MORPHOLOGICAL AND ULTRASTRUCTURAL ANALYSIS OF THE TONGUE OF Gracilinanus microtarsus ( WAGNER , 1842 )

The G. microtarsus is a small marsupial belonging to the Didelphidae family and may be found mainly in the Atlantic Rainforest of the Brazilian Southeastern region. G. microtarsus has important ecological importance in the dispersion of seeds of several plant species. Current research characterizes morphologically six tongues of adult male and female specimens, which are fixed in 10% formaldehyde. The tongues were analyzed, measured and photographed "in situ" and "ex situ". Anatomy, light microscopy and ultrastructural analysis were performed. Macroscopically the tongue has root, a fixed body and a free apex. The tongue has fungiform papillae and high conical and acute filiform papillae across the dorsal surface. It also featured three vallate papillae located at the root and marginal fungiform papillae at the apex. Microscopically, the tongue is composed of longitudinal and transversal muscle fibers. Blood vessels, nerves, serous, mucous and mixed acini are found in non-modeled connective tissue. Ventrally, the tongue is lined by a keratinized stratified pavement epithelium. The morphology of the tongue of G. microtarsus resembles that described in carnivores and marsupials Didelphidae regarding its microscopic structure, differing only in the distribution and number of papillae.


INTRODUCTION
Marsupials, such as Gracilinanus microtarsus are included among the commonly called opossums, cuícas and catitas, all of which belong to the family Didelphidae (ORR, 1986) and is a very important family among marsupials due to its high number of species (GONÇALVES et al., 2009).Gracilinanus microtarsus is one of the smallest marsupials on Earth, measuring between 20 and 25 cm long, with the tail making more than a half of its length (LIMA et al., 2013).Specimens of the species have body mass average between 10 and 44 grams and exhibit nocturnal and arboreal habits (EISENBERG; REDFORD, 1999;CÁCERES et al., 2008;LOBO et al., 2014).They have frugivorousomnivorous feeding habits with a very varied diet, consisting of insects, fruits and small vertebrates.Since it feeds on small fruits, G. microtarsus is a species of considerable importance in dispersing seeds of trees (LOBO et al., 2014).
It is known that animals need raw material and energy for growth, maintenance and reproduction process.The processing of raw material derived from food requires several organs which constitute the digestory apparatus (LOBO et al., 2014).The tongue is an organ of the digestory apparatus and is responsible for moving food in the mouth in mastication and swallowing.It is also related to taste due to the taste buds (MANÇANARES et al., 2012).The tongue is also associated with speech in humans.Moreover, the tongue also participates in the functional stimuli that act on the growth and development of the face (BEZERRIL et al., 2005).
Current study describes anatomically, histologically and ultrastructurally the tongue of G. microtarsus for a deeper knowledge of the biology of this species which is widely distributed in Brazil.In fact, the species is highly interesting for studies related to natural selection, anatomical and physiological adaptations and ecological studies of mammals in general.

Animals
For this study, six adult male and female specimens of G. microtarsus were used and fixed in 10% formaldehyde solution.The specimens were provided by the Research Laboratory of Morphological Sciences of the University Center of Education Foundation Octavio Bastos, authorized by the Brazilian Institute of the Environment 02001.007176/03-69.

Macroscopic analysis and measurements
For the macroscopic analysis of the tongue, the jaws of the animals were moved for better in situ measurement, description and photographic documentation.
The macroscopic photo documentation was performed with a Sony Mavica digital camera 3.2 MP.Measurements of total length, width of root, apex, body and thickness were taken.

Light Microscopy (LM)
For microscopic analysis, the tongue was sectioned into root, body and apex, and the fragments were fixed in 10% formaldehyde solution.Samples were then dehydrated in an ethanol series at increasing concentrations (from 70 to 100%) and diaphanized in xylene, followed by paraffin embedding.
Samples were cut at 5µm thickness by microtome LEICA 2165 and the sections were stained with hematoxylin and eosin (HE), Picrosirius and Masson's trichrome.After staining, slides were mounted with cover slips and glue.The material was analyzed, mapped and photographed with a light microscope Nikon Eclipse E-400.

