryne carolinensis),with non-differentiated
squamousal shape of the epidermal cells; somedifferentiation of cell morphology showing slightlyelevated cuboidal cells on the toe (Rana clamitans)toa further development of pad type was seen at thetip of the toe, although they were still not bounded
by any grooves or distinct margins, to the highlydeveloped digital pad cells (columnar) with groovesand channels in arboreal species and even in somenon-arboreal species.
Several studies have been published on thestructure of the digital pads, notably using scanningelectron microscopy. In addition, many usedtransmission electron microscopy and it issomewhat surprising that there has been so littledetailed light microscopy. Many studies also
focused on the physiological aspects of toe-padadhesion rather than concentrating on their
histology.
Although the toe pad epithelium is, indeed,different from most areas of skin, the epithelium of
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IJST (2013) 37A4: 491-499 492
subarticular tubercles, which also have an adhesivefunction, often has a very similar morphology. Thecytological analysis of the toe-pad area by electronmicroscopy revealed modified and adhesivecorresponding to those in surface morphology.
The greatest difference between the adhesive andnon-adhesive epithelia concerned the outermost cell
layer. Nevertheless, the surface cells were firmlyinterdigitated by finger-like processes withdesmosomes at the lateral lower two-thirds andclearly separated from each other at their upperthird (free apices). Different methods, includingtransmission electron microscopy and scanningelectron microscopy [21] have revealed that the
tops of the epithelial cells are not flat, but composeof a dense array, that they named nanopillars.
Green [5] reported two types of mucous poresclassed as type I (simple) and type II (modified).
Type II pores were found in most hylid speciesincludingLitoria caerulea.
The typically structured dermis containednumerous nerve fibers and was highly vascularized
at the toe pad area and consisted bundles ofcollagen, and mucoid glands [3, 10, 22].
Among the literature on tree frog toe-pad, it isshown thatLitoria is becoming a model organismfor the study of wet adhesion in amphibians, andthough there are data on aspects of toe padmorphology on this species [19, 20, 23], a studythat presents a more complete picture of toe pad
morphology in this species is lacking.
The main purpose of this article is to study thesurface and internal structure of toe-pads ultra-structurally and by semi-thin section in the speciesLitoria caerulea (White, 1790), Family HylidaeRafinesque, (1815) with emphasis on dermalstructure and some aspects of epidermal structure
including mucous glands and pores. We will alsolook at differences between fore and hind toe pads,
something that has not been attempted in any study,and that we will put the findings into the context ofprevious studies of toe pad morphology andultrastructure.
2. Materials and methods
Whites tree frogs (Litoria caerulea, family
Hylidae) were purchased from commercialsuppliers and maintained in glass vivaria at 2024C, using heat mats. The vivaria containedfoliage, dishes of Cu-free fresh water to maintain ahigh humidity, branches on which the frogs couldclimb and sphagnum moss for the frogs to burrow
into, all on a gravel base. They were fed on livehouse crickets dusted with a calcium balancer andmulti-vitamin supplement (Nutrobal, purchasedfrom Peregrine Live Foods, Ongar, Essex, England)
twice weekly.
Toe pad study was carried out on three fully adultfrogs with average snout-vent length 73 mm 1.2mm. I used the largest toe pads, namely those onthe third and fourth digits of the fore limbs and hindlimbs. Frogs were killed via a lethal dose of
Benzocaine. The toe pads were immediately cutfrom the dead frogs, washed and then fixed in 2.5%
glutaraldehyde in phosphate buffer for 24 hr at pH7.4.
2.1. Scanning and transmission electron microscopy
The glutaraldehyde-fixed samples were rinsed inphosphate-buffered sucrose, post-fixed in buffered
1% osmium tetroxide, and stained in 0.5% aqueousuranyl acetate.
For scanning electron microscopy (SEM),specimens were then dehydrated in an acetone
series and critical point-dried. Samples weremounted and gold-coated before viewing with aPhilips SEM 500 scanning electron microscope.
