Comparative Morphology of the Lingual Papillae and Their Connective Tissue Cores in the Tongue of the American Mink, Neovison vison Author(s): Ken Yoshimura, Yuko Fukue, Ryosuke Kishimoto, Junji Shindo and Ikuo Kageyama Source: Zoological Science, 31(5):292-299. 2014. Published By: Zoological Society of Japan DOI: http://dx.doi.org/10.2108/zs130214 URL: http://www.bioone.org/doi/full/10.2108/zs130214
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ZOOLOGICAL SCIENCE 31: 292–299 (2014)
¤ 2014 Zoological Society of Japan
Comparative Morphology of the Lingual Papillae and Their Connective Tissue Cores in the Tongue of the American Mink, Neovison vison Ken Yoshimura1*, Yuko Fukue2, Ryosuke Kishimoto3, Junji Shindo4, and Ikuo Kageyama1 1
Department of Anatomy, Faculty of Life Dentistry, The Nippon Dental University at Niigata, Niigata 951-8580, Japan 2 NPO Institute for Biodiversity Research and Education “Earthworm”, Karuizawa, Nagano 389-0115, Japan 3 Nagano Environmental Conservation Research Institute, Nagano, Nagano, 381-0075 Japan 4 Laboratory of Wildlife Science, Department of Environmental Bioscience, School of Veterinary Medicine, Kitasato University, Towada 034-8628, Japan
We observed the morphology of the lingual papillae (filiform, conical, fungiform, and vallate papillae, and lateral organ) and their connective tissue cores (CTCs) in the American mink (Neovison vison) using light and scanning electron microscopy. Filiform papillae were distributed on the apex linguae and rostral regions of the corpus linguae. Conical papillae were distributed over the caudal region and absent in the radix linguae. Numerous ridges were present in the radix linguae. Four to six vallate papillae were situated at the border between the corpus and radix linguae. Instead of foliate papillae, a pair of lateral organs was situated on the caudal edge of the corpus. The epithelial surface of each filiform papilla consisted of a single main process and 10–12 accessory processes. Notably, filiform papillae in the apex linguae exhibited morphological variation, and some were dome-like and lacked processes. In contrast, filiform papillae on the rostral part were not variable, were extended to a sharp tip, were associated with an eosinophilic stratum corneum, and lacked nuclei. The CTCs of the filiform papillae consisted of a main core and slender accessory cores surrounding a concavity. Those in the apex linguae were similar in appearance and consisted of main and adjacent accessory cores. The fungiform papillae had a dome-like epithelial surface and their CTCs were columnar, with upper concavities and flanges. The simplified lingual morphology of the American mink, particularly in the filiform papillae in the apex linguae, may be influenced by its diet and semiaquatic lifestyle. Key words:
American mink, tongue, lingual papillae, scanning electron microscopy, morphological diversity
INTRODUCTION The mammalian tongue exhibits morphological diversity among species, in terms of its shape, the size of the lingual body, and the distribution of the lingual papillae (Sonntag, 1920). Besides being determined by their phylogeny, morphological traits of the tongue and lingual papillae are influenced by dietary habits and living environment. The morphological diversity of the lingual papillae is evident in their connective tissue cores (CTCs) after removal of the overlying epithelium (Kobayashi et al., 1988, 1989, 1992). The mammalian family Caniformia, within the order Carnivora, contains well-known living representatives. Among carnivore species, feliforms are better optimized to eating * Corresponding author. Tel. : +81-25-267-1500; Fax : +81-25-267-1134; E-mail: [email protected] doi:10.2108/zs130214
meat than caniforms, which tend to be more omnivorous and opportunistic feeders (Ewer, 1973). Weasel-like carnivores, the Mustelidae, are caniforms and terrestrial hunters. They feed on small rodents, rabbits, birds, insects, lizards, and frogs, and most are purely carnivorous. Their teeth are highly adapted to killing and cutting up prey (Macdonald, 1984). The living environment of Mustelidae is highly varied and is associated with particular adaptations; although many species are terrestrial, the sea otter, for example, is highly aquatic. A number of studies have investigated the lingual papillae and their CTCs in Caniformia and have described morphological variability of both mechanical and gustatory papillae of several Mustelidae: the ferret (Emura, 2008; Takemura et al., 2009), Japanese marten (Emura et al., 2007), Siberian weasel (Emura, 2008), Japanese weasel (Furubayashi et al., 1989), Japanese badger (Yoshimura et al., 2009), and the sea otter (Shimoda et al., 1996). The American mink Neovison vison is a semi-aquatic
Lingual papillae of American mink
carnivorous mustelid, which preys on crayfish, crabs, and fish (Macdonald, 1984). In particular, Shimatani et al. (2008) reported a fecal DNA analysis of the American mink that revealed that it preys on the signal crayfish. The phylogenetic and taxonomic positions of the American mink have long been unclear (Kurose et al., 2008). Abramov (2000) and Kurose et al. (2008) suggested that it should be placed in a distinct mustelid genus, Neovison. Sato et al. (2012) investigated the phylogeny of Mustelidae using molecular techniques and concluded that the Neovison and Mustela are polyphyletic genera. However, there is a lack of information concerning the lingual papillae and their underlying CTCs in the American mink. The aim of this study was to analyze in detail the surface morphology of the lingual papillae on the dorsal surface of the tongue of the American mink before and after exfoliation of the epithelium of their CTCs, to compare them with descriptions of other terrestrial and aquatic Mustelidae. MATERIALS AND METHODS Animals and tissue preparation Nine American minks (six males and three females; body weights, males 1180–1720 g, females 645–900 g), which were culled as part of an extermination program of invasive alien species that took place in the areas of Saku city and Sakuho town in Minami-Saku county, Nagano Prefecture, Japan, were used in this study. The sampling procedure was carried out by Minami-SakuNanbu and Saku fishery cooperatives. The animals were fixed with 10% formalin (Wako Chemicals, Tokyo, Japan) for autopsy and, shortly afterward, tissue blocks were excised from various regions of the tongue. Age determinations were carried out following the method of Fukue and Kishimoto (2010) using teeth annuli analysis (Scheffer, 1950; Kleinenberg, 1966), morphological analysis of skull (Churcher, 1960) and baculum (Elder, 1951), and animals were categorized as either ‘sub-adult’ (less than 10 months after birth) or ‘adult’ (more than 11 months old, equivalent to sexually mature). Light microscopy The tissue samples were dehydrated with a graded ethanol series, embedded in paraffin wax, and sectioned at 4 μm. The sections were stained with hematoxylin-eosin (H-E) (Wako Chemicals) and observed using bright-field microscopy (BH-2, Olympus, Tokyo, Japan). Scanning electron microscopy Tissue samples were immersed in 3.5 N HCl (Wako Chemicals) for five days at room temperature (25–28°C). The epithelium was then exfoliated from the underlying CTCs. The specimens were washed with tap water and treated with a 0.5% tannic acid (SigmaAldrich, St. Louis, USA) solution. Post-fixation was accomplished by immersion for 10 min in 1% OsO4 (Merck, Darmstadt, Germany). The tissue was then washed and dehydrated with a graded ethanol series. After dehydration, specimens were freeze-dried using t-butyl alcohol (Inoue and Osatake, 1988), coated with Pt-Pd, and observed with a scanning electron microscope (SEM; S-800, Hitachi-Hi-Technologies, Tokyo, Japan). Representative micrographs shown in the figures are from a 23-month-old adult male specimen of body weight 1150 g.
RESULTS Macroscopic overview Macroscopically, the tongue of the American mink (Fig.
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Fig. 1. (A) Overview of the dorsal surface of the tongue of the American mink Neovison vison. Scale bar, 5 mm. (B) Diagram of the tongue. A, apex linguae; B, rostral; and C, caudal regions of the corpus linguae; D, radix linguae; Fu, fungiform papillae; Fi, filiform papillae; VP, vallate papillae; Lat, lateral organ; CP; conical papillae.
