Surface Ciliation of Anuran Amphibian Larvae: Persistence to Late Stages in Some Species but Not...

Surface Ciliation of Anuran Amphibian Larvae:Persistence to Late Stages in Some Species but NotOthersM. Nokhbatolfoghahai,1* J.R. Downie,2 and V. Ogilvy2

1Biology Department, Faculty of Sciences, University of Shiraz, Shiraz 71345, Iran2Division of Environmental and Evolutionary Biology, Graham Kerr Building, University of Glasgow, Glasgow G128QQ, Scotland, UK

ABSTRACT Scanning electron microscopy was used toexamine the surfaces of 21 species of tadpoles from sixfamilies, from Gosner Stage 25/26 until close to metamor-phosis. Contrary to most previous reports, ciliated epider-mal cells persisted until late stages in many but not allspecies and not at all locations examined. The commonestlocation for ciliated cells was around the nostrils, suggest-ing a role in chemosensation. Ciliated cells also occurredaround the circumference of the eye, suggesting a cleaningrole. Several species had ciliated cells on the tail. Thedensest, most regular arrays of ciliated cells occurred inspecies that tend to hang motionless in still-water pools,suggesting a respiratory function for these cells. J. Mor-phol. 267:12481256, 2006. 2006 Wiley-Liss, Inc.

KEY WORDS: anuran; tadpole; surface ciliation; interspe-cic differences; respiration; chemosensation

Many kinds of invertebrate embryos and larvaepossess surface ciliated cells, which function in lterfeeding and locomotion (Nielsen, 1987). In the ver-tebrates, abundant embryonic/larval surface cilia-tion has been reported only in lungsh (Whiting andBone, 1980) and amphibians (Kessel et al., 1974),although cells with isolated, transient cilia havebeen found in dogsh (Nokhbatolfoghahai andDownie, 2003), the chick blastoderm (Bancroft andBellairs, 1974), the early mouse embryo (Sulik et al.,1994), and Xenopus laevis and zebrash embryos(Essner et al., 2002). At least in some cases, tran-sient embryonic monociliated cells have a role inleftright patterning (Brueckner, 2001).

Surface ciliation on the embryos and early larvaeof urodele and anuran amphibians has been knownsince the late 19th century, with the rst detailedinvestigation of their distribution and functions be-ing Asshetons (1896) study on Rana temporaria.Scanning electron microscopy made it feasible tomap the distribution of surface ciliation stage bystage in different species (Steinman, 1968: Xenopuslaevis; Billett and Courtenay, 1973; Lofberg, 1974;Landstrom, 1977: Ambystoma mexicanum; Kessel etal., 1974: Rana pipiens; Nokhbatolfoghahai et al.,2005: a 20-species study across six anuran families).

Several functions, not necessarily mutually exclu-sive, have been suggested for amphibian embryo andearly larval ciliation. These are: preventing micro-organisms and debris from attaching to the epider-mis; movement of the body, including prehatchingrotation and posthatching gliding; respiratory gasexchange; movement of surface mucus lms; andgeneration of water currents providing chemical in-formation about the surrounding environment.

Nokhbatolfoghahai et al. (2005) used the datafrom their 20-species comparison to assess thesepossible functions. They concluded that respirationis the main function of surface ciliation, but thatposthatching gliding over the substratum may alsobe important. In addition, their discovery of persis-tent dense ciliation around the external nares sup-ported a chemosensory role.

Kessel et al. (1974) reported that Rana pipienssurface ciliation rst appeared during neurulationand disappeared soon after hatching. However, As-sheton (1896) claimed that ciliation on the tails of R.temporaria persisted until just before metamorpho-sis. Based solely on morphological observations,Kessel et al. (1974) suggested that the ciliated epi-dermal cells in R. pipiens lost their cilia graduallythrough the conversion of ciliated cells to nonciliatedmucus-secreting epidermal cells. This pattern of de-velopment primarily suggests that the surface ciliahave a functional role restricted to early develop-ment.

Nokhbatolfoghahai et al. (2005) described cilia-tion patterns for most species up until Gosner (1960)Stage 27, which is well past hatching in most cases

Contract grant sponsors: Carnegie Trust; University of Glasgow (toJ.R.D.).