For transmission electron microscopy (TEM),
specimens were dehydrated in an alcohol (ratherthan acetone) series. Samples were rinsed twice in
propylene oxide to remove the alcohol, embeddedin Araldite resin and polymerised at 70C. Ultra-thin sections (6070nm) were cut on a Reichertultramicrotome. These were then mounted oncopper grids, stained with uranyl acetate (2%aqueous solution) and lead citrate, and examined
using a Philips TEM 301 transmission electron
microscope.
2.2. Light microscopic study (Semi-thin sections)
Toluidine blue staining: the glutaraldehyde-fixedtoe pads were post-fixed in 1% osmium tetroxide,
stained in 0.5% aqueous uranyl acetate, dehydratedusing an ethanol series, and embedded in Araldite
resin; sections were cut at 0.5 to 1 m, then stainedusing 1% Toluidine blue in 1% borax.
Periodic acid/Schiff staining: The semi-thinsections were deresinised in saturated sodiumethoxide for 15-20 minutes [24]. They were then
hydrated through two changes of absolute alcohol,90 %, 70% and finally washed in running water for10 minutes. Sections were treated in 1% periodic
acid for 10 minutes, and then washed in runningwater for 10 minutes. The sections were transferredto Schiffs regent for 20-30 minutes then washed inrunning water for 15-30 minutes. They were stainedin Weigerts haematoxylin for 5-10 minutes, thendehydrated, cleared in xylene and mounted in D. P.
X.Toe-pad sections were examined over a range of
magnifications using a Wild compoundphotomicroscope and selected images were
recorded using Photo-Shop v. 7 software (Adobe
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493
Systems,
3. Result
3.1. Toe-caerulea
3.1.1. Ge
Toe pa
distal enand dist
shows aIn add
lateral grpossesssubdivid
areas. Tdevelopecompare
Fig. 1. G
view). clg=lateral
margin; st
Fig. 2. Tfurrow attsample,
transverse
San Jose, C
s
ad ultrastru
neral feature
ds in Litoria
s of each diglly by a circ
eneral plan oition to theooves, toe paone or mothe toe pad s
hese furrowd in the toto those of t
neral organiza
g=circumferengroove; pp
=subarticular t
e pad with oached to glassind limb, cg
margin; vf=ve
).
tural feature
of toe pad (S
are located
t, and are delmferential g
fLitoriatoecircumferents ofLitoria
re verticalurface into m
s were seee pads ofe fore-limbs
ion of tree fro
ial groove;proximal pa
bercles; vf=ve
e large andplate. Photogr circumferen
tical furrow
in adult Lito
M)
entrally on
ineated lateraroove. Figur
ad.ial grooveay additiona
furrows whiedial and late
to be betthe hind-li(Fig. 2 and 3
g toa pad (ven
dp=distal pt; tm=transve
rtical furrow
ne small vertiphed from a lial groove; t
ia
he
lly1
ndllychral
terbs.
ral
rt;rse
calive
=
F
vgea
topa
rip
dip
tu
e
o(fdic
3.
e
th
chreoh
arc
aroc
sib
fl
g. 3.Scannin
ntral view. A:ooves have dech of the pads
e of fore limborly-developed flat.cg=circ
ge; dp=distalrt; tm=transveThere is alsostal part ofoximal part
bercles also eThe distal paidermal epit
cupied by nissures) are vstal area, whlls in the pro
1.2. Columna
Two basic sidermal cells
e surface of
lls are also oxagonal cellsgions of thecupies the mxagonal cell
e separatedannels betwe
e highly varithe toe, theannels reducA high magngle hexagon covered
attened tips, a
I
electron micr
3rd toe of hindeloped in thento a medial a
ad; lateral gror absent andmferential g
part; vf=vertise margin
a transversethe pad (th(non-adhesi
xist.t of the pad ihelia, while
rmal epiderisible betweele there is aimal area.
