Fine Structure of the Dorsal Lingual Epithelium of the Juvenile Hawksbill Turtle, Eretmochelys Imbricata Bissa

THE ANATOMICAL RECORD 244437-443 (1996)

Fine Structure of the Dorsal Lingual Epithelium of the Juvenile Hawksbill Turtle, Eretmochelys imbricata bissa

SHIN-ICHI IWASAKI, TOMOICHIRO ASAMI, AND CHAITIP WANICHANON Department of Histology (SJ.) and Department of Anatomy (TA.), The Nippon Dental University School of Dentistry at Niigata, Niigata, Japan; and Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand (C.W,) ABSTRACT Background: Various species of turtles are adapted to different environments, such as freshwater, seawater, and terrestrial habitats. Comparisons of histological and ultrastructural features of the tongue of the juvenile Hawksbill turtle, Eretmochelys imbricata bissa, with those of freshwater turtles should reveal some aspects of the relationship between the structure of the lingual epithelium and the environment. Methods: The light microscope, scanning electron microscope and transmission electron microscope were used. Results: Light microscopy revealed that the mucosal epithelium of the tongue was of the keratinized, stratified squamous type. Under the scanning electron microscope, no lingual papillae were visible on the dorsal surface of the tongue. Micropits and the thickening of cell margins were clearly seen on the surface of cells located on the outermost side. The transmission electron microscope revealed that the cells in the intermediate layer were gradually flattened from the basal side to the surface side, as were their nuclei. In the shallow intermediate layer, the cells were significantly flattened, and their nuclei were condensed or had disappeared. The cytoplasm contained keratohyalin granules, tonofibrils, free ribosomes, mitochondria, and rough endoplasmic reticulum. Numerous free ribosomes were attached to the surface of small keratohyalin granules. The cells of the keratinized layer were significantly flattened, and their nuclei had completely disappeared. Most of cytoplasm was filled with keratin fibers of high electron density. Keratin fibers of the shedding cells, which were located on the outermost side of the keratinized layer, appeared looser, and each fiber, which was somewhat thicker than the tonofibrils and tonofilaments, was clearly distinguishable. Conclusions: The lingual epithelium of the juvenile Hawksbill turtle dif- fers significantly from that of the adult freshwater turtle, in spite of the similarity in gross morphology of the tongues of these species. 0 1996 Wiley-Liss, Inc. Key words: Hawksbill turtle, Tongue, Epithelium, Keratinization, Kerato- hyalin granules, Histology, Ultrastructure

Many studies have demonstrated that the dorsal sur- papillae are located on the surface of the anterior bi- face of the reptilian tongue is rich in lingual papillae furcated area of the tongue of the adult Japanese lizard (Iwasaki and Miyata, 1985a; Rabinowitz and Tandler, (Iwasaki and Miyata, 1985a). 1986; Schwenk, 1986; Iwasaki, 1990, 1992; Iwasaki et There are many differences in the features of the al., 1992a), as is the case in mammals also (Farbman, dorsal lingual epithelium in reptiles. These differences 1966,1970; Cane and Spearman, 1969; Hume and Pot- can be categorized phylogenetically and, at the same ten, 1976; Steflik et al., 1983; Iwasaki and Miyata, time, they reflect the adaptation of reptiles to their 1985b; Iwasaki et al., 1987a,b; Boshell et al., 1989). It environments to a certain extent. For example, the lin- has been also shown that, together with variations in the shape of the tongue itself, the form and the pattern of distribution of the lingual papillae exhibit signifi- cant variations among different species of reptiles. Received August 7, 1995; accepted October 25, 1995. Only snakes are known, at present, to have no lingual Address reprint request to Dr. Shin-ichi Iwasaki, Department of papillae on the entire surface of the tongue (Mao et al., Histology, The Nippon Dental University School of Dentistry at Nii- 1991; Iwasaki and Kumakura, 1994), and no lingual gata, 1-8 Hamaura-cho, Niigata 951, Japan.

0 1996 WILEY-LISS, INC. 438 S.-I. IWASAKI ET AL.

