HERPETOLOGICAL JOURNAL, Vol. 8, pp. 47-50 (1998)

HITHERTO UNDESCRIBED ULTRASTUCTURAL FEATURES IN THE EPIDERMIS OF TWO AFRICAN

M. R . WARBURG *', MIRA ROSENBERG1 AND K. E. LiNSENMAIR2

1 Department of Biology, Te chnion- Israel Institute of Te chnology, Haifa, 32000, Israel

2Theodor-Boveri-lnstitutfur Biowissenschaften, Zoologie Ill, Biozentrum, Am Hub/and D- 97074, Wurzburg, Germany

* Author fo r correspondence

The epidermis of nitidulus (Peters, 1875) () consists of three strata: stratum corneum, stratum granulosum and stratum germinativum. The stratum corneum of both H. nitidu/us and Hemisus marmoratus (Peters, 1854) (Hemisiidae), contains 1-2 replacement layers. The granular cells of H. nitidu/us are arranged in 2-3 rows. No granular cells typical of the stratum granulosum could be seen in H. marmoratus. This is a unique situation for any .. Very large germinative cells were observed in both anurans. In H. nitidulus a unique, long and slender 'pillar cell' is situated under the stratum corneum, extending through the stratum germinativum, and the basement membrane deep into the dermis. This cell contains abundant tonofilaments, and appears to functionas a pillar suppporting the frequentlymoulting stratum corneum.

INTRODUCTION In the present study we describe some of the novel The ultrastructure of the amphibian epidermis cells ultrastructural features in the ventral epidermis of these has been described in about 12 anuran . No epi­ two African amphibian species: H. nitidulus and dermis of an African amphibian has previously been Hemisus marmoratus (Hemisiidae). studied. A number of epidermal cell types have been MATERIALS AND METHODS described (Fox, 1986; Warburg et al., 1994). Among were collected in the Ivory Coast (by one of these are cells, typical only of the larval stages, which us: KEL) and dissected shortly after capture. Between ultimately disappear; others first appear near the com­ capture and dissection they were kept under humid con­ pletion of metamorphic climax, and persist in the ditions and fed on either fl ies (Hyperolius) or ants juvenile and adult stages (Warburg & Lewinson, 1977; (Hemisus). Pieces of ventral epidermis of three adult H. Rosenberg & Warburg, 1978, 1992, 1993, 1995; nitidulus and H. marmoratus were examined Warburg et al., 1994). histologically and ultrastructurally using light and Hyperolius nitidulus (D & B) (Hyperoliidae) is a transmission electron microscopy. The methods are de­ unique in many ways (Linsenmair, 1998). It is a scribed in greater detail in Warburg et al., (1994). short-lived frog in which only the juveniles aestivate For light microscopy, tissues were fixed in Bouin's successfully, by clinging to vegetation exposed to the fluid for 24 hrs. Sections were stained with harsh ambient conditions persisting during the dry sea­ haematoxylin and eosin. Epon sections 2 µm thick, son. Two phases can be recognized in this frog:the wet were stained with toluidin blue. season phase and the dry season phase. These phases For Transmission Electron Microscopy (TEM), differ in both their water balance and dermal skin struc­ pieces of skin were fixed cold in 3% glutaraldehyde in ture (Geise & Linsenmair, 1986, 1988; Kobelt & 0.05 M cacodylate buffer for2-4 hrs, and then rinsed in Linsenmair, 1986, 1992). The dry season frog has a thin buffer containing 8% sucrose. Post-fixation was in 1% layer of desiccated mucus sealing the body surfaceand OsO containing 1.5% potassium ferrocyanidefor 1 hr, thus reducing water loss (Geise & Linsenmair, 1986). 4 and dehydrated in graded ethanols. Finally, the material The main barrier against desiccation seems to be the was embedded in Epon 812. Unstained sections 70- stratum comeum (Geise & Linsenmair, 1988). Moreo­ 90A thick were mounted on copper grids 300 mesh, and ver, the dermis contains several layers of iridiophore were examined in a Jeol 100 B (TEM) operated at 60 filledwith purine platelets that are partly arranged par­ KV. allel to the surface and thus cause a high reflection (up RESULTS to 70%) ofthe sun's radiation in the visible and infrared spectrum (Kobelt & Linsenmair, 1986, 1992). In H. nitidulus, under the upper layer of the stratum comeum there are 2-3 replacement layers composed of In contrast, H. marmoratus is a long-lived, fossorial frog that rarely ventures onto the surface, and then only flattened, tightly packed stratum corneum cells under very humid conditions. There is little published (Fig. lA,B). ln H. marmoratus, the epidermis appears to information on this frog (it is presently studied by one consist only of a stratum comeum with wide intercellu­ of us: KEL). lar spaces in the ventral epidermis (Fig. 3A,B), and 48 M. R. WARBURG, M. ROSENBERG AND K. E. LINSENMAIR

FIG. I (TOP LEFT). Sections through the epidermis of Hyperolius nitidulus. A, section through the entire depth of the ventral epidermis to the dermis (D) viewed in light microscope. Note the long pillar cells (p) situated perpendicular to the surface (arrow indicates moult) and stratum corneum (s), penetrating through the basement layer into the dermis (D)(x 420; scale bar= I 0 µm). B, same as in A but an ultramicrograph. Note the several stratum corneum layers (asterisk, 1,2), the granular cells (gr), and the large germinative cells (g) situated on the basement layer (arrows) (n-nucleus) (x 6060; scale bar=lµm). C, granular cell containing granules viewed in EM (arrowhead; n-nucleus) (x 10 500; scale bar=! µm).

