HISTOLOGICAL AND ULTRASTRUCTURAL CHANGES OF THE TONGUE IN RED-EARED TURTLE (TRACHEMYS SCRIPTA ELEGANS) AFTER HIBERNATION

Krzysztof Marycz¹, Joanna Klećkowska-Nawrot², Krzysztof Maksymowicz³, Krzysztof Kaliński^4
1 Electron Microscope Laboratory, Departament of Animal Hygiene and Ichthyology, Wrocław University of Environmental and Life Sciences, Poland
² Department of Anatomy and Histology, University of Environmental and Life Sciences in Wrocław, Poland
³ Department of Forensic Medicine, Medical University of Wrocław, Poland
⁴ Electron Microscope Laboratory, University of Environmental and Life Sciences in Wrocław, Poland

ABSTRACT

Hibernation is a key process conditioning breeding in turtles. It is also an intrinsic part of reptiles' life: the period in which animals do not ingest food and the way to survive unfavourable temperature conditions. Until now, the research has solely focused  on the morphological structure of the turtle's tongue in definite physiological conditions. The purpose of this study was to detect ultrastructural changes in hibernation in the red-eared turtle (Trachemys scripta elegans). The material was collected between the years 2004-2006 and came from the Zoological Garden in Poznan, Poland and from private breeders. The examined material included four red-eared turtle females 2–4 years old. The conducted research showed distinct morphological changes related to the distribution of papillae and microvilli on the tongue surface. during hibernation. It was found that loose interpapillary areas had formed and the loss of small, strongly degenerated, cuboidal cells was observed. The hibernation process led to closure of columnar mucous channels that had been open before. The disuse of the tongue for 3–4 months resulted in local destruction or apoptosis of the examined structures. The cuboidal cells lost their cellular capsule  which had been partly or entirely digested. As a result, only the skeleton of these cells remained. Microvilli that are found directly on the tongue papillae surface have decreased. They are differentiated in structure and often of irregular size. The macroscopic picture of the tongue of the red-eared turtle (T. scripta elegans) after the hibernation period exhibits a considerable hypertrophy of the connective tissue, though the position of the tongue in respect to the mandilular bones remains unchanged. After introducing turtles into hibernation the tongue does not show histophysiological activity, and cubicoidal cells are empty inside.

Key words: hibernation, red-eared turtle, lingual papillae, microvilli.

INTRODUCTION

The studies conducted (macroscopical, light microscopical and SEM) on reptiles' tongues [1,3,4,6,7,9,10,13,16,18,20,21] show great variability of the tongue as far as  the morphological structure is concerned. The research results are in large measure affected by  introducing  some reptiles into hibernation which is often an intrinsic part of their life and the way to survive unfavorable environmental conditions.

Hibernation slows down the metabolic rate and stops physiological functions of particular tongue parts.

The morphological structure of the reptiles' tongue is often a result of their adaptation to living in specific environmental conditions [8]. It largely depends on the kind of ingested food. In chameleons, for instance, the tongue serves as a prehensile organ [15]. This animal thrusts its long tongue forward at a distance equal to its body length. A club-like, sticky tongue catches the prey and it is instantly pulled back. The tongues of snakes and lizards are very agile. Their surface is covered by taste buds. In these reptiles the tongue plays an important role in orientation in the open area, hunting and identification of objects by transferring chemical substances to the vomeronasal organ (the Jacobson organ) [6,16,17].

In turtles the function of the tongue is considerably limited due to its little agility which is caused by the reduction of a substantial part of the tongue's muscles [5]. The aim of the present study was to analyze the tongue's ultrastructure in the red-eared turtle (T. scripta elegans) both before and after hibernation. The study also attempts to explain degradation of tongue cells during this process.

MATERIALS AND METHODS

Animal. The study was conducted on four red-eared turtle females 2–4 years old (family: Emydidae, subfamily: Deirochelyinae, genus: Trachemys, species: Scripta – subspecies: Elegans) with body weight ranging from  0.65–1.79 kg. The study material came from the Zoological Garden in Poznan and from private breeders. The turtles have been hibernated for four months.

Histology. The material was fixed in 4% formaldehyde solution (POCH) and next washed in water for 12h. Then it was dehydrated in a graded series of ethyl alcohol (30–100%). The material was paraffin-embedded and cut by means of HM 340E ultramicrotome (Zeiss, Walldort, Germany). HE staining was used. The specimens were examined under an Axio Imager A1LED light microscope (Zeiss, Oberkochen, Germany) at magnification 40x and 100x. The pictures of fungiform lingual papillae were taken with a Canon Power Shot A640 camera (Canon, Canada, USA).

