toe pad litoria article

7/27/2019 Toe Pad Litoria Article

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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).


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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,

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th

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m

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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


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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


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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


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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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IJST (2013) 37A4: 491-499 498

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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