1) was elongated rostrocaudally, and the apex linguae was rounded. The torus linguae could not be distinguished. A dense distribution of filiform papillae (Fig. 1B; Fi) was observable over the entire dorsal surface of the tongue between the apex linguae (Fig. 1B; A) and the rostral region of the lingual body (Fig. 1B; B). A transitional distribution of conical papillae (Fig. 1B; CP) was evident between the caudal end of the corpus linguae (Fig. 1B; C) and the radix linguae (Fig. 1B; D). However, conical papillae became gradually diminished toward the radix linguae (Fig. 1B; D), and a weakly folded appearance was observable only proximal to the epiglottis. Fungiform papillae (Fu) were scattered over the apex linguae (A) and in the rostral region (B); a rather dense distribution of fungiform papillae was identified in the caudal region (C) of the corpus linguae (Fig. 1B). Neither a sulcus medianus linguae nor sulcus terminalis linguae could be discerned. Four to six vallate papillae (Fig. 1B; VP) were located in single V-shaped line at the boundary between the caudal part of the corpus linguae (Fig. 1B; C) and the radix linguae (Fig. 1B; D). Structures resembling lateral organs (Sonntag, 1920) could be distinguished at the lateral edge of the mid-end region (Fig. 1B; C) of the corpus linguae. We examined both female and male specimens; no evident sexual dimorphism was found. Microscopic observation Filiform papillae Light microscopy revealed that the epithelium of the filiform papillae exhibited weak keratinization. It was noted that the filiform papillae distributed on the apex linguae retained nuclei on the superficial layer (Fig. 2D). The filiform papillae on the rostral and caudal regions of the body exhibited an acidophilic stratum corneum on the uppermost and rostral aspects of the papillae (Fig. 4C). However, keratohyalin granules, which were generally evident on the posterior aspect of the papillae of filiform papillae, could not be observed throughout the dorsal surface of the tongue of this species.
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Fig. 2. Group of lingual papillae distributed on the apical part of the corpus linguae of N. vison (adult male, 23 months). (A) Light micrograph of a sagittal section of the fungiform papillae situated on the apex linguae (H-E stain). A taste bud (arrow) is present on the top of the papilla. Scale bar, 50 μm. (B) Scanning electron micrograph (SEM) of the external surface of the fungiform papillae distributed on the apex linguae. A flattened dome-like fungiform papilla is present among the fungiform papillae. Scale bar, 100 μm. (C) SEM of the connective tissue core (CTC) of a fungiform papilla situated on the apex linguae after epithelial exfoliation. At the top of the cylinder-like CTC, there is a flattened flange. Six concavities are observable. Scale bar, 50 μm. (D) Light micrograph of a sagittal section of filiform papillae distributed on the apex linguae. The uppermost layer of the filiform papillae retain nuclei; keratohyalin granules, usually evident on the caudal aspect of the filiform papillae, could not be distinguished. R, rostral. Scale bar, 100 μm. (E) SEM of the apex linguae exhibiting the epithelial surface of the filiform papilla. The rounded triangular processes of the filiform papillae are inclined caudally. A relatively thick ‘main’ process in the central region of the apex together is surrounded by approximately 12 short ‘accessory’ processes at the margin of the filiform papilla. Scale bar, 100 μm. (F) CTC of filiform papillae distributed on the apex linguae after removal of the epithelium. A rather long main process and approximately 12 accessory processes surround the concavity in front of the papilla. Scale bar, 50 μm.
The external surface view with the SEM revealed that each filiform papilla was associated basically with several accessory processes arranged around the rim of each main
Fig. 3. (A–F) A series of micrographs illustrating the morphological variation of the epithelial surface of filiform papillae distributed on the apex of the corpus linguae of N. vison (see Figs. 1B, 3A, Table 1). Although the external surface morphology of the filiform papillae linguae varied, the CTC of dome-like filiform papillae were identical in appearance (as in Fig. 2F). (D–F) show robust and slender main protrusions. Fu: Fungiform papilla. Scale bars, (A, C, F) 200 μm; (B, D, E) 100 μm.
protrusion. The length of these processes varied depending on the region: in the rostral region of the corpus linguae, the main process of the filiform papillae was elongated vertically to a sharp tip (Fig. 4D), which was clearly distinguishable from the neighboring accessory processes. However, it was notable that the filiform papillae varied among different specimens, particularly on the apex linguae (Figs. 2E, 3A–F); some had a dome-like appearance (Fig. 3A), while others were finger-like, with a robust or slender main protrusion (3D–F). Moreover, one specimen exhibited a mixed type of arrangement comprising both domed and finger-like filiform papillae (Fig. 3B). In another transitional pattern of domed and finger-like types of filiform papillae, the main process was short and quite similar to the neighboring accessory processes (Figs. 2E, 3C). Interestingly, these morphological variations of filiform papillae were observable only within the apex linguae. Furthermore, age-analysis of the teeth annuli and the morphology of the skull and bacu-
Lingual papillae of American mink Table 1.
a b c d e f
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Details presented in Fig. 3.
Gender
Body weight
male female female male male female
1180 g 645 g 810 g 1260 g 1200 g 900 g
Results of age determination analysis Adult Adult Subadult Adult Subadult Subadult
10–11 months 7 years 10 months 22–23 months 7 months 10 months
form papillae basically reflected the structure of the overlying epithelial surface; each CTC of the filiform papillae on the apex linguae consisted of finger-like protrusions that surrounded the rim of each main core (Fig. 2F). The CTC had a concavity in the center of the main core. Despite the morphological variation of the filiform papillae distributed on the surface of the apex linguae, the morphology of the corresponding CTCs was relatively uniform, consisting of fingerlike protrusions that surrounded the rim of each main core (e.g. Fig. 3A). On the apex linguae, the main CTC processes were rather thick and comparable with the neighboring accessory cores (Fig. 2F). By contrast, the main CTC processes in the rostral region were more prominent and the accessory processes were short (Fig. 3E). The filiform papillae were approximately 180–700 μm long and 180–380 μm wide.
Fig. 4. Set of fungiform and filiform papillae distributed on the rostral (see Figs. 1B; 4B) part of the corpus linguae of N. vison (adult male, 23 months). (A) SEM of the epithelial surface of the fungiform papillae in the rostral region of the corpus. The fungiform papillae in this area are somewhat compressed and present a trapezoid appearance. Scale bar, 100 μm. (A) SEM of the CTCs of the fungiform papillae of the rostral part of the corpus after removal of the epithelium. The CTCs of the fungiform papillae in this area possess numerous frill-like longitudinal processes at the side of the CTC. Several concavities are also observable at the top of the CTCs. Scale bar, 100 μm. (C) Light micrograph of a sagittal section of the filiform papillae distributed on the rostral region of the body. The filiform papillae have an overlying stratum corneum in the posterior aspect of the papillae (arrow). However, no keratohyalin granules were evident in the posterior aspect of the papillae. R, rostral. Scale bar, 100 μm. (D) SEM of the epithelial surface of the filiform papillae situated in the rostral region of the corpus. The filiform papillae in this area have one obvious main process and four or five neighboring accessory processes. Scale bar, 100 μm. (E) SEM of the CTCs of the filiform papillae in the rostral region observed after epithelial exfoliation. The marginal ridges of the CTCs of the filiform papillae and main and accessory cores are relatively thin. Scale bar, 100 μm.