Figs. 1-4. EPITHELIUM OF THE JUVENILE HAWKSBILL TURTLE 439 gual epithelium of completely terrestrial reptiles has a ter rinsing in 0.1 M cacodylate buffer, samples for scan- tendency to be karatinized (Mao et al., 1991; Iwasaki ning electron microscopy were post-fixed in a phos- and Kumakura, 1994), while that of the reptiles that phate-buffered solution (pH 7.4) of 1% osmium live in or near ponds or small rivers has a tendency to tetroxide at 37°C for 2 hours. These samples were then be nonkeratinized (Iwasaki, 1992a; Iwasaki et al., treated with 8 N hydrochloric acid at 60°C for 30 min in 1992a). The tongue of reptiles that live under amphib- order to remove the mucus from the surface of the tis- ious circumstances tend to have both keratinized and sue. This procedure was followed by dehydration, nonkeratinized epithelia in different parts of the freeze-drying in tertiary butanol, and sputter-coating tongue (Schwenk, 1988; Iwasaki, 1990; Iwasaki and with platinum and palladium ions. The specimens were Kobayashi, 1992). examined under a scanning electron microscope (S-800; The purpose of the present study was to examine the Hitachi, Tokyo). histological and ultrastructural features of the lingual epithelium of one type of sea turtle, the Hawskbill tur- RESULTS tle, by light, scanning electron, and transmission elec- Scanning Electron Microscopy tron microscopy, and to compare the results of the ob- The tongue was triangular with a slightly round servations with those reported for the tongues of other apex when viewed dorsally under the scanning electron reptiles, in particular, freshwater turtles. The eventual microscope but it appeared flattened when viewed lat- goal of our comparative ultrastructural studies is to erally. No lingual papillae were observed on the dorsal clarify the relationship between the ultrastructural surface of the tongue. Instead, numerous plicae were features of the lingual epithelium and the life style of seen on the surface of the body and the radix (Fig. 1). A turtles that live in different environments. narrow prominence was located along the midline of the anterior third of the dorsal surface. Protrusions of MATERIALS AND METHODS the shedding epithelia were frequently distributed over The Hawksbill turtle, Eretmochelys imbricata bissa, the dorsal surface (Fig. 2). Numerous fine plicae of ep- is widely distributed in the tropical and temperate wa- ithelium were seen over the entire surface. Each cell ters of the Pacific Ocean. Three male and three female had a clearly polygonal profile (Fig. 3). Scanning elec- juveniles of four months of age were obtained commer- tron microscopy at higher magnification revealed cially on Tarutao Island, Thailand. The length of their widely spread micropits on the surface of cells located carapaces ranged from 8.5 cm to 9.3 cm, and the width on the outermost side. The thickening of cell margins ranged from 7.4 cm to 8.3 cm. These juvenile animals was clearly visible (Fig. 4). had been reared on the Island after hatching. Under ether anesthesia, turtles were killed by de- Light Microscopy captation. Specimens for light microscopy were pre- Light microscopy revealed that the mucosal epithe- pared at Mahidol University, Thailand, by fixation in lium of the tongue was of the stratified squamous type. 10% formalin, paraffin-embedding, and hematoxylin- Lamina propria was located between the mucosal epi- eosin staining. Specimens for electron microscopy were thelium and the lingual muscle. In the basal layer and fixed in 2.5% glutaraldehyde for 8 hours and trans- deep intermediate layer, the cells were almost spheri- ferred to 0.1 M phosphate-buffered solution (pH 7.4) at cal and their nuclei were also spherical and large. The Mahidol University. After the specimens had been sent deep intermediate layer was composed of a few layers to the Nippon Dental University, Japan, they were of cells. In the shallow intermediate layer, cells became fixed in half-strength Karnovsky solution that con- suddenly flattened, and there was a rapid transition to tained 2% paraformaldehyde and 1.25% glutaralde- the keratinized layer. There was a layer of shedding hyde (pH 7.4). After rinsing in 0.1 M cacodylate buffer, cells on the outermost side of the keratinized layer some of the samples for transmission electron micros- (Fig. 5). copy were postfixed in a phosphate-buffered