*Correspondence to: Dr. M. Nokhbatolfoghahai, Biology Depart-ment, Faculty of Sciences, University of Shiraz, Shiraz 71345, Iran.E-mail: [email protected]

Published online 2 August 2006 inWiley InterScience (www.interscience.wiley.com)DOI: 10.1002/jmor.10469

JOURNAL OF MORPHOLOGY 267:12481256 (2006)

2006 WILEY-LISS, INC.

(Stages 1721 in the majority of species described)but also well before metamorphosis (onset at Stage42). They found that in most cases surface ciliationwas greatly reduced by Stages 2627 over most bodyregions, except around the external nares. However,their sampling of these later stages was incomplete,and a few species showed late persistence of ciliationon the tail and other regions.

The aim of the work reported here was to examinemore systematically the occurrence of surface cilia-tion in anuran larvae up until metamorphosis, inorder to assess whether ciliation may have a role inthese later stages.

In this present work the epidermal ciliary patternwas examined in 21 species belonging to six familiesof anurans (Table 1). Larvae were studied fromStage 26 (after the operculum closed) to stages closeto metamorphosis. Ciliated cell patterns at nine lo-cations on the body surface were examined and re-corded using scanning electron microscopy (SEM).

MATERIALS AND METHODSEgg Collection, Incubation, and Fixation

All spawn and tadpoles were collected from eld sites in Trin-idad, Scotland, and Iran. The exception was Xenopus laevis, pro-vided from a captive population maintained at the University ofSt. Andrews.

The 21 species examined are shown in Table 1. Ecologicaland tadpole type data on Trinidad species were extracted from

Kenny (1969) and Murphy (1997) or personal observations.Most tadpoles were of the standard polliwog type: globose body;oral disc with beak and tooth rows; and tail tapering posteri-orly. Unusual features found in some species were reduction ofthe oral disc, a more leaf-like overall body form, and the pres-ence of an elongated posterior extension to the tail, the taillament. Spawn and tadpoles were maintained in the labora-tory at temperatures appropriate to the normal habitat (Trin-idad 2729C; Scotland 1517C; Iran 2527C; Xenopus lae-vis, 25C) in dechlorinated, aerated tap water. Tadpoles werefed daily on sh food akes (except for X. laevis, fed on algapellets). At appropriate times, specimens were selected forxation and killed by immersion in a lethal concentration ofBenzocaine (0.1 g per liter).

Most specimens were xed in 2.5% glutaraldehyde in phos-phate buffer (PB) for about 5 h, then rinsed in 0.1 M PB (pH 7.4)and stored in buffer at 5C until further preparation for electronmicroscopy. Some specimens were xed and stored in Bouinsuid or in buffered neutral formalin (BNF) and kept at roomtemperature until required for electron microscopy.

Specimen Identication and StagingIn most species tadpoles were reared from spawn produced by

identied adults; where tadpoles were collected in the eld, Trinidadspecies were identied using features listed by Kenny (1969).

The specimens were staged using Gosners (1960) system. Weuse Gosner staging throughout this article, and when referring tostages using other systems in the literature have converted themto Gosner, using the table in McDiarmid and Altig (1999).

Specimen Preparation and ExaminationGlutaraldehyde-xed specimens were postxed in 1% osmium

tetroxide, stained in 0.5% aqueous uranyl acetate, dehydrated

TABLE 1. Classication, country of origin, and tadpole type and habitat of the 21 species used in this study

Family Species Country Tadpole habitatTadpoletype Special features

Bufonidae Bufo beebei Gallardo Trinidad Temporary pools P Bufo bufo Linnaeus United Kingdom Pools P Bufo marinus Linnaeus // Pools, stream, ditches P Forms loose shoalsBufo viridis Laurenti Iran Pools P