Scanning electron microscopy. The research was conducted on the fresh material fixed in 2.5% glutaraldehyde on phosphate buffer of pH 7.4 The specimens were then dehydrated in a graded series of acetone 50–100%. Next the specimens were mounted on stages and sputtered with gold using a Scancoat 6 sputter (Edwards, London, England). The ultrastructure of the studied material was analyzed using a LEO ZEISS 435VP scanning microscope (Zeiss, Oberkochen, Germany). The morphometric examinations of papillae were carried out with the application of an AXIO VISION programme by ZEISS (ZEISS, Oberkochen, Germany).

RESULTS

The research conducted on the tongue of the red-eared turtle (T. scripta elegans) proved that hibernation results in 20% total body lost in all the examined individuals. It is caused by inhibition of metabolism and lack of food during hibernation.

The mean length of turtle tongues is 2.5 cm (the apex of the tongue – 0.4 cm, the body of the tongue – 1.5 cm, the root of the tongue – 0.6 cm) (Fig. a). In order to detect changes caused by  introduction of the reptiles into hibernation the cuboidal cells were measured. Before hibernation these cells are uniformly distributed on the entire tongue surface and are 12–24 µm long. Their mean width is 6–12 µm (Fig. b, g). Fungiform papillae situated on the apex of the tongue are on the average about 200 µm high. The length of glandular cells is 3–4 µm and the width – 1–3 µm (Fig. c). During hibernation considerable numbers of cuboidal cells are lost; their length is 3–12 µm, and width – 3–4 µm (Fig. f). On the surface of the fungiform papilla microvilli are well visible (Fig. e). Fungiform papillae situated on the apex of the tongue are closely packed leaving no free interpapillary spaces. Their size decreased due to disuse of the organ; after hibernation their length was 3–6 µm (Fig. d).

Fig. a. Red-Eared Turtle tongue before hibernation. x18

Fig. b. Surface of tongue before hibernation. x5000

Fig. c. Cross-section of turtles tongue before hibernation, fungiform papillae. x300

Fig. d. Glandular cell on the epithelial surface before hibernation. x8000

Fig. e. Ultarstructure of cross-section turtles epithelium before hibernation. x2000

Fig. f. Degenerated cuboidal cells after hibernation. x1000

Fig. g. Fungiform papilla before hibernation. HE. x40

Fig. h. Fungiform papilla after hibernation. HE. x40

Before hibernation slight concentration of mucus secreted by open columnar mucous cells is noticeable on the papillae surface (Fig. b). Small cuboidal cells are subject to degradation, marking distinctly the boundaries of intercellular space. Within these boundaries single sporadically occurring drops of fat are visible. Numerous microvilli of unknown function are found on the surface of fungiform lingual papillae.

On the tongue's cross-section four layers are clearly marked: papilla, connective tissue penetrating deep each fungiform papilla, muscular tissue covering the papilla and cartilage built in the tongue. It was found that in  turtles after hibernation the space between papillae became loose. A substantial loss of small, highly degenerated cuboidal cells was observed. Another observation was that salivary channels closed down. After introducing the turtles into hibernation one could also observe visible necrotic areas.

During hibernation the disuse of the tongue for 3–4 months leads to local destruction or apoptosis of the examined structures. The disuse of the organ results in lowering of microvilli on the surface of fungiform papillae. They are often of irregular size and different morphological appearance (Fig. f).

The macroscopic picture of the tongue after hibernation shows a considerable connective tissue hypertrophy. However, the position of the tongue remains unchanged in respect to the mandibular bones. It is important to note that the introduction of the reptiles into hibernation results in the loss of cuboidal cells (Fig. h). These structures lose their cellular capsule. They are partly or entirely digested which leads to the formation of the cells' skeleton (Fig. f). It proves that during hibernation the tongue does not display histophysiological activity and cuboidal cells are empty inside, which is confirmed by the morphological analysis under the scanning microscope and histological pictures.

DISCUSSION

The morphological structure of reptiles' tongue is extremely interesting. Until now many researchers have focused  on the ultrastructure [1,3,6,7,13] of the tongue of reptiles living in normal environmental conditions at relatively constant temperature (for most turtles – 25–35°C) and unchanging lifestyle.

The tongue of most reptiles is characterized by a well-developed system of muscles, which ensures its high agility (eg. chameleons). In lizards and snakes this organ serves as a sense organ enabling hunting and identification of objects by transferring chemical substances to the Jacobson organ. The tongue-vomeronasal complex functions in an extremely precise way, allowing the localization of some objects even at large distances. The monitor lizard, for instance, may detect an object from 8 km away on the basis of chemical compounds in the air [2,7,20]. In turtles and crocodiles the function of the tongue is considerably limited, due to the fact that a substantial part of lingual muscles is reduced limiting thus the mobility of the tongue [5].