lum (Table 1) showed that the above-mentioned morphological variations in the filiform papillae (Fig. 3A–F) were not systematically related to age. After exfoliation of the epithelium, the CTCs of the fili-
Conical papillae Conical papillae (Fig. 1B; CP) were distributed between the caudal end of the rostral part (Fig. 1B; C) of the corpus linguae and radix linguae (Fig. 1B; D). Under light microscopy, the conical papillae had a stratum corneum, which lacked nuclei in the uppermost part of the posterior portion (Fig. 6A), and keratohyalin granules could not be distinguished. By contrast, a thickened epithelium was evident in the anterior aspect of the papilla. In SEM observations, the epithelial surface of each conical papilla appeared smooth and conical, with a sharp tip (Fig. 6B). After exfoliation of their epithelium, the CTCs were seen to be conical or fingerlike, and there were numerous protrusions in the basal twothirds of the core (Fig. 6C). The conical papillae were approximately 460–650 μm long and 320–380 μm wide. Fungiform papillae Light microscopy revealed the fungiform papillae as dome-like (Fig. 2A). Keratinization of these papillae was weak. A few taste buds were evident in the epithelium at the tips of the fungiform papillae. In SEM observations, the external surfaces of the fungiform papillae distributed on both the apex linguae (Fig. 2B) and the rostral part of the corpus linguae (Fig. 4A) were smooth and dome-like. After epithelial exfoliation, the CTCs of the fungiform papillae on the apex linguae (Fig. 2C) were columnar and those of the rostral part of the corpus linguae (Fig. 4A) had a peduncular appearance. The CTCs of both types of fungiform papillae had several vertically aligned ridges and a concavity on the uppermost surface (Figs. 2C, 4B). The fungiform papillae were approximately 120–300 μm in diameter. Lateral organ Structures resembling lateral organs (Sonntag, 1920)
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Fig. 5. Set of lateral organs distributed on the caudal section of the corpus linguae (see Figs. 1B, 5C) and radix linguae (see Figs. 1B, 5D) of N. vison (adult male, 23 months). (A) Sagittal section of a lateral organ on the caudal area of the corpus. Taste buds are not observable in this area. Mucus-rich lingual glands (G) are present in the lamina propria. Scale bar, 500 μm. (B) SEM of the CTCs of the lateral organ situated on the caudal area of the corpus after epithelial exfoliation. Orifices of the mucus-rich lingual gland (arrows) are distributed at the posterior part of the lateral organ. Numerous shallow grooves run laterally from the lateral side of the lateral organ. Scale bar, 1000 μm. (C) A sagittal section of a vallate papilla stained by H-E. Numerous taste buds (arrows) are present in the inner wall of the epithelium of the circumferential furrow. Lingual glands similar to von Ebner’s glands (G) are located in the lamina propria beneath the papilla. Scale bar, 300 μm. (D) SEM of the epithelial surface of the vallate papillae. A circumferential furrow and weak ridge are present around the papillae. Scale bar, 300 μm. (E) SEM of the vallate papillae after epithelial exfoliation. The CTCs of the circumferential furrow were more evident than were visible on the epithelial surface. Numerous CTC protrusions are distributed on the circumferential furrow and ridge. Scale bar, 300 μm.
Fig. 6. Group of conical papillae distributed on the lingual mucosa of the radix linguae (see Figs. 1B, 6D) of N. vison (adult male, 23 months). (A) Sagittal section of the conical papillae stained by H-E. An eosin-intensive stained stratum corneum is observable at the posterior of the papilla. Scale bar, 200 μm. (B) SEM of the epithelial surface of the conical papillae. The surface is smooth and has a conical appearance. R, rostral. Scale bar, 200 μm. (C) SEM of the conical papillae after removal of the epithelium. Numerous thumblike CTCs are distributed on the surface. The lower two-thirds of each conical CTC has a rough appearance and is covered by densely studded short protrusions. Scale bar, 200 μm. (D) Sagittal section of the radix linguae stained by H-E. The epithelial surface is rather wavy. Mucus-rich lingual glands (G) can be seen. Scale bar, 200 μm. (E) SEM of the radix linguae after partial epithelial exfoliation. Some orifices of mucus-rich lingual glands (arrows) are present. Numerous ridges are observable at the lateroposterior end. Scale bar, 200 μm.
were observable on the lateral edge of the mid-end region (Fig. 1B; C) of the corpus linguae. Light microscopy revealed a broader ridge-like structure (Fig. 5A). Taste buds could not be identified. Mucus-rich lingual glands were present in the lamina propria. SEM observation of the CTCs showed a serrated structure with shallow horizontal grooves.
Vallate papillae Four or five vallate papillae (Fig. 1B; VP) were situated at the boundary between the caudal section of the corpus (Fig. 1B; C) and the radix linguae (Fig. 1B; D). Light microscopy showed numerous taste buds distributed vertically in the inner epithelial wall of the circumferential furrow. Gusta-
Several orifices of the lingual glands were distributed on the posterior part of the serrated edge (Fig. 5B).
Lingual papillae of American mink
tory glands could be seen in the lamina propria (Fig. 5C; G), with their orifices opening into the base of the circumferential sulcus. In SEM preparations, the external surfaces of the vallate papillae were round or obliquely elongated in the rostrolateral direction. Each dome-like papilla was surrounded by a circumferential ridge and furrow. After exfoliation of the epithelium, the CTCs of the vallate papillae appeared domelike and were surrounded by a deep ridge-like core in the circumferential furrow (Fig. 5E). The vallate papillae were approximately 1020–1500 μm in diameter. Radix linguae The radix linguae exhibited a weakly folded appearance (Fig. 1B; D). Light microscopy revealed large mucus-rich mixed glandulae linguales in the lamina propria (Fig. 6D; G). After removal of the epithelium, the surface of the CTCs of the radix linguae exhibited a mesh-like ridge (Fig. 6E). Orifices of glandulae linguales were observed among the mesh-like CTCs. DISCUSSION Our observations indicate that the lingual papillae of the semiaquatic American mink possess a number of unique morphological features while having morphological similarity to those of terrestrial species of Mustelidae. The external morphology of the filiform papillae, particularly those on the apex linguae, was variable and some exhibited a very simplified (reduced) structure in comparison with those of previously studied terrestrial mustelids. The dome-like filiform papillae of the apex linguae of some American mink resembled the filiform papillae in the apex linguae of newborn sea otters, as reported by Shimoda et al. (1996). Fukue and Kishimoto (2010) made external measurements of large numbers of adult and subadult American minks (125 males 400–2397 g; 66 females 592–1086 g) captured in the same area. Based on these measurements, the individuals in our study that exhibited dome-like filiform papillae in the apex linguae clearly were not newborn, in contrast to the sea otters studied by Shimoda. Furthermore, our analyses of tooth annuli and of skull and baculum morphology indicated that the variation among the filiform papillae in our study was not related to age. These data suggest that the dome-like external morphology of filiform papillae, which was sometimes present in adults, depends on the individual. On other epithelial surfaces of the tongue of the American mink, the filiform papillae exhibited transitional morphology between ‘dome’ and ‘finger’ types; some were comparatively short processes and others exhibited clear a finger-like morphology. It could be speculated that the morphological variation among the individuals investigated in this study resulted from crossbreeding. The American mink population has expanded in the Nagano region during the last 25 years, and many individuals are likely to have escaped 15 years ago when the fur farm was closed (Shimatani et al., 2010). However, Shimatani et al. (2010) investigated the genetic variation of American mink captured in this area and concluded that the genetic diversity of present individuals was relatively low. Therefore, the morphological variation of the filiform papillae found in the apex linguae may be less influenced by genetic factors than by the dietary diversity associated with their semi-