Hylidae Hyla punctata (Schneider) Trinidad Rich muddy sediments P Hyla boans (Linnaeus) // Shallow streams P Hyla crepitans Wied-Neuwied // Pools P Hyla minuta Peters // Pools L, TF, OR Suspension feeder,

head upHyla geographica Spix // Rivers, pools P Forms tight shoalsHyla microcephala Fouquette // Pools L, TF, OF Suspension feeder,

head upHyla savignyi Audouin Iran Pools P Scinax ruber* (Laurenti) Trinidad Pools L, TF Browser and

suspensionfeeder, head up

Phrynohyas venulosa (Laurenti) // Pools P Phyllomedusa trinitatis Mertens // Pools L, TF Browser and

suspensionfeeder, head up

Leptodactylidae Leptodactylus fuscus (Schneider) // Temporary pools P Leptodactylus bolivianus Boulenger // Pools P Forms tight shoalsPhysalaemus pustulosus (Cope) // Temporary pools P

Ranidae Rana temporaria Linnaeus United Kingdom Pools P Rana ridibunda Pallas Iran Pools P

Pipidae Xenopus laevis Daudin Africa Pools TF, OF Suspension feeder;head down

Dendrobatidae Mannophryne trinitatis (Garman) Trinidad Mountain streams P

*Scinax given as rubra by Murphy (1997) but now regarded as a masculine generic name (Caramaschi, 2004). Tadpole type code:P, standard polliwog tadpole; L, leaf-like body form; TF, tail extended as a long lament; OR, oral disc reduced; OF, mouth as a funnel.

1249TADPOLE SURFACE CILIATION

Journal of Morphology DOI 10.1002/jmor

using an acetone series, then critical-point-dried and coated withgold using a Polaron SC 515. They were then examined using aJSM 6400 scanning electron microscope. Images were examinedover a magnication range of 243,200 and recorded by Imag-eslave for Windows (Meeco Holdings, Australia). Bouins andformalin-xed specimens were dehydrated using an acetone se-ries, then processed as for the glutaraldehyde-xed samples.

Ciliated cell density was assessed as described in Nokhbatol-foghahai et al. (2005). We assessed the presence/absence anddensity of ciliated cells at eight locations on the body surface oflarvae: nostril, dorsal side of head (including the eyes, wherepossible), ventral side of head, dorsal side of trunk, ventral side oftrunk, proximal part of tail, mid-part of tail, distal part of tail,and hindlimb buds. The relative area taken up by ciliated cellscompared to nonciliated cells was measured and given a value:very dense (ciliated cells to nonciliated cell ratio 3:1); dense(between 2:3 and 3:1); intermediate (between 1:3 and 2:3); dis-persed (between 1:5 and 1:3); very dispersed (1:5); 0 (no ciliatedcells). In the data presentation tables, we use a star system todenote the different density categories. We were able to examinebetween one and three specimens at each stage.

RESULTSCiliated Cell Patterns and Densities:Species Accounts, Ordered by Family

The occurrence of ciliated cells in the 21 species ofposthatching larvae examined is shown in Table 2.Selected images to show ciliated cell patterns atdifferent regions in different species are shown inFigure 1 (limb and tail regions) and Figure 2 (nostriland eye regions). We were unable to collect everystage for every species. For consistency, missingstages are shown as no case in the table. Where wewere able to examine more than one specimen froma species at any particular stage, we found no sig-nicant variability between specimens.

Bufonidae

Table 2(a) shows detailed ciliary pattern resultsfor four bufonid species: Bufo beebei, B. bufo, B.marinus, and B. viridis. Nostril ciliation persistedwell past Stage 26 but was greatly reduced or absentin the two temperate region species we examined (B.bufo, B. viridis) at later stages (Fig. 2B). On otherbody regions, ciliation was greatly reduced: only inB. marinus did tail ciliation persist beyond Stage 30or so. In B. bufo and B. viridis, some ciliated cellswere found on the leg-bud (Fig. 1B). Ciliated cells onthe ventral side of the head in all species were dis-persed to very dispersed and no cilia were presentthere after Stage 27/28.

Hylidae

A more complex pattern of results emerged fromthe hylids. Table 2(b) shows detailed ciliary patternresults for 10 hylid species: Hyla punctata, H. boans,H. crepitans, H. minuta, H. geographica, H. micro-cephala, H. savignyi, Scinax ruber, Phrynohyasvenulosa, and Pyllomedusa trinitatis.