solution (pH 7.4) of 1% osmium tetroxide at 4°C for 1.5 hours. Transmission Electron Microscopy This procedure was followed by dehydration, epoxy- Under the transmission electron microscope, the resin embedding, ultrathin sectioning, and double- cells of the basal and deep intermediate layers of the staining with lead citrate and uranyl acetate. The spec- epithelium were elliptical in shape. A large elliptical imens were then observed under a transmission nucleus lay in the central region of each epithelial cell. electron microscope (JEM-1200 EX; Jeol, Tokyo). Af- The basal lamina was intercalated between the basal cells of the epithelium and the lamina propria (Fig. 6). Hemidesmosomes were frequently located between the basal cells and the basal lamina. The cytoplasm of these cells contained mitochondria, free ribosomes, Fig. 1. Scanning electron micrograph of the dorsal lingual surface of a juvenile Hawksbill turtle, Eretmochelys imbricata bissa. Bo, lingual rough endoplasmic reticulum, and bundles of tono- body; Ph, pharynx; arrow, lingual apex. X 18. fibrils (Fig. 7). Fine cellular processes were visible all around the epithelial cells. Desmosomes were interca- Fig. 2. Scanning electron micrograph of the narrow prominence lated between the processes of adjacent cells. The nu- along the midline of the anterior part of the dorsal surface. x 110. cleus in each cell contained well-developed heterochro- Fig. 3. Scanning electron micrograph of the epithelial surface of the matin (Fig. 6). The intermediate layer was relatively body of the tongue. Arrow; thickening of the cell margin. X 1,100. thin and was composed of somewhat more than ten layers of cells. The cells of the intermediate layer be- Fig. 4. Higher-magnification scanning electron micrograph of the cells located on the outermost side of the lingual epithelium. Arrow; came abruptly flattened, and in the shallow interme- thickening of the cell margin. x 3,400. diate layer, the cells were significantly flattened. The 440 S.-I. IWASAKI ET AL.

Figs. 5-8. EPITHELIUM OF THE JUVENILE HAWKSBILL TURTLE 441 nuclei were elliptical in the deep intermediate layer. In juvenile Hawksbill turtle was of the keratinized, strat- the shallow intermediate layer, some of the nuclei were ified squamous type. This type of epithelium has pre- still large and elliptical, while other nuclei were flat- viously been recognized only on the anterior bifurcated tened or had disappeared. The cytoplasm contained area of the tongue of the Japanese lizard (Iwasaki and many bundles of tonofibrils, free ribosomes, rough en- Miyata, 1985a) and over the whole of the lingual dor- doplasmic reticulum, mitochondria, and keratohyalin sum of snakes (Ma0 et al., 1991; Iwasaki and Ku- granules. Numerous free ribosomes were attached to makura, 1994). As mentioned above, the area of kera- the surface of small keratohyalin granules throughout tinized stratified squamous epithelium coincides with the intermediate layer. The cell membranes of the in- the region in which no lingual papillae are found in termediate-layer cells were smooth in some cases and these animals. Indeed, most epithelium with lingual slightly undulated in others. Desmosomes were fre- papillae is of the nonkeratinized, stratified cuboidal quently intercalated between adjacent cells (Figs. type. Most or a large part of the lingual dorsum in G. 8-10). A keratinized layer was located on the apical reevesii (Iwasaki, 1992a), C.japonica (Iwasaki et al., side of the shallow intermediate layer and appeared 1992a), and T. tachydromoides (Iwasaki and Koba- with abrupt transition from the shallow intermediate yashi, 1992) is composed of cells that contain secretory layer (Fig. 11).The cells in this layer were significantly granules. By contrast, in S. punctatus (Schwenk, 19861, flattened, and their nuclei had completely disappeared. A. carolinensis (Rabinowitz and Tandler, 1986), and G. Most of the cytoplasm was filled with keratin fibers japonicus (Iwasaki, 19901, cells containing secretory with high electron-density. Hardly any other or- granules are fewer in number in the dorsal lingual ganelles were visible. Very fine processes composed of epithelium than in G. reevesii, C. japonica, and T. ta- cell membrane were still observed and intercalated chydromoides. Instead, a large part of the dorsal epi- desmosomes were also seen in this layer (Figs. 11, 12). thelium is stratified and lacks secretory granules. The The cells located on the extreme free-surface side of the former animals live in or near ponds or small rivers keratinized layer were of the shedding type. In this while the latter are terrestrial. Iwasaki et al. (1992a) layer of shedding cells, keratin fibers became looser, postulated that the composition of the dorsal epithe- and each fiber, which was somewhat thicker than the lium of the tongue might reflect an animal's environ- tonofibrils and tonofilaments, was clearly distinguish- ment. Thus, it is suggested that, when animals move able. The rugged surface of cell membranes originated from a freshwater to a terrestrial habitat, the dorsal from cellular processes in the underlying layer and co- lingual epithelium begins to lose secretory cells, which incided with the micropits seen under the scanning are replaced by stratified squamous cells. The latter electron microscope (Fig. 11). type of cell appears to be more suitable for the dry circumstances of terrestrial life. The present study in- DISCUSSION dicates that the same hypothesis is also applicable Scanning electron microscopy failed to reveal any when animals move from freshwater to seawater hab- lingual papillae at all over the entire dorsal lingual itats. In this case, however, the dorsal lingual epithe- surface of the Hawskbill turtle, although they were lium of the sea turtle is composed of stratified squa- clearly visible on the dorsal lingual surface of the mous cells, which are more protective of the inside of freshwater turtles Geoclemys reevesii (Iwasaki, 1992a) this organ than are stratified cuboidal cells. In fact, the and Clemmysjaponica (Iwasaki et al., 1992a). This dif- composition of the lingual epithelium of the sea turtle ference might be related to the role of the lingual epi- is very similar to that of terrestrial vertebrates, irre- thelium as a salivary gland. Almost all of the epithelial spective of whether or not lingual papillae are present cells of the lingual dorsum of freshwater turtles con- (Cane and Spearman, 1969; Farbman, 1970; Hume and tain mucous granules or other types of secretory gran- Potten, 1976; Iwasaki and Miyata, 1989, 1990; ule (Iwasaki, 1992a; Iwasaki et al., 1992a), while those Iwasaki, 1992b; Iwasaki et al., 1992b). Further obser- of the juvenile Hawskbill turtle do not contain any type vations in numerous species are necessary to confirm of secretory granule. the validity of our hypothesis. From our observations by light microscopy and The pattern of keratinization of the lingual dorsal transmission electron microscopy, it was apparent that epithelium of the juvenile Hawksbill turtle exhibits the whole of the dorsal epithelium of the tongue of the some similarities to that of mammals. First, the epithelium of this species has a relatively thick layer of keratinized cells. Second, keratohyalin granules, often accompanied by surrounding free ribosomes, can be found in the intermediate layer. The same kind of kera- Fig. 5. Light micrograph of dorsal lingual mucosa. Lp, lamina pro- tohyalin granule was also found in the epithelium of pria; Ep, lingual epithelium. x 1,350. the anterior bifurcated area of the tongue of Japanese Fig. 6. Transmission electron micrograph of the basal and deep in- lizard (Iwasaki and Kobayashi, 1992). The lingual dor- termediate layers. N, nucleus; M, mitochondria; rER, rough endoplas- sal epithelium of mammals is generally composed of mic reticulum; R, free ribosomes; Tb, tonofibrils; B1, basal lamina; Lp, regularly ordered columns of cells with different de- lamina propria; arrow, desmosome. x 15,000. grees of keratinization, such as the anterior cell col- Fig. 7. Transmission electron micrograph of the cytoplasm of a basal umn of the filiform papillae, the posterior cell column cell. B1, basal lamina; M, mitochondria; Tb, tonofibrils; Lp, lamina of the filiform papillae, and the interpapillar cell col- propria; arrow, hemidesmosome. x 20,000. umn (Farbman, 1970; Iwasaki and Miyata, 1989, Fig. 8. Transmission electron micrograph of the deep intermediate 1990). Some authors have proposed a more complex layer. R, free ribosomes; K, keratohyalin granule; arrow, desmosome. composition for the dorsal epithelium of the mamma- x 20,000. lian tongue (Cane and Spearman, 1969; Hume and Pot- 442 S.-I. IWASAKI ET AL.

