OkajimasLingual Folia PapillaeAnat. Jpn., of the87 (2):Common 65–73, raccoon August, 201065 Light and Scanning Electron Microscopic Study on the Tongue and Lingual Papillae of the Common raccoon, Procyon lotor By Yoshiko MIYAWAKI1, Ken YOSHIMURA1, Junji SHINDO2, and Ikuo KAGEYAMA1 1 Department of Anatomy, Faculty of Life Dentistry, The Nippon Dental University at Niigata, Niigata, Japan 2 Laboratory of Wildlife Science, Dept. of Environmental Bioscience, School of Veterinary Medicine, Kitasato University, Towada, Japan –Received for Publication, June 4, 2010– Key Words: Common raccoon, carnivore, lingual papillae, comparative anatomy, scanning electron microscopy Summary: We observed the external surface and connective tissue cores (CTCs), after exfoliation of the epithelium of the lingual papillae (filiform, fungiform, foliate and vallate papillae) of the common raccoon (Procyon lotor) using scanning electron microscopy and light microscopy. The tongue was elongated and their two-third width was almost fixed. Numer- ous filiform papillae were distributed along the anterior two-thirds of the tongue and fungiform papillae were distributed between the filiform papillae. Eight vallate papillae that had a weak circumferential ridge were distributed in a V-shape in the posterior part of the tongue and numerous taste buds were observable in the circumferential furrows of vallate papil- lae. Weak fold-like foliate papillae were observable at the lateral edge in the posterior part of the tongue and a few salivary duct orifices were observable beneath the foliate papillae. An islet-like structure with numerous taste buds, was observable on the deep part of the salivary duct of foliate papillae. Large conical papillae were distributed at the posterior part and root of the tongue. After removal of epithelium, filiform papillae of CTCs were appeared to be a thumb or cone-like main core and associating several finger-like short accessory cores. These cores were surrounded an oval concavity. The main core was situated behind the concavity and associated with accessory cores. CTCs of fungiform papillae were cylinder-like with numerous vertically running ridges and with a few concavities seen at the top of the cores. CTCs of vallate papillae and their surrounded circumferential ridge were covered with numerous pimple-like protrusions. The lingual papillae of Common raccoon’s tongue had morphological feature of carnivore species. The tongue of mammalian species has morphological composed of several families of bear-like carnivores, diversity i.e. distribution of the different types of lingual Arctoidea. Arctoid families include: Ursidae (bears) and papillae (Sonntag, 1925). In particular, variations in the Pinnipedia [Phocidae (true or earless seals), Otariidae three-dimensional structures of the lingual papillae and (eared seals), and Odobenidae (walruses)] and Musteroid; their connective tissue cores (CTCs) after removal of Ailuridae (Red Panda), Mephitidae (skunks), Procyoni- the overlying epithelium. The morphological diversity dae (raccoons) and Mustelidae (weasels) (Delisle and of mammalian species depends on the dietary habits and Strobeck, 2005; Flynn et al., 2005; Fulton and Strobeck, their living environment (Kobayashi et al., 1988a, 1989, 2006). 1992). There have been a number of morphological reports of Among mammalian families, Carnivora are widely lingual papillae and their connective tissue cores (CTCs), distributed and are traditionally divided at the suborder including several reports among Caniformia: Canidae level into Caniformia, dog-like carnivores, and Felifor- (Domestic dogs; Kobayashi et al., 1988b, 1992), (Rac- mia, cat-like carnivores. Dog-like carnivores, Caniformia coon dogs; Emura et al., 2006, 2008), Ursidae (Japanese are futher subdivided into two infraorders: Cynoidea (or black bears; Inatomi et al., 1999), Phocidae (Spotted Canoidea) comprising a single family, Canidae (dogs seals; Yoshimura et al., 2007) and Otariidae (California and foxes), sister to Arctoidea, a more diverse infraorder sea lion; Yoshimura et al., 2002), Musteroidea; Ailuridae Corresponding author: Ken Yoshimura, Dept. of Anatomy, Faculty of Life dentistry, The Nippon Dental University at Niigata, 1-8 Hamaura-cho, Chuo-ku, Niigata City, Niigata, 951-8580 Japan. E-mail: [email protected] 66 Y. Miyawaki et al. (Red Panda; Emura et al., 2009) and Mustelidae (Japanese badgers; Yoshimura et al., 2009, Japanese Marten; Emura et al., 2007), however, investigations of lingual papillae of the Procyonidae’s tongue have yet to be reported. The aim of this study was to investigate both the three-dimensional structures on the surface of the lingual papillae and their CTCs after exfoliation of the epithe- lium on the dorsal surface of