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aquatic lifestyle (e.g., consumption of freshwater crayfish) (Shimatani et al., 2008). In terrestrial Mustelidae, the filiform papillae on the apex linguae exhibit a distinct morphology in which main and accessory processes are present. In the Japanese weasel (Furubayashi et al., 1989), Siberian weasel (Emura et al., 2007), and ferret (Emura, 2008; Takemura et al., 2009), which are closely related taxa in the Mustelinae (Sato et al., 2012), the epithelial surface of these papillae has a long main process and associated small accessory processes. In less closely related taxa, such as the Japanese marten (Guloninae, Mustelidae), the filiform papillae on the apical surface of the tongue have a main process and long hairy accessory processes (Emura et al., 2007). In other less closely related taxa such as the Japanese badger (Melinae, Mustelidae), the external surface of the filiform papillae on the apical surface of the tongue has an even longer main process and long accessory processes (Yoshimura et al., 2009). The present observations indicate that the external form of the filiform papillae in the American mink also conforms to this basic plan of main and accessory processes, which is observable among the filiform papillae distributed on both apical and rostral surfaces. Among aquatic mammals, particularly the pinnipeds, the external surface of the lingual papillae is usually smooth and without accessory protrusions. Yoshimura et al. (2002) reported that the outer surface of the filiform papillae of a pinniped, the California sea lion, consisted of a smooth conical main process and lacked accessory processes. In another pinniped, the spotted seal, the external surface of the filiform papillae was clear and cone-shaped (Yoshimura et al., 2007). A smooth, simplified external appearance of the filiform papillae has also been observed among the highly aquatic Mustelidae. Shimoda et al. (1996) observed the epithelial structure of the lingual papillae distributed over the lingual mucosa of the tongue of newborn sea otters. Micrographs from that study show the surface texture of the filiform papillae epithelium to be conical, completely smooth, and without accessory protrusions. Furthermore, simple dome-like filiform papillae were observable in the anterior part of the tongue. Macdonald (1984) described the American mink as semiaquatic; the animal has a carnivorous diet and preys on crayfish, crabs, and fish. The specimens in the present study were living close to rivers in mountainous regions and were associated with a semiaquatic environment. However, the structure of the CTCs of the filiform papillae on the apex linguae of the American mink resembled that of the more terrestrial Mustelinae and Guloninae. As in the present study of the American mink, SEM micrographs of the filiform CTCs on the apex linguae of other Mustelinae and Guloninae species exhibited rod-like cores, consisting of main and accessory cores surrounding a cavity. In contrast, in the less closely related Japanese badger (Melinae), the filiform CTCs on the apex linguae have a somewhat different morphology in which both main and accessory cores are elongated (Yoshimura et al., 2009). We observed 4–6 vallate papillae located in single Vshaped line at the boundary between the caudal part of the corpus linguae and the radix linguae. In a previous account of mustelid species, Takemura et al. (2009) reported that the
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ferret had 8–12 vallate papillae, uniquely arranged in two or three V-shaped lines. A diagram by Furubayashi et al. (1989) shows 4–5 vallate papillae arranged in a double Vshape in the Japanese weasel. In contrast, in the Japanese marten (Emura et al., 2007) there are four, in the Siberian weasel (Emura et al., 2007) and Sea Otter (Shimoda et al., 1996) six, and in the Japanese badger (Yoshimura et al., 2009) seven vallate papillae arranged in a single V-shaped line. In the present study, instead of foliate papillae, a pair of lateral organs was found situated on the caudal edge of the corpus linguae. In our observations of their CTCs, we identified a serrated structure with shallow horizontal grooves. Several orifices of the lingual glands were distributed at the posterior end of the serrated edge and mucus-rich lingual glands were present in the lamina propria. Generally, under the sulci of foliate papillae lie serous gustatory glands (similar to von Ebner’s glands in humans), the ducts of which empty into the sulci (Eurell and Frapper, 2006). The orifices of mucus-rich lingual glands in this region are a unique finding and their contribution to gustatory function is unclear. In a previous description of mustelids (Takemura et al., 2009), no ridges and furrows equivalent to the foliate papillae were recognized. However, in that study a micrograph of the lateral margin of the posterior tongue body shows parallel shallow grooves and serrated structures. Although they did not recognize taste buds, a slit-like groove related to foliate papillae is evident in micrographs of the Japanese marten (Emura et al., 2007). The Siberian weasel (Emura et al., 2007) also had similar shallow grooves in this region in spite of being described as lacking foliate papillae. Moreover, Furubayashi et al. (1989) described the Japanese weasel as having foliate papillae that consisted of four to five slit-like grooves on each side of the lateral margin of the radix linguae, and several taste buds were observed in the cleft epithelium. The serous lingual glands (similar to von Ebner’s gland) were situated beneath the foliate papillae. Therefore, foliate papillae may be present in other weasel species but not in the American mink. However, information about the vallate papillae, lateral organs and foliate papillae remains incomplete and further study, including a detailed histological analysis, is required. The unique features of the lingual morphology of the American mink described above imply that these traits may have been influenced more by the semiaquatic living environment of this species than by its phylogeny. However, the differences appear to relate only to the epithelia and the structures of the underlying CTCs in the American mink are similar to those among other Mustelinae. The oral cavity is the entrance to the digestive tract (Bloom and Fawcett, 1994) and it is important in the prehension, mastication, and deglutition of food (Eurell and Frapper, 2006). This implies that the oral cavity could be influenced by dietary habits and living environment as well as morphological traits that arise from their phylogenetic position. Therefore, it is important to understand the comparative morphology of the oral cavity among other mammal species. Further detailed morphological studies coupled with developmental and molecular investigations are required to fully elucidate the basis of the diversity of lingual papillae of the American mink.
ACKNOWLEDGMENTS We are grateful to members of Minami-Saku-Nanbu and Saku fishery cooperatives and Nagano Prefecture for supplying autopsy materials. We also thank Mr. Narumi Kouno (Nagano Prefectural Fisheries Experimental Station Saku Branch) and Ms. Miyuki Sato for their support in sampling specimens. This work was supported by a research promotion grant-in-aid (NDUF-09-14) from the Nippon Dental University.