FIG. 2 (BOTTOM LEFT). The large, slender pillar cell located perpendicular to the stratum corneum (s, I) and the basement layer (arrow), containing a nucleus (n) and mitochondria (m), is separated from neighbouring cells through tight junctions (flowers). A germinative cell (g) is situated on the basement layer (arrow) (x 9000; scale bar=l µm).

FIG. 3 (ABOVE). Sections through the ventral (A,B,D) and dorsal (C,E) epidermis of Hemisus marmoratus. A, section through the entire width of the ventral epidermis from the stratum corneum (asterisk), through its replacement layers (1,2) separated from each other by a wide intercellular space (flower), to a stratum germinativum cell (g) situated on the basement layer (arrow; n, nucleus). (x 7500; scale bar=l µm). B, as in A but enlarged. The stratum corneum (asterisk) and its two replacement layers (1,2) separated from each other and from the stratum germinativum by wide intercellular spaces (flowers). Arrow indicates the basement layer (x 10 500; scale bar= ! µm). C, section through the entire width of the dorsal epidermis. Note the compact layers of stratum corneum (asterisk, 1,2), and the stratum germinativum cell (g) situated on the basement layer (arrow; n-nucleus) (x 7500; scale bar=l µm). D, germinative cell situated on the basement membranes (arrows; n-nucleus). Note the abundance of tonofillaments (x 15000; scale bar= 1 µm). E, enlarged section through the dorsal stratum cornem (SC) showing the microvilli covered by fuzz(arrowhead) (x 15 OOO; scale bar= 1 µm). UL TRASTRUCTURE OF AFRICAN AMPHIBIAN EPIDERMIS 49 tightly packed cells in its dorsal epidennis (Fig.3C,E), found in deeper layers, possibly indicating a role in as well as a stratum germinativum (Fig. 3D). The stra­ water conservation (Navas et al., 1980). tum granulosum appears to be missing in this frog The large pillar cells described here for the fi rst time (Fig.3A-C). This is the first time that a stratum in Hyperolius, may play a role in supporting the epider­ granulosum could not be foundin an amphibian. On the mis of this unique frog during its frequent moultings. other hand, in H. nitidulus the stratum granulosum, These frequentand rather costly moultings are a means which is situated under the stratum corneum, is com­ of preventing the formation of cocoon-like structures posed of 1-2 rows of large, oval-shaped cells found in some other xeric-inhabiting arboreal containing large granules around the large oval nucleus (Warburg, 1997). The reason could be that the forma­ (Fig. IB,C). tion ofa cocoon will impede this frog' s free functioning The stratum germinativum in both species is com­ during the dry season (Linsenmair, unpublished). posed of large cells, fully packed with tonofilaments ACKNOWLEDGEMENTS and mitochondria in a perinuclear arrangement, which are situated on the basement membrane (Figs. I .B; 3 A­ Partial financial support to one of us (KEL) came D). from the Deutsche Forschungsgemeinschaft(SFB 25 1, In H. nitidulus, a unique new cell type was seen for Project No B 3). the firsttime. This is a very long, slender, pillar-shaped cell, situated under the stratum corneum, with its root­ Budtz, P. E. ( 1977). Aspects of moulting in anurans and its shaped base penetrating through both the stratum control. Sy mp. Zoo/. Soc. Land 32, 317-334. germinativum and the basement membrane deep into Budtz, P. E. & Larsen, L. 0. ( 1975). Structure of the toad the dermis (Figs. IA; 2). This cell contains numerous epidermis during the moulting cycle. II. Electron tonofilaments,thus possibly indicating its function asa microscopic observations on Bufo bufo (L.). Cell. Tiss. supporting cell. We suggest naming this cell "pillar Res. 159, 459-483. cell" because of its pillar-like shape and its vertical po­ Fox, H. ( 1977). The anuran tadpole skin: changes sition in the tissue, indicative of a putative fu nction in occurring in it during and some supporting the stratum corneum. It seems to be unique comparisons with that of the adult. Sy mp. Zoo/. Soc. tp H. nitidulus, and was never previously observed in Land. 39, 269-289. the epidermis of any other amphibian species. Fox, H. (1986). Epidermis. In:_Bio/ogy of the Integument vol 2 Ve rtebrates, 78-110. Bereiter-Hahn, J ., DISCUSSION Matoltsy, A. G., Richards, K. S. (Eds). Springer, Epidermal cell layers of amphibians increase in Berlin. number until the completion of metamorphosis Geise, W. & Linsenmair, K. E. (1986). Adaptations of the (Rosenberg & Warburg, 1978; Robinson & reed frog Hyperolius viridijl.avus (Amphibia, Anura, Heintzelman, 1987; Warburg et al., 1994). Thus, in the Hyperoliidae) to its arid environment. II. Some aspects juvenile Pelobates syriacus there are three epidermal of the water economy of Hypero/ius viridijl.avus cell layers, whereas in the adult between three and four nitidu/us under wet and dry season conditions. layers. The epidermis of the two African anurans stud­ Oeco/ogia 68, 542-548. ied here differs in the number of layers. The epidermis Geise, W. & Linsenmair, K. E. (1988). Adaptations of the of H. marmoratum appears to be composed of only two reed frog Hypero/ius viridiflavus (Amphibia, Anura, layers. Hyperoliidae) to its arid environment. IV. Ecological At metamorphic climax, the process of cell flatten­ significance of water economy with comments on ing and keratinization reaches its peak, culmunating in thermoregulation and energy allocation. Oecologia 77, the formation of the stratum corneum (Budtz, 1977; 327-338. Warburg & Lewinson, 1977). There are a number of Kobelt, F. & Linsenmair, K. E. ( 1986). Adaptations of the stratum corneum replacement layers found in reed frog Hypero/ius viridiflavus (Amphibia, Anura, Hyperolius. These may be related to the high frequency Hyperoliidae) to its arid environment. I. The skin of of moulting taking place in this species (Kobelt & Hypero/ius viridiflavus nitidu/us in wet and dry season Linsenmair, 1986). The replacement layer of the stra­ conditions. Oeco/ogia 68, 533-541. tum corneum contains mucous granules (Fox, 1977). Ko belt, F. & Linsenmair, K. E. ( 1992) Adaptations of the The presence of granules in epidermal surfacecells ap­ reed frog Hypero/ius viridijl.avus (Amphibia, Anura, pears to be a feature of the keratinization process Hyperoliidae) to its arid environment. VI. The (Budtz & Larsen, 1975). We have observed in H. iridiophores in the skin as radiation reflectors. J. nitidulus an ouststanding richness in granules in these Comp. Physio/. B 162, 314-326. cells. Linsenmair, K. E. ( 1998). Risk spreading and risk In the stratum granulosum of Hyperolius, the granu­ reducing tactics of West African anurans in an lar cells are most abundant. In Rana ridibunda these unpredictable and stressful environment. Brit. Eco/. granules were apparently situated closer to the stratum Soc. Sy mp . (in press). corneum, possibly related to the process of Navas, P., Lopez-Campos, J. L. & Diaz-Flores, L. ( 1980). keratinization, whereas in Bufo calamita they were Diferencias epidermicas del estrato granular entre 50 M. R. WARBURG, M. ROSENBERG AND K. E. LINSENMAIR