r epidermal c
apes, pentag are arrange
the pad. Ver
cupied someare dominantoe; while
ost distal partpoint slightl
y deep inteen the colum
ble in width.ells are tightd (Fig. 4).ification vie
al/pentagonalith nanopi
nd peg-like p
ST (2013) 37A4:
ographs of toe
limb pad; twohind limb pad,nd two lateral
oves in fore lithe pad surfacoove; cr=circ
cal furrow; p
margin, sep adhesion p
on part). S
s occupied bythe proxim
al epithelia.n columnar ctight link be
ells of the toe
onal and hex very regula
y dispersed
regions of tht and occupythe pentagonof the toe.y backwards.
cellular chanar cells of t
. In the moster and the wi
w of the su cell showsllars with
rojections (Fi
491-499
pads from
prominentseparatingarts. B: 3rd
b pad aree is smoothmferential
=proximal
rating theart) frombarticular
columnarl part is
Channelsells in thetween the
s
agonal, ofrly across
eptagonal
e toe. Thealmost allal mainlyost of theThe cells
nels. Thee toe pad
distal partths of the
face of ahe cell tosomewhat
g. 5).
7/27/2019 Toe Pad Litoria Article
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IJST (2013)
Fig. 4.Sc
pad (3rd to
toe, showi
distal toe
channels s
toward pro
Fig. 5.A:from the t
fore limb).
hexagonal
showing t(nanopillar
37A4: 491-499
nning electron
e of fore limb).
ng tight epithel
ad, indicating e
eparating the ce
ximal. mp=muc
Medium resolue-pad surface c
B: High resolu
pentagonal cell
at the cell iss)
micrograph of
A: from the m
ial cells. B: fr
pithelial cells
lls. The arrow i
ous pore
ion scanning eells (examined
ion view of the
from the 3rd
covered with p
skin surface of
ost distal part o
m the rest of
ith well-develo
figure B point
ectron microgrfrom the 3rdtoe
surface of a sin
toe of fore li
eg-like projecti
toe
f a
the
ed
ing
phof
gle
b,
ons
3.
b
mth
cp
vro
cto
Ehi
F
th
sc
m
F
frA
II
1.3. ucous
Three typestween the epiType I: por
odification; te cell faci
mparison tores which haThe type Iry tip of thendomly andthe toe, whi
nsiderable ne (Fig. 7).
ithelial cellghly micropo
g. 6. Low rese very tip of t
owing type I=circumferenti
pI=mucous po
g. 7. High r
om mucous po: type II and B:
I=mucous pore
ores
of mucous-sthelial cells.s which ar
pe II: poresg the lum
the normal se their ownores are mo distal part oery dispersedle types II a
mbers over tore size an
on the cirrated.
lution scannie distal part fr
ucous poresal groove;
es
solution scan
es (examinedtype III, mp II
type III
creting pore
simple, wi
in which then are mo
triations, anucts.
re concentratf the toe (Fily within thed III are dist
he entire sur shape were
cumferential
g electron miom a 3rdtoe o
concentrated ir=circumferen
ing electron
from 3rdtoe of=mucous pore
494
are seen
hout any
e sides ofdified in
type III:
ed on theg. 6), anddistal partributed in
ace of thevariable.
ridge are
rograph offore limb,
this area,tial ridge;
icrograph
fore limb).type II; mp
7/27/2019 Toe Pad Litoria Article
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495
3.2. Toe-
Semi-tpad epidThe out
cuboidalcolumna
epidermiThe ou
epithelialcolumnaboth fornon-livin
limbs thdenser
nanopillnot locatundernea
translocaunder thenonopillbasal par
Fig. 8. SeA: Low
showing tthe upper
sides ofshowing
area (noteLow magthe same
reductionD: Highlayers of
which icoc=colucell; em
gland; vf
The kelayer of
ad histology
in sectioninrmis in hindrmost epith
in shape an epithelial
s consists of 3termost epithcells and th epithelial climbs and hg, but stains
n in the foronofilament
rs (the distaled radially toth, where t
ted somewhacell layer; thr regions iss of the same
i-thin transveagnification
wo different spart of the pa
he pad. B: Hifferent layers
the outermostification from
structure of hi
in vertical furrmagnificationepithelial tissu
s columnar),nar epithelial
=empty muc
vertical furro
atinised layekin is seen i
in Litoria ca
(Fig. 8) shlimbs consistlial cell lay
d the innercells. In
layers.elial cell layeinnermost llls. The outnd limbs is
darker in the
elimb regionin the hi
part of the epthe proximaley extend
t spirally orerefore, the cnot in the licells.