REFERENCES Abramov AV (2000) A taxonomic review of the genus Mustela (Mammalia, Carnivora). Zoosyst Rossica 8: 357–364 Bloom W, Fawcett DE, Raviola E (1994) A Textbook of Histology 12th ed, Chapman & Hall, New York, pp 559–561 Churcher CS (1960) Cranial variation in the North American red fox. J Mammal 41: 349–360 Elder WH (1951) The baculum as an age criterion in mink. J Mammal 32: 43–50 Emura S (2008) SEM studies on the lingual papillae and their connective tissue cores of the ferret and Siberian weasel. Medicine and Biology 152: 48–56 Emura S, Okumura T, Chen H (2007) Morphology of the lingual papillae in the Japanese marten. Okajimas Folia Anat Jpn 84: 77–82 Eurell JA, Frapper BL (2006) Dellmann’s Textbook of Veterinary Histology 6th ed, Blackwell, Oxford, pp 174–177 Ewer RF (1973) The Carnivores. Cornell University Press, New York, pp 139–189 Fukue Y, Kishimoto R (2010) The external measurements of American mink Neovision vision controlled in the basin of the Chikuma river, Nagano Prefecture. Bull Nagano Conserv Res Inst 6: 35–43 Furubayashi R, Sato E, Ishibashi T (1989) Histological pattern of the tongue in the Japanese weasels, Mustela itatsi, with special reference to the morphology and distribution of papillae, taste buds and lingual glands. Kaibogaku Zasshi. 64: 210–214 Inoue T, Osatake H (1988) A new drying method of biological specimens for scanning electron microscopy: The t-butyl alcohol freeze-drying method. Arch Histol Cytol 51: 53–59 Kleinenberg SE, Klevezal GA (1966) Age determination in mammals by the structure of tooth cement (in Russian) Zool Shur 45: 717–724 Kobayashi K (1992) Comparative anatomical studies on the tongues with special reference to the connective tissue cores of the lingual papillae. Odontology 80: 661–678 Kobayashi K, Iwasaki S (1989) Comparative studies on the stereo architecture of the connective tissue papillae in some mammalian tongues. In “Cell and Tissue: A Three-Dimensional Approach by Modern Techniques on Microscopy” Ed by PM Motta, Alan R. Liss, New York, pp 303–308 Kobayashi K, Miyata K, Iwasaki S, Takahashi K (1988) Threedimensional structure of the connective tissue papillae of cat lingual papillae. Jpn J Oral Biol 30: 719–731 Kurose N, Abramov AV, Masuda R (2008) Molecular phylogeny and taxonomy of the genus Mustela (Mustelidae, Carnivora), inferred from mitochondrial DNA sequences: New perspectives on phylogenetic status of the back-striped weasel and American mink. Mammal Study 33: 25–33 Macdonald DW (1984) The Encyclopedia of Mammals. Facts on File Publications, New York, pp 108–132 Sato JJ, Wolsan M, Prevosti FJ, D’Eli’a G, Begg C, Begg K, et al. (2012) Evolutionary and biogeographic history of weasel-like carnivorans (Musteloidea). Mol Phylogenet Evol 63: 745–57 Scheffer VB (1950) Growth layers on the teeth of Pinnipedia as an indication of age. Science 112: 309–311
Lingual papillae of American mink Shimatani Y, Takeshita T, Tatsuzawa S, Ikeda T, Masuda R (2006) Genetic identification of mammalian carnivore species in the Kushiro wetland, eastern Hokkaido, Japan, by analysis of fecal DNA. Zool Sci 25: 714–720 Shimatani Y, Fukue Y, Kishimoto R, Masuda R (2010) Genetic variation and population structure of the feral American mink (Neovison vison) in Nagano, Japan, revealed by microsatellite analysis. Mammal Study 35: 1–7 Shimoda T, Nakanishi E, Yoshino S, Kobayashi S (1996) Light and scanning electron microscopic study on the lingual papillae in the newborn sea otter Enhydra lutris. Okajimas Folia Anat Jpn 73: 65–74 Sonntag CF (1920) The comparative anatomy of the tongues of the mammalia. I. General description of the tongue. Proc Zool Soc Lond 1920; IX: 115–129 Takemura A, Uemura M, Toda I, Fang G, Hidaka M, Suwa F (2009)
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Morphological study of the lingual papillae in the ferret (Mustela putorius furo). Okajimas Folia Anat Jpn 86: 17–24 Yoshimura K, Shindoh J, Kobayashi K (2002) Scanning electron microscopy study of the tongue and lingual papillae of the California sea lion (Zalophus californianus californianus). Anat Rec 267: 146–153 Yoshimura K, Shindo J, Miyawaki Y, Kobayashi K, Kageyama I (2007) Scanning electron microscopic study on the tongue and lingual papillae of the adult Spotted seal, Phoca largha. Okajimas Folia Anat Jpn 84: 83–97 Yoshimura K, Shindo J, Kageyama I (2009) Light and scanning electron microscopic study on the tongue and lingual papillae of the Japanese badgers, Meles meles anakuma. Okajimas Folia Anat Jpn 85: 119–127 (Received October 24, 2013 / Accepted January 21, 2014)
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ZOOLOGICAL SCIENCE 31: 292–299 (2014)
¤ 2014 Zoological Society of Japan
Comparative Morphology of the Lingual Papillae and Their Connective Tissue Cores in the Tongue of the American Mink, Neovison vison Ken Yoshimura1*, Yuko Fukue2, Ryosuke Kishimoto3, Junji Shindo4, and Ikuo Kageyama1 1
Department of Anatomy, Faculty of Life Dentistry, The Nippon Dental University at Niigata, Niigata 951-8580, Japan 2 NPO Institute for Biodiversity Research and Education “Earthworm”, Karuizawa, Nagano 389-0115, Japan 3 Nagano Environmental Conservation Research Institute, Nagano, Nagano, 381-0075 Japan 4 Laboratory of Wildlife Science, Department of Environmental Bioscience, School of Veterinary Medicine, Kitasato University, Towada 034-8628, Japan
We observed the morphology of the lingual papillae (filiform, conical, fungiform, and vallate papillae, and lateral organ) and their connective tissue cores (CTCs) in the American mink (Neovison vison) using light and scanning electron microscopy. Filiform papillae were distributed on the apex linguae and rostral regions of the corpus linguae. Conical papillae were distributed over the caudal region and absent in the radix linguae. Numerous ridges were present in the radix linguae. Four to six vallate papillae were situated at the border between the corpus and radix linguae. Instead of foliate papillae, a pair of lateral organs was situated on the caudal edge of the corpus. The epithelial surface of each filiform papilla consisted of a single main process and 10–12 accessory processes. Notably, filiform papillae in the apex linguae exhibited morphological variation, and some were dome-like and lacked processes. In contrast, filiform papillae on the rostral part were not variable, were extended to a sharp tip, were associated with an eosinophilic stratum corneum, and lacked nuclei. The CTCs of the filiform papillae consisted of a main core and slender accessory cores surrounding a concavity. Those in the apex linguae were similar in appearance and consisted of main and adjacent accessory cores. The fungiform papillae had a dome-like epithelial surface and their CTCs were columnar, with upper concavities and flanges. The simplified lingual morphology of the American mink, particularly in the filiform papillae in the apex linguae, may be influenced by its diet and semiaquatic lifestyle. Key words:
American mink, tongue, lingual papillae, scanning electron microscopy, morphological diversity
INTRODUCTION The mammalian tongue exhibits morphological diversity among species, in terms of its shape, the size of the lingual body, and the distribution of the lingual papillae (Sonntag, 1920). Besides being determined by their phylogeny, morphological traits of the tongue and lingual papillae are influenced by dietary habits and living environment. The morphological diversity of the lingual papillae is evident in their connective tissue cores (CTCs) after removal of the overlying epithelium (Kobayashi et al., 1988, 1989, 1992). The mammalian family Caniformia, within the order Carnivora, contains well-known living representatives. Among carnivore species, feliforms are better optimized to eating * Corresponding author. Tel. : +81-25-267-1500; Fax : +81-25-267-1134; E-mail: [email protected] doi:10.2108/zs130214
meat than caniforms, which tend to be more omnivorous and opportunistic feeders (Ewer, 1973). Weasel-like carnivores, the Mustelidae, are caniforms and terrestrial hunters. They feed on small rodents, rabbits, birds, insects, lizards, and frogs, and most are purely carnivorous. Their teeth are highly adapted to killing and cutting up prey (Macdonald, 1984). The living environment of Mustelidae is highly varied and is associated with particular adaptations; although many species are terrestrial, the sea otter, for example, is highly aquatic. A number of studies have investigated the lingual papillae and their CTCs in Caniformia and have described morphological variability of both mechanical and gustatory papillae of several Mustelidae: the ferret (Emura, 2008; Takemura et al., 2009), Japanese marten (Emura et al., 2007), Siberian weasel (Emura, 2008), Japanese weasel (Furubayashi et al., 1989), Japanese badger (Yoshimura et al., 2009), and the sea otter (Shimoda et al., 1996). The American mink Neovison vison is a semi-aquatic
Lingual papillae of American mink
carnivorous mustelid, which preys on crayfish, crabs, and fish (Macdonald, 1984). In particular, Shimatani et al. (2008) reported a fecal DNA analysis of the American mink that revealed that it preys on the signal crayfish. The phylogenetic and taxonomic positions of the American mink have long been unclear (Kurose et al., 2008). Abramov (2000) and Kurose et al. (2008) suggested that it should be placed in a distinct mustelid genus, Neovison. Sato et al. (2012) investigated the phylogeny of Mustelidae using molecular techniques and concluded that the Neovison and Mustela are polyphyletic genera. However, there is a lack of information concerning the lingual papillae and their underlying CTCs in the American mink. The aim of this study was to analyze in detail the surface morphology of the lingual papillae on the dorsal surface of the tongue of the American mink before and after exfoliation of the epithelium of their CTCs, to compare them with descriptions of other terrestrial and aquatic Mustelidae. MATERIALS AND METHODS Animals and tissue preparation Nine American minks (six males and three females; body weights, males 1180–1720 g, females 645–900 g), which were culled as part of an extermination program of invasive alien species that took place in the areas of Saku city and Sakuho town in Minami-Saku county, Nagano Prefecture, Japan, were used in this study. The sampling procedure was carried out by Minami-SakuNanbu and Saku fishery cooperatives. The animals were fixed with 10% formalin (Wako Chemicals, Tokyo, Japan) for autopsy and, shortly afterward, tissue blocks were excised from various regions of the tongue. Age determinations were carried out following the method of Fukue and Kishimoto (2010) using teeth annuli analysis (Scheffer, 1950; Kleinenberg, 1966), morphological analysis of skull (Churcher, 1960) and baculum (Elder, 1951), and animals were categorized as either ‘sub-adult’ (less than 10 months after birth) or ‘adult’ (more than 11 months old, equivalent to sexually mature). Light microscopy The tissue samples were dehydrated with a graded ethanol series, embedded in paraffin wax, and sectioned at 4 μm. The sections were stained with hematoxylin-eosin (H-E) (Wako Chemicals) and observed using bright-field microscopy (BH-2, Olympus, Tokyo, Japan). Scanning electron microscopy Tissue samples were immersed in 3.5 N HCl (Wako Chemicals) for five days at room temperature (25–28°C). The epithelium was then exfoliated from the underlying CTCs. The specimens were washed with tap water and treated with a 0.5% tannic acid (SigmaAldrich, St. Louis, USA) solution. Post-fixation was accomplished by immersion for 10 min in 1% OsO4 (Merck, Darmstadt, Germany). The tissue was then washed and dehydrated with a graded ethanol series. After dehydration, specimens were freeze-dried using t-butyl alcohol (Inoue and Osatake, 1988), coated with Pt-Pd, and observed with a scanning electron microscope (SEM; S-800, Hitachi-Hi-Technologies, Tokyo, Japan). Representative micrographs shown in the figures are from a 23-month-old adult male specimen of body weight 1150 g.