Although our dataset is somewhat incomplete forsome species, interesting patterns emerge. In Hylaboans, the only species in the set whose normalhabitat is fast-owing rivers, ciliated cells were al-most entirely absent even in the nostril region. Inmost other species, nostril ciliation, sometimes athigh density, persisted until advanced stages. Phry-nohyas venulosa was an exception, with no nostrilciliation by Stage 29. Elsewhere on the head andbody, ciliation was generally reduced or absent dur-ing most stages, although pond-living species, espe-cially those that spend much of their time suspendedin mid-water (Hyla minuta, H. microcephala, Scinaxruber, Pyllomedusa trinitatis) tended to have more,and more persistent, ciliation than those in streamhabitats or with more active habits (e.g., P. venu-losa, H. crepitans).

Tail ciliation was particularly prominent and per-sistent in some of the suspension feeders, notablyScinax ruber, Hyla microcephala, and H. minuta(Fig. 1CF). Ciliated cells were not at particularlyhigh density but were regularly arranged, coveringmost of the tail n surface, although not over most ofthe muscular part of the tail. Ciliated cells occurredon the proximal part of the tail lament, but we didnot nd them more distally.

Leptodactylidae

Table 2(c) shows detailed ciliary pattern results forthree leptodactylid species, Leptodactylus fuscus, L.bolivianus, and Physalaemus pustulosus. While densenostril ciliation persisted up to at least Stage 38 in P.pustulosus (Fig. 2C,D), no ciliated cells were presentaround the nostrils after Stage 26 in L. fuscus and L.bolivianus. Tail ciliation disappeared very early, be-fore Stage 26 in L. bolivianus, and did not persist pastStage 27 in L. fuscus, but persisted at very disperseddensity at Stage 39 in P. pustulosus. Ciliation on therest of the body regions was greatly reduced beyondStage 26, with only P. pustulosus showing body cilia-tion up to at least Stage 37.

Ranidae

Table 2(d) shows detailed ciliary pattern resultsfor two ranid species: Rana temporaria and R. ridi-bunda. Dispersed density nostril ciliation persistedwell past Stage 26. Tail ciliation disappeared afterStage 30. On other body regions, ciliation wasgreatly reduced. We did, however, nd a few ciliatedcells around the periphery of the eye (Fig. 2E) at latestages.

Pipidae

Table 2(e) shows detailed ciliary pattern resultsfor one pipid species, Xenopus laevis. Dense nostrilciliation persisted well after Stage 26 (Fig. 2A), but

1250 M. NOKHBATOLFOGHAHAI ET AL.


TABLE 2. Surface ciliated cell occurrence by species, stage, and body regions a) Bufonidae

a) Bufonidae

species stage nostril dorsal headventralhead

dorsaltrunk

ventraltrunk tail (proximal)

tail(mid-part)

tail(distal)

B. beebei 25/26 *** ** * * 0 * 27 ** 0 28 **** *** (eye ***) ** 0 29 **** ** ** 0 ** 33 *** 0 0 0 0 0

35/36 *** 0 0 0 0 0 0 037 *** 0 0 0 0 0 0 0

B. bufo 26 ***** *** * ** 0 27 ***** *** 0 ** 0 **

27/28 ***** *** * ** * ** (legbud **) * 030 ***** ***(eye *) 0 * * 0 036 0 0 0 0 0 0 0

B. marinus 26 ***** **** * ** 0 ** * 27 **** *** * * 0 28 **** **** * ** * ** * 030 *** * ** ** ** ** 031 * * ** 0 ** ** 032 **(eye ***) * ** 0 ** ** *34 * ** * 035 ** * * 0 0

36/37 * 0 0B. viridis 26 ***** * ** *** ** *** ** 0

26/27 ** **** ** *** ** 027 **** ** ** **** * *** ** 028 * * *** (legbud *) ** 029 * 0 0 0 0 *** * 031 0 0 0 0 0 0 0 034 0 0 0 0 0 0 0 035 0 0 0 0 0 0 0 038 0 0 0 0 0 0 0 0

b) Hylidae


dorsaltrunk

ventraltrunk

tail(proximal)

tail(mid-part)

tail(distal)