rse sections frofrom the 3rd
apes of mucod and very de
igh magnificaof epithelial tis
cell layer whithe 3rdtoe of fd limb, apart
w in the ventof figure C,e (note the ou
cg=circumf cell; cuc=cus gland; f
as an outerall areas of
rulea (sectio
ws that thes of four layeer is large
ost layer isforelimbs,
r is of columayer is of feermost layerkeratinisedsections in hi
, demonstratid limb.
ithelial cell)part of the cfrom, but
obliquely frannels betwe
ne between
m toe-pad regioe of hind li
s glands closep groove in b
ion of figuresue of ventral
h is cuboidal).relimb indicat
from a lack o
al part of toe phowing differermost cell la
erential grooboidal epitheg=filled muc
ost non-cellua digit, but it
s)
oers.ut
ofhe
arerofndnd
nghe
areellare
menhe
on.mb
tooth
A,ad
C:ingor
ad.enter,
ve;lialus
laris
sa
cb
th
thth(
F
thth
sles
mu
sothd
gl
th
idc
filoap
glluolafmfou
own to be mdition, there
annels in thrder. In forel
e toe pad co
e outermostis area consig. 9).
g. 9. Semi-thi
e 3rd toe-pads.e epithelium
owing a largding to the
owing the st
agnification phderneath the
me cavitated ge hind limb, sdermis; emg=
and; md=muco
The underlyie ventral pa
entified by tlls within th
led and anotcated dorsalld the preseriodic acid/SThe TEM st
and maturatimen glandsten polymorrge electron tll of transparucous glandsund, generall-myelinated
I
ch thicker iis lack of
skin surfacimbs, the ski
sists of 4-5 l
pithelial cellsts of 5-6 l
transverse se
A: Low magnand dermis
vesiculatedsurface. B: Hucture of ep
otograph fromhe 3rd toe pa
ands.D: Highowing the strmpty mucous
us duct
g dermis ist of the to
e presencem. Two type
her with a l, just beneatce of mucohiff staining.dy clearly sh
n. The glandre seen to chic secretioansparent ve
ent vesicles a, a number oy containingerve fibres (
ST (2013) 37A4:
the toe-padonopillar re
e areas out on of the dor
ayers, with fl
layers. In hayers, almost
ction from do
ification photonderneath th
gland with aigh magnificaithelial tissue
the epitheliumd of hind lim
magnificationcture of epithgland; fmg=fil
highly vasc, blood ves
of nucleateds of mucous
rge centralh the dorsalus was conows differen
ular cells of sontain either
granules orsicles; howere also foundf nerve bundseveral myeliFig. 10).
491-499
region. Ingions and
f toe padal side of
at cells in
nd limbs,cuboidal
sal part of
graph fromforelimb,
clear ducttion of A,. C: Low
and dermis, showing
underneathlial tissue,ed mucous
larised inels being
red bloodland, one
avity, arepidermis,irmed by
stages of
ome widelarge andrelativelyer, glands. Near thees can benated and
7/27/2019 Toe Pad Litoria Article
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IJST (2013)
Fig. 10.Tventral pa
beside amp=muco
Many
clear dumucousepithelialwill tranalthough
mucousglands.