RESULTS Macroscopic overview Macroscopically, the tongue of the American mink (Fig.
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Fig. 1. (A) Overview of the dorsal surface of the tongue of the American mink Neovison vison. Scale bar, 5 mm. (B) Diagram of the tongue. A, apex linguae; B, rostral; and C, caudal regions of the corpus linguae; D, radix linguae; Fu, fungiform papillae; Fi, filiform papillae; VP, vallate papillae; Lat, lateral organ; CP; conical papillae.
1) was elongated rostrocaudally, and the apex linguae was rounded. The torus linguae could not be distinguished. A dense distribution of filiform papillae (Fig. 1B; Fi) was observable over the entire dorsal surface of the tongue between the apex linguae (Fig. 1B; A) and the rostral region of the lingual body (Fig. 1B; B). A transitional distribution of conical papillae (Fig. 1B; CP) was evident between the caudal end of the corpus linguae (Fig. 1B; C) and the radix linguae (Fig. 1B; D). However, conical papillae became gradually diminished toward the radix linguae (Fig. 1B; D), and a weakly folded appearance was observable only proximal to the epiglottis. Fungiform papillae (Fu) were scattered over the apex linguae (A) and in the rostral region (B); a rather dense distribution of fungiform papillae was identified in the caudal region (C) of the corpus linguae (Fig. 1B). Neither a sulcus medianus linguae nor sulcus terminalis linguae could be discerned. Four to six vallate papillae (Fig. 1B; VP) were located in single V-shaped line at the boundary between the caudal part of the corpus linguae (Fig. 1B; C) and the radix linguae (Fig. 1B; D). Structures resembling lateral organs (Sonntag, 1920) could be distinguished at the lateral edge of the mid-end region (Fig. 1B; C) of the corpus linguae. We examined both female and male specimens; no evident sexual dimorphism was found. Microscopic observation Filiform papillae Light microscopy revealed that the epithelium of the filiform papillae exhibited weak keratinization. It was noted that the filiform papillae distributed on the apex linguae retained nuclei on the superficial layer (Fig. 2D). The filiform papillae on the rostral and caudal regions of the body exhibited an acidophilic stratum corneum on the uppermost and rostral aspects of the papillae (Fig. 4C). However, keratohyalin granules, which were generally evident on the posterior aspect of the papillae of filiform papillae, could not be observed throughout the dorsal surface of the tongue of this species.
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Fig. 2. Group of lingual papillae distributed on the apical part of the corpus linguae of N. vison (adult male, 23 months). (A) Light micrograph of a sagittal section of the fungiform papillae situated on the apex linguae (H-E stain). A taste bud (arrow) is present on the top of the papilla. Scale bar, 50 μm. (B) Scanning electron micrograph (SEM) of the external surface of the fungiform papillae distributed on the apex linguae. A flattened dome-like fungiform papilla is present among the fungiform papillae. Scale bar, 100 μm. (C) SEM of the connective tissue core (CTC) of a fungiform papilla situated on the apex linguae after epithelial exfoliation. At the top of the cylinder-like CTC, there is a flattened flange. Six concavities are observable. Scale bar, 50 μm. (D) Light micrograph of a sagittal section of filiform papillae distributed on the apex linguae. The uppermost layer of the filiform papillae retain nuclei; keratohyalin granules, usually evident on the caudal aspect of the filiform papillae, could not be distinguished. R, rostral. Scale bar, 100 μm. (E) SEM of the apex linguae exhibiting the epithelial surface of the filiform papilla. The rounded triangular processes of the filiform papillae are inclined caudally. A relatively thick ‘main’ process in the central region of the apex together is surrounded by approximately 12 short ‘accessory’ processes at the margin of the filiform papilla. Scale bar, 100 μm. (F) CTC of filiform papillae distributed on the apex linguae after removal of the epithelium. A rather long main process and approximately 12 accessory processes surround the concavity in front of the papilla. Scale bar, 50 μm.
The external surface view with the SEM revealed that each filiform papilla was associated basically with several accessory processes arranged around the rim of each main
Fig. 3. (A–F) A series of micrographs illustrating the morphological variation of the epithelial surface of filiform papillae distributed on the apex of the corpus linguae of N. vison (see Figs. 1B, 3A, Table 1). Although the external surface morphology of the filiform papillae linguae varied, the CTC of dome-like filiform papillae were identical in appearance (as in Fig. 2F). (D–F) show robust and slender main protrusions. Fu: Fungiform papilla. Scale bars, (A, C, F) 200 μm; (B, D, E) 100 μm.
protrusion. The length of these processes varied depending on the region: in the rostral region of the corpus linguae, the main process of the filiform papillae was elongated vertically to a sharp tip (Fig. 4D), which was clearly distinguishable from the neighboring accessory processes. However, it was notable that the filiform papillae varied among different specimens, particularly on the apex linguae (Figs. 2E, 3A–F); some had a dome-like appearance (Fig. 3A), while others were finger-like, with a robust or slender main protrusion (3D–F). Moreover, one specimen exhibited a mixed type of arrangement comprising both domed and finger-like filiform papillae (Fig. 3B). In another transitional pattern of domed and finger-like types of filiform papillae, the main process was short and quite similar to the neighboring accessory processes (Figs. 2E, 3C). Interestingly, these morphological variations of filiform papillae were observable only within the apex linguae. Furthermore, age-analysis of the teeth annuli and the morphology of the skull and bacu-
Lingual papillae of American mink Table 1.
a b c d e f
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Details presented in Fig. 3.