Scanning Electron Microscopy (MEV)
For the analysis in scanning electron microscopy, fragments of tongues were fixed in glutaraldehyde 1M (EM Grade Propylene oxide, Polysciences Inc., USA), followed by washing of samples with 0.1 M phosphate buffer at pH 7.4 and post-fixed in osmium tetroxide 1% (Spurr Spurr's kit, Electron Microscopy Sciences, USA).Tissue fragments were fixed in metal supports (stubs) and coated with gold ("sputtering" Emitech 550 K).The analysis was performed with Electron Microscope Leo 435 VP.

Macroscopic analysis
The tongue of G. microtarsus extends from the oropharynx and occupies most of the oral cavity.It is laterally limited by the teeth, caudally by the pharyngeal arch and dorsally by the hard and soft palates.The tongue has a root, a fixed body and a free apex, trapped to the floor just by the lingual frenulum.The tongue measures 1.85 ± 0.06 cm in length; it is 0.45 ± 0.06 cm wide at the base; 0.48 ± 0.05 cm wide in the body; 0.38 ± 0.05 cm wide at the apex; it is 0.3 ± 0 cm thick at the root; 0 ± 0.4 cm thick on the body and 0 ± 0.1 cm thick at the apex.The tongue has high conical and acute filiform papillae and fungiform papillae across the dorsal surface; marginal fungiform papillae at the margin of the apex; three vallate papillae, one central and two lateral at the root; and papillary projections at the lateral region of the root (Figure 1).

Light and scanning electron microscopy
The dorsal surface of the root revealed three vallate papillae, a central one and two lateral, surrounded by a groove, with numerous taste buds and serous salivary glands.The root surface also presented fungiform papillae and acute and high conical filiform papillae.The ventral surface was lined by non-keratinized stratified squamous epithelium.The tongue had striated transversal and longitudinal muscle, serous, mucous and mixed acini.In the lamina of the lingual papillae, there was a layer of stratified squamous epithelium and a basal layer of non-modeled dense connective tissue with vessels and nerves.Unilocular adipose tissue was also reported in this region (Figure 2).
Images of scanning electron microscopy showed the different types of lingual papillae: high conical and acute filiform papillae and fungiform papillae across the dorsal surface; marginal fungiform papillae located at the margin of the apex and three vallate papillae at the root.Papillary projections were also reported (Figure 3).

DISCUSSION
The tongue of mammal is divided into apex, body and root regions.As a rule, the dorsum of the tongue mucosa presents several types of papillae which may be mechanical-type as filiform and conical and gustatory-type as fungiform, foliate and vallate.These papillae become highly differentiated in shape, size, number, distribution and function among the various animal species (BUTENDIECK; VARGAS, 1998;MANÇANARES et al., 2012).
The dorsum of the tongue of G. microtarsus is divided into apex, body and root and presents acute and high conical filiform, fungiform and vallate papillae.Marginal papillae are present only at the apex, similar to those in Didelphis virginiana, D. albiventris (MARTINEZ et al., 1998) and D. marsupialis (MANÇANARES et al., 2012).
Table 1.Distribution of lingual papillae in mammalian species of different taxa.
The dorsum of G. microtarsus's tongue is lined with keratinized stratified squamous epithelium whereas in its interior non-modeled dense connective tissue and transversal and longitudinal striated muscles are found.Mucous and serous acini, lymphoid nodules, adipose tissue and nerves are present, as described in D. marsupialis (MANÇANARES et al., 2012), Nasua Nasua (SOUZA et al., 2012) and domestic animals such as cats and dogs (BUTENDIECK; VARGAS, 1998).
Since the G. microtarsus presents three vallate papillae, as reported in D. virginiana (MARTINEZ et al., 1998) andD. marsupialis (MANÇANARES et al., 2012), this characteristic is due to the fact that these animals belong to the same family Didelphidae (ORR, 1986).On the other hand, D. albiventris (MARTINEZ et al., 1998) has only two vallate papillae.
The morphology of the tongue of G. microtarsus resembles that described in carnivores and marsupials Didelphidae with regard to its microscopic structure and merely differs in the distribution and number of papillae.