H. punctata 25/26 ** ** ** ** * * * 027 ** ** ** * * 028 *** ** *** ** 0 * * 031 *** **(eye ***) ** ** *

H. boans 26 * 0 0 0 0 0 0 029 0 0 0 0 0 0 0 0

39/40 0 0 0 0 0 0 0 0H. crepitans 26/27 ** * * * ** ** ** *

38/39 0 0 (eye*) 0 0 0 0 0 0H. minuta 32 *** *** *** *** *** *** *** **

36/37 ** ** *** *** (leg **) *** **H. geographica 36/37 0 0 0H. microcephala 25/26 *** ** ** ** ** ** ** *

32 ** ** *** *** *** **35 ** *** *** *** **

H. savignyi 25 ** ** * * *26 ** * *** * ** ** 0 027 * * ** * ** 0 0

27/28 * ** ** * 0 ** 0 029 * * ** * 0 *** 0 0

35/36 0 0 0Scinax ruber 26 *** *** ***

27 *** *** *** *** *** 29 ***** *** 0 *** 0 *** ** *33 ***** *** 0 *** 0 34 ***** ** 0 0 0 ** * 036 *** 0 0 0 0 * * 038 *** * 0 * 0 0

P. venulosa 26 *** 0 0 0 0 0 0 027 *** 0 0 0 0 0 0 028 ** 0 0 0 0 0 0 029 0 0 0 0 0 0 0 038 0 0 0 0 0 0 0 0


TABLE 2. (Continued)

b) Hylidae

species stage nostril dorsal head ventralhead

dorsaltrunk

ventraltrunk

tail (proximal) tail(mid-part)

tail(distal)

P. trinitatis 26 *** ** * * * * * *27 *** ** * * * * * *

27/28 **** ** * * * 29 **** ** * ** ** 0

31/32 **** *** ** * ** * 0 037 * ** * ** * * * 0

41/42 0 0 0 0 0 0 0 0

c) Leptodactylidae


dorsaltrunk

ventraltrunk tail (proximal)

tail(mid-part)

tail(distal)

L. fuscus 26 * ** **** * ** ** ** **26/27 ** * 27 0 0 0 * (legbud *) * 0

27/28 0 0 0 0 0 0 0 030 0 0 0 0 0 0 0 0

31/32 0 0 0 0 0 0 0 0L. bolivianus 25/26 0 0 0 0 0 0 0 0

37 0 0 0 0 0 0 0 0P. pustulosus 26 **** ** **** ** *** ***(legbud *) *** *

27 **** * *** *** * ** ** 028 *** * * * 0 * 0 0

29/30 *** * * * * 0 031 *** * * * * 0 035 **** * * * * 0 037 ***** * * * 38 ***** * (eye **) ** 0 * * 0 039 0 0 * 0 0

d) Ranidae

species stage nostrildorsalhead

ventralhead

dorsaltrunk

ventraltrunk

tail(proximal)

tail(mid-part)

tail(distal)

R. temporaria 25/26 **** ** *** ** ** ** 27 *** 0 0 * 0 030 *** 0 0 * 0 0

32/33 *** * 0 0 0 0 0 036 ** (eye**) 0 0 0 0 0 038 ** 0 0 0 0 * 0 0

R. ridibunda 23 **** *** **** ** ** *** ** *24 **** *** **** ** ** *** ** *25 ***** ** *** *** ** *** ** *26 **** ** * ** 0 *** * 027 *** *** ** ** 0 ** * 029 ** 0 0 0 0 *** 0 0

30/31 * 0 0 0 0 * 0 036 0 0 0 0 0 0 0 0

e) Pipidae


ventralhead

dorsaltrunk

ventraltrunk

tail(proximal)

tail(mid-part)

tail(distal)

X. laevis 26 ***** * 0 0 0 * 0 027 ***** 0 0 0 0 0 0 028 **** 0 0 0 0 0 0 031 **** 0 0 0 0 0 0 0

f) Dendrobatidae


ventralhead

dorsaltrunk

ventraltrunk

tail(proximal)

tail(mid-part)

tail(distal)

M. trinitatis 25/26 **** 0 ** 0 ** ** ** *26 0 * 0 0 0 0

28/29 0 0 0 0 0 031 0 0 0 0 0 0 0 0

Species reported by Nokhbatolfoghahai et al. (2005) are shown here from Stage 27 onwards. For new species, or those where someearlier stages were not previously examined, stages 2526 are also shown where available. Where legbuds were examined, they aredescribed in the proximal tail column. Numbers of stars denotes ciliated cell density (5 very high; 4 high; 3 intermediate; 2 low and 1 very low). 0 no ciliated cells; , no case examined.

no ciliated cells were present after Stage 26 on theremaining body regions.