Gender
Body weight
male female female male male female
1180 g 645 g 810 g 1260 g 1200 g 900 g
Results of age determination analysis Adult Adult Subadult Adult Subadult Subadult
10–11 months 7 years 10 months 22–23 months 7 months 10 months
form papillae basically reflected the structure of the overlying epithelial surface; each CTC of the filiform papillae on the apex linguae consisted of finger-like protrusions that surrounded the rim of each main core (Fig. 2F). The CTC had a concavity in the center of the main core. Despite the morphological variation of the filiform papillae distributed on the surface of the apex linguae, the morphology of the corresponding CTCs was relatively uniform, consisting of fingerlike protrusions that surrounded the rim of each main core (e.g. Fig. 3A). On the apex linguae, the main CTC processes were rather thick and comparable with the neighboring accessory cores (Fig. 2F). By contrast, the main CTC processes in the rostral region were more prominent and the accessory processes were short (Fig. 3E). The filiform papillae were approximately 180–700 μm long and 180–380 μm wide.
Fig. 4. Set of fungiform and filiform papillae distributed on the rostral (see Figs. 1B; 4B) part of the corpus linguae of N. vison (adult male, 23 months). (A) SEM of the epithelial surface of the fungiform papillae in the rostral region of the corpus. The fungiform papillae in this area are somewhat compressed and present a trapezoid appearance. Scale bar, 100 μm. (A) SEM of the CTCs of the fungiform papillae of the rostral part of the corpus after removal of the epithelium. The CTCs of the fungiform papillae in this area possess numerous frill-like longitudinal processes at the side of the CTC. Several concavities are also observable at the top of the CTCs. Scale bar, 100 μm. (C) Light micrograph of a sagittal section of the filiform papillae distributed on the rostral region of the body. The filiform papillae have an overlying stratum corneum in the posterior aspect of the papillae (arrow). However, no keratohyalin granules were evident in the posterior aspect of the papillae. R, rostral. Scale bar, 100 μm. (D) SEM of the epithelial surface of the filiform papillae situated in the rostral region of the corpus. The filiform papillae in this area have one obvious main process and four or five neighboring accessory processes. Scale bar, 100 μm. (E) SEM of the CTCs of the filiform papillae in the rostral region observed after epithelial exfoliation. The marginal ridges of the CTCs of the filiform papillae and main and accessory cores are relatively thin. Scale bar, 100 μm.
lum (Table 1) showed that the above-mentioned morphological variations in the filiform papillae (Fig. 3A–F) were not systematically related to age. After exfoliation of the epithelium, the CTCs of the fili-
Conical papillae Conical papillae (Fig. 1B; CP) were distributed between the caudal end of the rostral part (Fig. 1B; C) of the corpus linguae and radix linguae (Fig. 1B; D). Under light microscopy, the conical papillae had a stratum corneum, which lacked nuclei in the uppermost part of the posterior portion (Fig. 6A), and keratohyalin granules could not be distinguished. By contrast, a thickened epithelium was evident in the anterior aspect of the papilla. In SEM observations, the epithelial surface of each conical papilla appeared smooth and conical, with a sharp tip (Fig. 6B). After exfoliation of their epithelium, the CTCs were seen to be conical or fingerlike, and there were numerous protrusions in the basal twothirds of the core (Fig. 6C). The conical papillae were approximately 460–650 μm long and 320–380 μm wide. Fungiform papillae Light microscopy revealed the fungiform papillae as dome-like (Fig. 2A). Keratinization of these papillae was weak. A few taste buds were evident in the epithelium at the tips of the fungiform papillae. In SEM observations, the external surfaces of the fungiform papillae distributed on both the apex linguae (Fig. 2B) and the rostral part of the corpus linguae (Fig. 4A) were smooth and dome-like. After epithelial exfoliation, the CTCs of the fungiform papillae on the apex linguae (Fig. 2C) were columnar and those of the rostral part of the corpus linguae (Fig. 4A) had a peduncular appearance. The CTCs of both types of fungiform papillae had several vertically aligned ridges and a concavity on the uppermost surface (Figs. 2C, 4B). The fungiform papillae were approximately 120–300 μm in diameter. Lateral organ Structures resembling lateral organs (Sonntag, 1920)
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Fig. 5. Set of lateral organs distributed on the caudal section of the corpus linguae (see Figs. 1B, 5C) and radix linguae (see Figs. 1B, 5D) of N. vison (adult male, 23 months). (A) Sagittal section of a lateral organ on the caudal area of the corpus. Taste buds are not observable in this area. Mucus-rich lingual glands (G) are present in the lamina propria. Scale bar, 500 μm. (B) SEM of the CTCs of the lateral organ situated on the caudal area of the corpus after epithelial exfoliation. Orifices of the mucus-rich lingual gland (arrows) are distributed at the posterior part of the lateral organ. Numerous shallow grooves run laterally from the lateral side of the lateral organ. Scale bar, 1000 μm. (C) A sagittal section of a vallate papilla stained by H-E. Numerous taste buds (arrows) are present in the inner wall of the epithelium of the circumferential furrow. Lingual glands similar to von Ebner’s glands (G) are located in the lamina propria beneath the papilla. Scale bar, 300 μm. (D) SEM of the epithelial surface of the vallate papillae. A circumferential furrow and weak ridge are present around the papillae. Scale bar, 300 μm. (E) SEM of the vallate papillae after epithelial exfoliation. The CTCs of the circumferential furrow were more evident than were visible on the epithelial surface. Numerous CTC protrusions are distributed on the circumferential furrow and ridge. Scale bar, 300 μm.
Fig. 6. Group of conical papillae distributed on the lingual mucosa of the radix linguae (see Figs. 1B, 6D) of N. vison (adult male, 23 months). (A) Sagittal section of the conical papillae stained by H-E. An eosin-intensive stained stratum corneum is observable at the posterior of the papilla. Scale bar, 200 μm. (B) SEM of the epithelial surface of the conical papillae. The surface is smooth and has a conical appearance. R, rostral. Scale bar, 200 μm. (C) SEM of the conical papillae after removal of the epithelium. Numerous thumblike CTCs are distributed on the surface. The lower two-thirds of each conical CTC has a rough appearance and is covered by densely studded short protrusions. Scale bar, 200 μm. (D) Sagittal section of the radix linguae stained by H-E. The epithelial surface is rather wavy. Mucus-rich lingual glands (G) can be seen. Scale bar, 200 μm. (E) SEM of the radix linguae after partial epithelial exfoliation. Some orifices of mucus-rich lingual glands (arrows) are present. Numerous ridges are observable at the lateroposterior end. Scale bar, 200 μm.
were observable on the lateral edge of the mid-end region (Fig. 1B; C) of the corpus linguae. Light microscopy revealed a broader ridge-like structure (Fig. 5A). Taste buds could not be identified. Mucus-rich lingual glands were present in the lamina propria. SEM observation of the CTCs showed a serrated structure with shallow horizontal grooves.
Vallate papillae Four or five vallate papillae (Fig. 1B; VP) were situated at the boundary between the caudal section of the corpus (Fig. 1B; C) and the radix linguae (Fig. 1B; D). Light microscopy showed numerous taste buds distributed vertically in the inner epithelial wall of the circumferential furrow. Gusta-
Several orifices of the lingual glands were distributed on the posterior part of the serrated edge (Fig. 5B).