Rana ridibunda y Buja ca/amita. Mor/a/. Normal arborea savignyi Aud. (Anura; Hylidae) throughout Pata/. 4, 313-321. metamorphosis. Acta Zoo/. 76, 217-227. . Robinson, D. H. & Heintzelman, M. B. ( 1987). Warburg, M. R. (1997). Ecophysio/ogy of amphibians Morphology of ventral epidermis of Rana catesbeiana inhabiting xeric habitats. Springer Verlag, during metamorphosis. Anat. Ree. 217, 305-3 17. Heidelberg, 182 pp. Rosenberg, M. & Warburg, M. R. (1978). Changes in Warburg, M. R. & Lewinson, D. (1977). Ultrastructure of structure of ventral epidermis of Rana ridibunda epidermis of Sa/amandra sa/amandra fo llowed during metamorphosis. Cell Tiss. Res. 195, 111-122. throughout ontogenesis. Cell Tiss. Res. 181, 369-393. Rosenberg, M. & Warburg, M. R. (I 992). Ultrastructure Warburg, M. R., Lewinson, D. & Rosenberg, M. (1994). and histochemistry of ventral epidermis in Pe/obates Ontogenesis of amphibian epidermis. in:_Amphibian syriacus (Anura; Pelobatidae) tadpoles. Biol. Struct. Biology vo/ I The Integument 33-43. Heatwole, H. and Morphogen. 4, 164-170. Barthalmus,G.T.(Eds). Surrey Beatty & Sons, Sydney. Rosenberg, M. & Warburg, M. R. (1993). The ventral epidermis of Pe/obates syriacus (Anura: Pelobatidae). Isr. J. Zoo/. 39, 235-243. Rosenberg, M. & Warburg, M.R. ( 1995). Changes in

structure and function of ventral epidermis in Hy /a Accepted: 14. 8.97