tongue in detail of a Procy- onidae’s species, namely the Common raccoon (Procyon lotor). Materials and Methods Tissue specimens and preparation Five adult tongues of Common raccoons (Procyon lotor; four males and one female.) (Body length: 97–140 cm, Body weight: 26–28 kg) that died in extermination programs of invasive alien species were used for this study. Specimens were fixed with 10% formalin for post- mortem autopsy. Shortly afterwards, blocks were then cut from various parts of the tongue. Fig. 1. a: Overview of the dorsal surface of the tongue of the Com- mon raccoon (Procyon lotor). Scale bar = 1 cm, b: Diagram of Light microscopy the tongue; A: Apex, B: Anterior/Middle part. C: Posterior part. For light microscopy, specimens were dehydrated D: Root. Fi: Filiform papilla, Fo: Foliate papilla, Fu: Fungiform with a graded series of alcohol. Following dehydration, papilla, VP: Vallate papilla. specimens were embedded with paraffin and sectioned four micrometers thick. Sections were stained with hematoxylin-eosin (H-E). These slides were observed with a light-field microscope (BH-2, Olympus, Japan). papillae [Fig. 1; Fu] were distributed in the apex [Fig.1b; A], anterior [Fig.1b; B] and posterior part [Fig.1b; C]. Scanning Electron Microscopy Eight vallate papillae [Fig. 1b; VP] were distributed in For scanning electron microscopy, specimens were a V-shape in the posterior part [Fig.1b; C]. Weak fold- immersed in 3.5N-HCl for five days at 32°C. After im- like foliate papillae were observable at the lateral edge in mersion of the specimens, the epithelium was exfoliated the posterior part [Fig.1b; C] where a few orifices were from the underlying CTCs at the boundary of epithelium- found. Large conical papillae were distributed at the connective tissue layer. Specimens were washed with tap posterior part [Fig.1b; C] and root of the tongue [Fig.1b; water, and then treated with a 0.5% tannic acid solution. D]. Toward the posterior, large conical papillae increased Post fixation was accomplished by a ten-minute immer- in their size. sion in a 1% OsO4 solution. Specimens were then washed and dehydrated with a graded series of ethanol. 2) Microscopic Observation After dehydration, specimens were freeze-dried with a a) Filiform Papillae t-butyl alcohol freeze-drying method (Inoue and Osatake, Under lightmicroscopy, the epithelium of filiform 1988). Before observation, specimens were coated with papillae exhibited ortho-keratinization and had a thick Pt-Pd, and then observed with a scanning electron micro- stratum corneum, especially on the anterior part of scope (S-800, Hitachi-Hi-Technologies, Japan). the papilla with keratohyaline granules being observ- able [Fig. 2d; arrow] in the anterior part. No lingual glands were observable in the lamina propria of the Results anterior/middle part of the tongue, however, serous- rich lingual glands were found in the posterior part. 1) Macroscopic Overview Under scanning electron microscopic observation, In the macroscopic images, tongues of the Common the external appearance of the filiform papillae had a raccoon [Fig. 1] were elongated and their two-third width thick main conical protrusion and was associated with was almost fixed. A lingual prominence was absent. 7–20 accessory protrusions. In front of the filiform Numerous filiform papillae [Fig. 1; Fi] were distributed papilla, main protrusion appeared to be somewhat along the anterior two-thirds of the tongue. Fungiform concaved. Among the accessory protrusions, long pro- Lingual Papillae of the Common raccoon 67 Fig. 2. A set of lingual papillae distributed on the apex (indicated as “A” on Fig. 1b) part of the tongue. a: Light micrograph of fungiform papil- lae distributed on the apex of tongue (sagittal section). Taste buds (indicated as arrows) were observable on the top of the papilla. Scale bar = 100 µm. b: Scanning electron micrograph of the external surface of fungiform papilla situated on the apical part of tongue. Scale bar = 100 µm. c: SEM micrograph of the connective tissue core (CTC) of a fungiform papilla after epithelial exfoliated. Cylinder-like CTC was observable after removal of the epithelium. Several concavities were also seen at the top of the CTC. Scale bar = 100 µm. d: Light micro- graph of filiform papillae situated on the apical part (sagittal section). Thick stratum corneum of filiform papillae was seen. Keratohyalin granules were observable at the front part of the papillae (arrow). Scale bar = 100 µm. e: SEM micrograph of the epithelial surface of filiform papillae situated on the apical part of tongue. Main conical protrusion of filiform papillae inclined towards the posterior direction. Accessory protrusions appeared to be surrounding each main protrusion. Scale bar = 100 µm. f: CTCs of