Lingual papillae of American mink
tory glands could be seen in the lamina propria (Fig. 5C; G), with their orifices opening into the base of the circumferential sulcus. In SEM preparations, the external surfaces of the vallate papillae were round or obliquely elongated in the rostrolateral direction. Each dome-like papilla was surrounded by a circumferential ridge and furrow. After exfoliation of the epithelium, the CTCs of the vallate papillae appeared domelike and were surrounded by a deep ridge-like core in the circumferential furrow (Fig. 5E). The vallate papillae were approximately 1020–1500 μm in diameter. Radix linguae The radix linguae exhibited a weakly folded appearance (Fig. 1B; D). Light microscopy revealed large mucus-rich mixed glandulae linguales in the lamina propria (Fig. 6D; G). After removal of the epithelium, the surface of the CTCs of the radix linguae exhibited a mesh-like ridge (Fig. 6E). Orifices of glandulae linguales were observed among the mesh-like CTCs. DISCUSSION Our observations indicate that the lingual papillae of the semiaquatic American mink possess a number of unique morphological features while having morphological similarity to those of terrestrial species of Mustelidae. The external morphology of the filiform papillae, particularly those on the apex linguae, was variable and some exhibited a very simplified (reduced) structure in comparison with those of previously studied terrestrial mustelids. The dome-like filiform papillae of the apex linguae of some American mink resembled the filiform papillae in the apex linguae of newborn sea otters, as reported by Shimoda et al. (1996). Fukue and Kishimoto (2010) made external measurements of large numbers of adult and subadult American minks (125 males 400–2397 g; 66 females 592–1086 g) captured in the same area. Based on these measurements, the individuals in our study that exhibited dome-like filiform papillae in the apex linguae clearly were not newborn, in contrast to the sea otters studied by Shimoda. Furthermore, our analyses of tooth annuli and of skull and baculum morphology indicated that the variation among the filiform papillae in our study was not related to age. These data suggest that the dome-like external morphology of filiform papillae, which was sometimes present in adults, depends on the individual. On other epithelial surfaces of the tongue of the American mink, the filiform papillae exhibited transitional morphology between ‘dome’ and ‘finger’ types; some were comparatively short processes and others exhibited clear a finger-like morphology. It could be speculated that the morphological variation among the individuals investigated in this study resulted from crossbreeding. The American mink population has expanded in the Nagano region during the last 25 years, and many individuals are likely to have escaped 15 years ago when the fur farm was closed (Shimatani et al., 2010). However, Shimatani et al. (2010) investigated the genetic variation of American mink captured in this area and concluded that the genetic diversity of present individuals was relatively low. Therefore, the morphological variation of the filiform papillae found in the apex linguae may be less influenced by genetic factors than by the dietary diversity associated with their semi-
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aquatic lifestyle (e.g., consumption of freshwater crayfish) (Shimatani et al., 2008). In terrestrial Mustelidae, the filiform papillae on the apex linguae exhibit a distinct morphology in which main and accessory processes are present. In the Japanese weasel (Furubayashi et al., 1989), Siberian weasel (Emura et al., 2007), and ferret (Emura, 2008; Takemura et al., 2009), which are closely related taxa in the Mustelinae (Sato et al., 2012), the epithelial surface of these papillae has a long main process and associated small accessory processes. In less closely related taxa, such as the Japanese marten (Guloninae, Mustelidae), the filiform papillae on the apical surface of the tongue have a main process and long hairy accessory processes (Emura et al., 2007). In other less closely related taxa such as the Japanese badger (Melinae, Mustelidae), the external surface of the filiform papillae on the apical surface of the tongue has an even longer main process and long accessory processes (Yoshimura et al., 2009). The present observations indicate that the external form of the filiform papillae in the American mink also conforms to this basic plan of main and accessory processes, which is observable among the filiform papillae distributed on both apical and rostral surfaces. Among aquatic mammals, particularly the pinnipeds, the external surface of the lingual papillae is usually smooth and without accessory protrusions. Yoshimura et al. (2002) reported that the outer surface of the filiform papillae of a pinniped, the California sea lion, consisted of a smooth conical main process and lacked accessory processes. In another pinniped, the spotted seal, the external surface of the filiform papillae was clear and cone-shaped (Yoshimura et al., 2007). A smooth, simplified external appearance of the filiform papillae has also been observed among the highly aquatic Mustelidae. Shimoda et al. (1996) observed the epithelial structure of the lingual papillae distributed over the lingual mucosa of the tongue of newborn sea otters. Micrographs from that study show the surface texture of the filiform papillae epithelium to be conical, completely smooth, and without accessory protrusions. Furthermore, simple dome-like filiform papillae were observable in the anterior part of the tongue. Macdonald (1984) described the American mink as semiaquatic; the animal has a carnivorous diet and preys on crayfish, crabs, and fish. The specimens in the present study were living close to rivers in mountainous regions and were associated with a semiaquatic environment. However, the structure of the CTCs of the filiform papillae on the apex linguae of the American mink resembled that of the more terrestrial Mustelinae and Guloninae. As in the present study of the American mink, SEM micrographs of the filiform CTCs on the apex linguae of other Mustelinae and Guloninae species exhibited rod-like cores, consisting of main and accessory cores surrounding a cavity. In contrast, in the less closely related Japanese badger (Melinae), the filiform CTCs on the apex linguae have a somewhat different morphology in which both main and accessory cores are elongated (Yoshimura et al., 2009). We observed 4–6 vallate papillae located in single Vshaped line at the boundary between the caudal part of the corpus linguae and the radix linguae. In a previous account of mustelid species, Takemura et al. (2009) reported that the
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ferret had 8–12 vallate papillae, uniquely arranged in two or three V-shaped lines. A diagram by Furubayashi et al. (1989) shows 4–5 vallate papillae arranged in a double Vshape in the Japanese weasel. In contrast, in the Japanese marten (Emura et al., 2007) there are four, in the Siberian weasel (Emura et al., 2007) and Sea Otter (Shimoda et al., 1996) six, and in the Japanese badger (Yoshimura et al., 2009) seven vallate papillae arranged in a single V-shaped line. In the present study, instead of foliate papillae, a pair of lateral organs was found situated on the caudal edge of the corpus linguae. In our observations of their CTCs, we identified a serrated structure with shallow horizontal grooves. Several orifices of the lingual glands were distributed at the posterior end of the serrated edge and mucus-rich lingual glands were present in the lamina propria. Generally, under the sulci of foliate papillae lie serous gustatory glands (similar to von Ebner’s glands in humans), the ducts of which empty into the sulci (Eurell and Frapper, 2006). The orifices of mucus-rich lingual glands in this region are a unique finding and their contribution to gustatory function is unclear. In a previous description of mustelids (Takemura et al., 2009), no ridges and furrows equivalent to the foliate papillae were recognized. However, in that study a micrograph of the lateral margin of the posterior tongue body shows parallel shallow grooves and serrated structures. Although they did not recognize taste buds, a slit-like groove related to foliate papillae is evident in micrographs of the Japanese marten (Emura et al., 2007). The Siberian weasel (Emura et al., 2007) also had similar shallow grooves in this region in spite of being described as lacking foliate papillae. Moreover, Furubayashi et al. (1989) described the Japanese weasel as having foliate papillae that consisted of four to five slit-like grooves on each side of the lateral margin of the radix linguae, and several taste buds were observed in the cleft epithelium. The serous lingual glands (similar to von Ebner’s gland) were situated beneath the foliate papillae. Therefore, foliate papillae may be present in other weasel species but not in the American mink. However, information about the vallate papillae, lateral organs and foliate papillae remains incomplete and further study, including a detailed histological analysis, is required. The unique features of the lingual morphology of the American mink described above imply that these traits may have been influenced more by the semiaquatic living environment of this species than by its phylogeny. However, the differences appear to relate only to the epithelia and the structures of the underlying CTCs in the American mink are similar to those among other Mustelinae. The oral cavity is the entrance to the digestive tract (Bloom and Fawcett, 1994) and it is important in the prehension, mastication, and deglutition of food (Eurell and Frapper, 2006). This implies that the oral cavity could be influenced by dietary habits and living environment as well as morphological traits that arise from their phylogenetic position. Therefore, it is important to understand the comparative morphology of the oral cavity among other mammal species. Further detailed morphological studies coupled with developmental and molecular investigations are required to fully elucidate the basis of the diversity of lingual papillae of the American mink.
ACKNOWLEDGMENTS We are grateful to members of Minami-Saku-Nanbu and Saku fishery cooperatives and Nagano Prefecture for supplying autopsy materials. We also thank Mr. Narumi Kouno (Nagano Prefectural Fisheries Experimental Station Saku Branch) and Ms. Miyuki Sato for their support in sampling specimens. This work was supported by a research promotion grant-in-aid (NDUF-09-14) from the Nippon Dental University.
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