4. Discu
We pres
normalfrog, Litindicatestoe-pads12]. Altpads ofthe padsimilar anon-arbo
developesimilar
morpholHowever
shape ofadapted tto be adwater fro
Variouto surfagenerategravity;animalclaws; (interlock
materialsbonding,
Function
37A4: 491-499
ansmission elert of the toe-pa
ucous gland.us pore; msv=
f the glandsts. In someduct can be
cells in theport mucousthere were
ducts with n
sion
nt in this art
dhesive toe-ria caerulea.that there isof differentough fine diifferent groepitheliumross the arbo
real species [
d similar adhenvironment
gical and e, Ohler [11] h
toe pad epitho living on wptations to alm under the p
mechanismsce and toforces in thr(1) by interith that of) by frictiong and int
at points ofbetween th
ally, these b
ctron micrograd; B: mucous
cf=collagen fucous secreti
lead to the dsections, theseen amongventral surf
to the surfac very few
o clear link
icle a detaile
ads in oneOur survey
wide range oamilies of frfferences inps and speciis nonethelereal species a11]. It is likel
esive toe-padl pressures,
cological coas observed d
lial cells amet and floodelow rapid reads.
are used byvoid fallinge differentlocking thethe support,, that involrmolecular
contact, e.g.e animal a
onding mec
ph of the 3rdtoland with muc
bres; gce=glag vesicles; nb
orsal surfacedistal part othe uppermce. Such duof the toe pentrally-loca
to the muco
d description
species of tof the literatf studies onogs [1, 3-8, 1structure ines are reportss significand even in so
y that they h
s in responsedemonstrati
vergence [2ifferences in
ng ranoid frod rocks, thou
oval of exc
animals to bo. Animals cays that oppsurface ofe.g. by usees both mi
forces betwe
rimates; (3)d its supp
anisms can
e of hind limbous secreting
d cell epithelnerve bundle
byf aostctsad,ed
us
of
eerehe0-he
ed,tly
eve
tong
5].he
gsht
ess
ndanseheofroen
byrt.
be
saachfl
asg
thfrhi
ainaluin
4.
sb
eci
reTcth
fthretho
fi
foO
o
showing A: a gesicles and su
um; md= mu
parated intohering throuhesion (tree
pillarity). Stiry adhesivees and beetl
tree frogs aass hoppers).Scherge andat the similarogs and graghly optimis
hesion. Howsects do nothough manye adhesion.cluding disk-
1. General m
Hertwig and
ecialisationtween each f
pansion ofrcumferential
lative contrierefore, tmparisons oey refer to
nction in theere are manports have nere is inter-dhind limb ofFurthermore,e, different
relimb and hmar et al. [28
adhesive toe
and duct leadiporting tissues
ous duct; mn
dry adhesiogh van der
frogs anducturally, thpads (geckos) and smoo
d insects su
Gorb [26] anty between tshoppers isd (by natural
ever, many s utilize a hclimbing aniA few aniinged bats a
rphology
Sinsch [3] su
aries withinger and toe.
he pad andgroove and
ution of eey arguedtoe morpholthe same to
compared sp comparativticed this pogital variatioan individualthere is a la structure
ind limb in t] in their stud
-pads in Phy
g to a pore lo; C: a nerve b
f=myelinated
(geckos aaals forcesinsects ad
y can be dis and insectth adhesive p
ch as stick i
d Persson [2e adhesive premarkable,selection) s
ooth adhesiexagonal pamals use claals also us
nd clingfish.
ggested that
the sameThey indicat
also the foridge are rel
ch digit tothat in
ogy are onlye as having
ecies. In oure works butint and consin in the sam.k of data shof toe-pads
he same indiies on the de
lomedusa,in
496
ated on thendle found
erve fiber;
d spidersand wet
ering byided into
s such asads (such
sects and
] indicateds of treeindicatingstems for
e pads intern, ands, otherssuction,
he digital
individuald that the
m of theted to the
climbing.erspecificreliable ifthe same
literature,very few
dered thatforelimb
wing anybetween
idual. Baelopment
vestigated
7/27/2019 Toe Pad Litoria Article
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497 IJST (2013) 37A4: 491-499
the toe-pads in both forelimbs and hind limbs, andalthough they have reported details of toe-paddevelopment for both locations, there was nocomparative investigation. They concluded thatdevelopment in forelimbs was slightly more
advanced than that of hind limbs.In the toe pads of Litoria caeruleaI investigated
the differences between hind limbs and fore limbsand found the vertical furrow were more developedin hind limbs than forelimbs (Fig. 3).