Dendrobatidae

Table 2(f) shows detailed ciliary pattern resultsfor one dendrobatid species, Mannophryne trinitatis.Ciliary cells disappeared after Stage 26 from allbody regions.

DISCUSSION

Foxs (1986) review of the amphibian integumentconcluded that ciliated cells disappeared from theepidermis before premetamorphosis, stated to be NFstage 46 ( Gosner 25/26). Altig and McDiarmid(1999) referred briey to the ciliated cells of amphib-ian embryos and hatchlings and their possible func-tions. Viertel and Richter (1999) made no mention ofciliated cells as a component of the tadpole epider-

Fig. 1. Ciliated cell patterns onthe tails and leg regions in someanuran tadpoles. A: Hyla savignyi,Stage 29, proximal part of tail withlimb-bud devoid of ciliated cells.B: Bufo viridis, Stage 32, leg-bud.C: H. microcephala, Stage 32, tail.D: H. microcephala, Stage 21, mid-part of tail. E: H. minuta, Stage 37,proximal part of tail. F: H. minuta,Stage 32, proximal part of tail. Scalebars 400 m in A; 20 m inB,F; 650 m in C; 20 m in D;1,200 m in E.



mis. The only previous report of ciliated cells per-sisting into late tadpole stages appears to be that ofAssheton (1896): he did not use a morphologicalstaging system for his study of surface ciliation inRana temporaria embryos and larvae, reportingonly on tadpole lengths. However, it is clear that hesaw active ciliated cells on larvae well past hatching.As tadpoles grew, ciliated cells disappeared from thebody, including the eyes, but persisted on the tail, at

low density, higher at the proximal end. He appearsto have seen ciliated cells till soon before metamor-phosis: on the development of the hind limbs anddiminution of the tail cilia disappear from the tail(Assheton, 1896, p. 475).

We report here on surface ciliation in 21 species ofanuran larvae from six families and over a widerange of posthatching stages. Our sample is incom-plete: we have not examined every stage for every

Fig. 2. Ciliated cell patternsaround the nostrils and eyes insome anuran tadpoles. A: Xeno-pus laevis, Stage 31, dorsal head.B: Bufo viridis, Stage 31, dorsalhead lacking ciliated cells.C: Physalaemus pustulosus,Stage 38, dorsal head. D: P. pus-tulosus, Stage 38, nostril.E: Hyla punctata, Stage 31.F: Rana temporaria, Stage 36,lateral head, eye region. G: Lep-todactylus bolivianus, Stage 25,dorsal head. CCs, ciliated cells;n, nostril; lls, lateral line system.Scale bars 500 m in AC,G;240 m in D; 225 m in E; 1,000m in F.



species. Some of the specimens were from our gen-eral collections, rather than taken specically forthis study, and only a limited range of stages wasavailable. However, we felt that it was useful, giventhe lack of previous reporting on this aspect of tad-pole morphology, to examine as wide a range ofspecies as possible. The species range overlaps con-siderably (16 species the same) with our study ofciliation patterns on embryos and early larvae (No-khbatolfoghahai et al., 2005).

Our basic result is that surface ciliation clearlydoes persist well past hatching in tadpoles, but notin all species and not at all locations on the body.Examination of Table 2 shows that the nostrils arethe location where ciliated cells occur most densely,most persistently and over the widest range of spe-cies. Nokhbatolfoghahai et al. (2005) similarly foundnostril ciliation to be the most persistent and wide-spread in their survey of embryos and larvae upuntil Stage 25. An exception was the microhylidElachistocleis ovalis, where the external nares closeafter initially forming, and the ciliation in the nos-tril region disappears (Nokhbatolfoghahai andDownie, 2005). Nokhbatolfoghahai et al. (2005) sug-gested, following Assheton (1896), that nostril cilia-tion might help in chemosensation, by drawing wa-ter currents into the nares. The widespreadpersistence of this feature in later stage tadpolessupports this hypothesis, and ts with the impor-tance of the olfactory sense, for kin and predatordetection, in many tadpole species (Hoff et al., 1999).