The conducted research shows significant differences in the morphological structure of the tongue before and after hibernation. The examinations carried by means of scanning microscope (SEM) revealed a uniform distribution of cuboidal cells on the entire tongue surface in the red-eared turtle (T. scripta elegans).

The lingual papillae differ in size and shape. Being closely packed they either do not leave free interpapillary areas or this area is very narrow [3]. On the fungiform papillae surface one can observe columnar mucous cells which according to our research results are open prior to hibernation. No such cells were found in the Reeve`s turtle (Geoclemys reevesii), the soft-shell turtle (Trionyx cartilaginous), Malayan snail-eating turtle (Malayemys subtrijuga) where only fine granule cells were found [11,13].

Gustatory papillae (taste buds), similarly to those in other reptiles, play the main role in distinguishing taste [19,21]. This is often associated with the lifestyle, kind of ingested food and environment.

The turtles that live in aquatic environment possess a small, not very mobile tongue poor in lingual papillae. Such a tongue – deprived of lingual papillae – allows a free transport of food with the water current through the oral cavity, which takes place in all  Cryptodira [3].

Some other turtles, e.g. the Reeve's turtle (G. reevesii), the Malayan snail-eating turtle (M. subtrijuga) and the Japanese turtle (Clemmys japonica), which are both land and aquatic, have a relatively differentiated diet [21] and are able to feed in both land and aquatic environments.

Tortoises, which are land reptiles, are characterized by a considerable number of fungiform lingual papillae whose surface is covered by microvilli of different length and unknown function.

On the lingual surface of the red-eared turtle (T. scripta elegans) one can observe four cellular layers: papillae, connective tissue penetrating deep into the centre of each papilla, muscular tissue covering the papilla and cartilage built in the tongue.  Similar pattern is found in the Reeve's turtle (G. reevesii) and the Japanese turtle (C. japonica) [12].

In the red-eared turtle (T. scripta elegans) two types of cells were observed: plasmatic cells and basal cells [3] situated in the basal line. The plasmatic cells are not found in the Chinese turtle (G. reevesii), the Malayan turtle (M. subtrijuga) and the Japanese tortoise (C. japonica), where all the basal line cells are in close contact with each other [3,12,13].

The morphology of the tongue looks extremely interesting just after hibernation. Loose interpapillary areas are formed and the loss of small, highly degenerated cuboidal cells is observed. It can also be seen that the hibernation process leads to closure of columnar mucous channels which were open before. The conducted research showed that the disuse of the tongue for 3–4 months leads to the local destruction or apoptosis of the examined structures. The cuboidal cells lose their cellular capsule which is partly or entirely digested and, as a result, only the cells skeleton remains. Microvilli situated directly on the surface of lingual papillae decrease. They are often of irregular size and different structure. The macroscopic picture of the tongue of the red-eared turtle (T. scripta elegans) after the period of hibernation shows a considerable hypertrophy of the connective tissue, though the position of the tongue remains unchanged in respect to the mandibular bones. After the introduction of the turtles into hibernation the tongue does not display histophysiological activity, and the cuboidal cells are empty inside.

However, it seems that after waking  the turtles up from hibernation the tongue regains its initial agility, the lost cells are regenerated and the morphological structure returns to its original state.

CONCLUSIONS

Hibernation slows down the metabolic rate and stops physiological functions of particular tongue parts. In consequence morphological structure of all kind of tongue papilla's is changing in different direction. Hibernation is rebuilding ultrastructure of Turtles tongue.

REFERENCES

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11. Iwasaki, S., 1992. Fine structure of the dorsal epithelium of the tongue of the freshwater turtle, Geoclemys reevesii (Chelonia, Emydinae). J. Morphol. 211, 125–135.

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Accepted for print: 8.05.2009

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Krzysztof Marycz
Electron Microscope Laboratory,
Departament of Animal Hygiene and Ichthyology,
Wrocław University of Environmental and Life Sciences, Poland
Kożuchowska 5b, 50-375 Wrocław, Poland
Phone: +48 71 320 58 88
email: krzysztofmarycz@interia.pl

Joanna Klećkowska-Nawrot
Department of Anatomy and Histology,
University of Environmental and Life Sciences in Wrocław, Poland
Kożuchowska 1/3, 51-631 Wrocław, Poland
Phone: +48 71 320 57 44
email: lestat_v@poczta.onet.pl

Krzysztof Maksymowicz
Department of Forensic Medicine,
Medical University of Wrocław, Poland
Mikulicza-Radeckiego 4, 50-368 Wrocław, Poland
email: maks@forensic.am.wroc.pl

Krzysztof Kaliński
Electron Microscope Laboratory,
University of Environmental and Life Sciences in Wrocław, Poland
Kożuchowska 5b, 51-631 Wrocław, Poland

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