Scholz et al. [20] also reported the circumferentialgroove running around the top and sides of each toepad. Although they mentioned that the twoprominent grooves develop which separate each of
the pads into a medial and two lateral parts, whichlimbs this structure (vertical furrow) is found on isunclear. Another study by Wttke, et al. [29] on thegrooves of toe pad of about 16 Litoria frogs of
different ages showed that some of them had novertical furrow but in others the number of grooves
varied from 1 to 5 and was correlated with frogsize.
Lee, et al. [9] compared the toe pads of 11 speciesof Rhacophoridae and Hylidae tree frogs. The
results indicated that the same pattern ofcircumferential groove and transverse groove on thetoe pads was found among all members ofRhacophorus, but the lateral groove was absent.The study on hyla species (Hyla chinensis) by Leeet al. [9] indicated that the transverse furrow and
lateral groove are absent and the circumferential
groove did not extend to and around the proximalmargin. Of interest was the presence of a shortvertical furrow on the hind limb, which was notseen inRhacophorus.
4.2. Epithelial layer
Figure 3 showed the most hexagonal epidermal
cells occupying the surface of the pad in theLitoriaspecies, elongated proximo-distally which pointedslightly backwards. Among the various ranoid frogsstudied by Ohler [11], some species had epidermalcell surface with regular outline on the digital pad,
but most of the ranid species had no regular outlinebut were elongated proximo-distally. Ohler [11]showed on the narrow distal side, the elongated
cells have more or less developed projections;specifically there were well developed projectionsin the genus Amolops.
Typically, the toe at the ventral side consists ofsix to eight cell layers in Hyla cinerea [2], whichdifferentiate gradually into columnar cells at the
top. The cytological analysis of the toe pad byHertwig & Sinsch [3] in marsupial frogs were alsocomprised of six cell layers.
The innermost four layers of epidermal cells of
the toe pad did not differ in structure from those of
normal skin, but in the fifth layer, the shape of cellschanged from cubical to prismatic.
However, Ba Omar [28] found the epithelial layerinPhyllomedusatrinitatisto have 12 or so layers inadult pads, which seems to be an exception among
toe-pad epithelial thicknesses.Our finding inLitoria caeruleais that the number
and the cell shape of the toe-pad epidermis layer inhind limbs are different from those of fore limbs.The hind limb consists of four layers. Theoutermost epithelial cell layer is cuboidal in shapeand the innermost layer is of columnar epithelialcells. In contrast, in forelimbs the epidermisconsists of three layers. The outermost epithelial
cell layer is columnar and the innermost layer is ofless columnar epithelial cells.
Further, we cannot exclude the possibility thatthis is related to pad size, i.e. smaller pads having
fewer layers, or it is perhaps dependent to the stageof development.
Although these variations exist between differentspecies as well as between the forelimbs and hind
limbs of any individual, there is no knownfunctional relationship for this feature either.
Green [5] suggested that if digital characters areuseful in the classification of the frogs, the accuracyof such data would be enhanced and morecharacters could be observed through the furtheruse of the scanning electron microscope. Green [5]argued that the size of the pad cells could thus be
used to distinguish between the two species with
high probability. My view is that even theoutermost columnar epithelial cells change at theirapices into peg-like projections with differentshapes (pentagonal, hexagonal or heptagonal tips)and the distribution of these columnar cells withdifferently-shaped tips can be computerised and
analysed, taking into consideration many othersurface characteristics. Hence, a way may be foundnot only to distinguish the two species, but also forpotentially distinguishing individuals in a speciesby fingertip examination. We may need to take thisin to consideration if there are minor changes in thedetailed pattern of cell shape every time the outer
cell layer is sloughed off at a molt.