A second function that has been widely suggestedfor amphibian embryonic surface ciliation is respi-ration. Burggren (1985) showed that the convectioncurrents generated by embryos prior to hatchingprevented hypoxia within the egg masses. Kessel etal. (1974) noted that hatching did not signal animmediate decline in surface ciliation and thathatchlings might still benet from surface-generated water currents; since their external gillswere in decline, hatchlings tended to remain quies-cent, and internal gills did not become functionaluntil around Stage 25.

Could this function persist in active-swimmingtadpole stages? There has been some disagreementover the role of cutaneous respiration in tadpoles.Viertel and Richter (1999) suggest that gills areessentially the sole respiratory exchange organs intadpoles, even when lungs develop early (as occursin most tadpoles except the bufonids); however, Hoffet al. (1999) suggest that many tadpole species use acombination of gills, lungs, and cutaneous respira-tion, with skin being a particularly important sur-face where it itself makes up much of the metaboli-cally active tissue, as in the tail ns. The activity oftadpoles might be thought to make redundant thegeneration of currents across the skin using ciliatedcells. However, many kinds of tadpoles spend muchof their time quiescent at the bottom of pools, possi-bly in quite hypoxic water; and a common lifestyle

for some species is to hang motionless in midwater,ltering the water for food by buccal pumping. Ourmost surprising result was the nding of ratherdense and regular arrays of ciliated cells on the tailns of three species until late stages, Scinax ruber,Hyla minuta, and H. microcephala. All are pond-living tropical hylids with tail laments, wheremuch of the time is spent suspended in midwater.This nding accords with the hypothesis that theciliated cells assist cutaneous respiration; however,it should be noted that another hylid which behavesin a similar fashion, Phyllomedusa trinitatis,showed much less tail ciliation.

Another nding that surprised us, especiallygiven Asshetons (1896) observations, was the occur-rence of ciliated cells around the periphery of the eyein several species. One of the long-suggested func-tions for surface ciliation is the removal of microor-ganisms and debris from the body surface. Given thekind of habitat, full of detritus, inhabited by manytadpoles, a means of cleaning the surface of the eyesin order to maintain good vision could be very im-portant. We have not surveyed this feature system-atically, and suggest that this could be worth furtherinvestigation.

Finally, our ndings differ in two respects fromAsshetons (1896). His study of Rana temporariashowed ciliated cells of fairly high density on the tailuntil very late stages; he also described these cellsas elongated. Among the species we report here, R.temporaria had comparatively few ciliated cells onthe tail; the ciliated cells we found on later-stagetadpoles tended to be fairly small and polygonal,with the cilia mainly attached to the middle part ofthe cell (Fig. 1D). Nokhbatolfoghahai et al. (2005),however, did describe the kind of elongated cellsreported by Assheton (1896) in other species at laterembryonic or early hatchling stages.

This discrepancy raises the possibility of variabil-ity in ciliation patterns, either between individualsor in relation to environmental conditions. It is wellknown that the external gills of urodele larvae de-velop differently in relation to the oxygen tension inthe surrounding water (Bond, 1960). We found nosignicant variability between same-stage speci-mens of the same species, but we did not rear tad-poles under variable conditions, so it may be worthinvestigating whether surface ciliation patterns areresponsive to factors such as oxygen tension.

ACKNOWLEDGMENTS

We thank Margaret Mullin for technical assis-tance. Students on University of Glasgow expedi-tions to Trinidad helped collect specimens. Studentsof Shiraz University, Iran, helped collect Iranianspecies. The research department, Shiraz Univer-sity, supported the collection of samples duringeldwork in Iran. The late Professor Peter Baconand colleagues at the University of the West Indies



kindly provided laboratory space, and the TrinidadGovernment Wildlife Section provided collectionpermits. J.R.D.s eldwork in Trinidad was sup-ported by the Carnegie Trust and the University ofGlasgow.

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Surface Ciliation of Anuran Amphibian Larvae: Persistence to Late Stages in Some Species but Not...

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