Figs. 9-1 2. EPITHELIUM OF THE JUVENILE HAWKSBILL TURTLE 443 Fig. 9. Transmission electron micrograph of the shallow intermedi- tongue of the freshwater turtle, Geoclemys reeuesii (Chelonia, ate layer. N, nucleus; M, mitochondria; R, free ribosomes; Tb, Emydinae). J. Morphol., 211:125-135. tonofibrils; K, keratohyalin granule; arrow, desmosome. x 10,000. Iwasaki, S. 1992b Fine structure of the dorsal lingual epithelium of the domestic, newborn kitten, Felis catus. Ann. Anat., 174:293- Fig. 10. Transmission electron micrograph of the cytoplasm of cells 300. of the shallow intermediate layer. M, mitochondria; rER, rough en- Iwasaki, S. and K. Kobayashi 1992 Fine structure of the dorsal lin- doplasmic reticulum; R, free ribosomes; Tb, tonofibrils; K, keratohy- gual epithelium of the Japanese lizard, Takydromus tuchydro- alin granule; arrow, desmosome. x 20,000. moides. Acta Anat. Nippon., 67:214-225. Iwasaki, S. and M. Kumakura 1994 An ultrastructural study of the Fig. 11. Transmission electron micrograph of the border area be- dorsal lingual epithelium of the rat snake, Eluphe quadriuirgutu. tween the shallow intermediate and the keratinized layers (Kl). Ar- Ann. Anat., 176:455-462. row, desmosomes. x 20,000. Iwasaki, S. and K. Miyata 1985a Scanning electron microscopy of the lingual dorsal surface of the Japanese lizard, Takydromus tuchy- Fig. 12. Transmission electron micrograph of the keratinized layer dromoides. Okajimas Folia Anat. Jpn., 62:15-26. that contained shedding cells. Kf, keratin fibers. x 20,000. Iwasaki, S. and K. Miyata 1985b Light and transmission electron microscopic studies on the lingual dorsal epithelium of the musk shrew, Suncus murinus. Okajimas Folia Anat. Jpn., 6267-88. Iwasaki, S. and K. Miyata 1989 Fine structure of the filiform papilla of beagle dogs. J. Morphol., 201:235-242. Iwasaki, S. and K. Miyata 1990 Fine structure of the dorsal epithe- ten, 1976). In most mammals, keratohyalin granules lium of the mongoose tongue. J. Anat., 172201-212. are recognized only in the anterior area of the filiform Iwasaki, S., K. Miyata, and K. Kobayashi 1987a Comparative studies of the dorsal surface of the tongue in three mammalian species by papillae (Farbman, 1970; Iwasaki and Miyata, 1989; scanning electron microscopy. Acta Anat., 128:140-146. Iwasaki, 199213; Iwasaki et al., 1992b). Iwasaki, S., K. Miyata, and K. Kobayashi 19871, The surface structure This study clearly indicated that the lingual epithe- of the dorsal epithelium of tongue in the mouse. Acta Anat. Nip- lium of the juvenile Hawskbill turtle is significantly pon., 6269-76. Iwasaki, S., T. Asami, Y. Asami, and K. Kobayashi 1992a Fine struc- different from that of the adult freshwater turtle. How- ture of the dorsal epithelium of the tongue of the Japanese ter- ever, because this study was limited to juveniles, fur- rapin, Clemmys juponicu (Chelonia, Emydinae). Arch. Histol. Cy- ther investigations are necessary to identify the simi- tol., 55:295-305. larities and differences between tongues of juvenile Iwasaki, S., H. Yoshizawa, and K. Suzuki 1992b Fine structure of the dorsal lingual epithelium of the Japanese monkey Macaca fus- and adult animals. cuta fuscata. Acta Anat., 144~267-277. Mao, S.H., J.J. Wang, S.C. Huang, C.F. Chao, and C.C. Chen 1991 LITERATURE CITED Ultrastructure of the tongue and anterior process of the sublin- Boshell, J.L., W.H. Wilborn, and B.B. Singh 1982 Filiform papillae of gual plica in four species of venomous snake. J. Morphol., 208: cat tongue. Acta Anat., 114:97-105. 279-292. Cane, A.K. and R.I.C. Spearman 1969 The keratinized epithelium of Rabinowitz, T. and B. Tandler 1986 Papillary morphology of the the house mouse (Mus musculus) tongue: its structure and his- tongue of the American chameleon, Anolis carolinensis. Anat. tochemistry. Arch. Oral Biol., 142329-841. Rec., 216483-489. 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