4.3. Mucous pores and mucous glands
Green [5] showed that there is remarkablevariation in the numbers of mucous poresassociated with the toe-pads of various species. Lee[9] reported no mucous visible on the outermostepidermis of any tree frog he examined. Ohler [11]
argued that the prismatic cells, the channels and themucous glands are required in the humidificationmechanism necessary for sticking. On the otherhand, Nachtigall [30] showed that even distilled
water has sufficient adhesive ability as an adequate
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intermediary fluid to affect adhesion. Thus, heconcluded that mucous, or even particularly stickymucous, need not be mandatory for the functioningof the pads. Ernst [22] believed that the mucousglands present in the dermis of the digital pads are
not fundamentally different from similar glandsfound elsewhere in the skin of tree frogs. We found
three types of mucous pore (type I, II and III)dispersed on the surface of toe pads, but the specificfunction for each type of pore has not beeninvestigated so far. Although mucous pore type IIIis visible in some published micrographs, forexample, Rivero, et al. [31], Figs. 7, 8, 10, in thetoe-pad of species Eleutherodactylus coqui, this
feature has not been noted before. We also noticed asignificant number of mucous glands on the dermisof the digital pads, and it is somewhat interestingthat during the serial sectioning of the toe pad,
many of these glands were found to have their ductspointing to the dorsal surface rather than to the
ventral surface of the digital pads. For the rest ofthe glands, no clear link between the glands and the
ducts was seen. The very close location of theglands in the dermis to the dorsal surface of the toe
pad suggests that the rest of the glands may also berelated to the upper part of the pad. Noble andJaeckle [1] reported that these glands originate inthe central or upper part of the pad, continuingventrally into shorter and narrower ducts whichperforate the epidermis at an acute angle. Our PAS
staining showed some mucous-secreting cells and
micro ducts on the ventral surface of the toe pad,which can support the source of mucous secretionto the ventral surface of the toe pad, but we did notfind any link to the main glands in the dermis.
I found a dense network of capillaries and lymphsacs in the dermis beneath the toe pad. These
sinuses are believed to play an important role asshock absorbers during landing after a jump [19].
These structures produce a hydrostatic pressure fora safe landing, but may also make the toe padsufficiently flexible to transform the pad to asuction form for better adhesion when the blooddrains away to the main circulatory system by
pressing the toe pad to the surface.SEM and semi-thin sections indicate that the hind
limb digital toe-pad is deeply grooved in lateral
parts as a marker to diagnose hind limb fromforelimb digital pads, but it also increases thesurface of the digital pad in hind limbs, probablyfor stronger adhesion. Having such grooves allowslarger pads to conform better to surfaceirregularities.
Hydrostatic pressure induced by the sinus couldalso help to expand these, folding when necessary.Apart from this suggested role, the grooves beingthe same as other characters on the toe pad could be
considered as species recognition.
5. Conclusion
Fine differences in structure exist between the pads
of forelimbs and hind limbs in Litoria caeruleaspecies in the extent and existence of the
circumferential groove, and in the organisation ofthe surface-cell epithelium.The number, type and distribution of mucous
pores, and the number of epithelial layer are alsodifferent from those of other hylid species.
Together, all these studies indicate that anuranadhesive pads are a remarkable example of
convergent evolution, but some prominentcharacteristics of the toe pads still need to beclarified in terms of the relation between theirforms and their functions. In general, apart fromdifferent structure and function of anuran adhesivepad, there is various adhesive systems and
mechanisms among the animal kingdom, someexamples have been given in the discussion part.
Acknowledgements
The author thanks Dr. Jon Barnes at the Universityof Glasgow, UK for his help in accessing andstudying specimens, also for very usefulcommenting on the draft of this paper. The author
also thanks Margaret Mullin for technical assistanceand advice. Finally thanks to Diana Samuel for helpwith photographing. MNs field work wassupported by Shiraz University.
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