Comparative Morphology of the of Natrix tessellata (Family: Colubridae) and (Family: ) Author(s): Rasha E. Abo-Eleneen and Ahmed A. Allam Source: Zoological Science, 28(10):743-748. Published By: Zoological Society of Japan DOI: http://dx.doi.org/10.2108/zsj.28.743 URL: http://www.bioone.org/doi/full/10.2108/zsj.28.743

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Comparative Morphology of the Skin of Natrix tessellata (Family: Colubridae) and Cerastes vipera (Family: Viperidae)

Rasha E. Abo-Eleneen1 and Ahmed A. Allam1,2*

1Department of Zoology, Faculty of Science, Beni-suef University, Beni-Suef 65211, 2King Saud University, College of Science, Zoology Department, Riyadh 11345,

We studied beneficial difference of the skin of two . Two snakes were chosen from two dif- ferent habitats and two families: Colubridae (Natrix tessellata) and Viperidae (Cerastes vipera). The investigations were performed by light and electron microscopy. Histologically, the skin of the stud- ied show pronounced modifications that correlated with functional demands. The scales in Natrix tessellata overlapped slightly, while in Cerastes vipera they were highly overlapped. SEM shows that scales of Natrix tessellata had bidentate tips while the scales of Cerastes vipera were keeled. Histochemically, in both studied species, melanocytes and collagenous fibres were distrib- uted throughout the . Polysaccharides were highly concentrated in the and dermis of both species while proteins were highly concentrated only in the epidermis. Transmission elec- tron microscopy (TEM) showed that the skin of both snakes consisted of keratins located in the epidermis. Some lipids and mucus were incorporated into the outer scale surfaces such that lipids were part of the fully keratinised hard layer of the snakes’ . Lipids are probably responsible for limiting water loss and ion movements across the skin. Melanosomes from epidermal melano- cytes were present only in Cerastes vipera. In aggregate, these results indicate that snakeskin may provide an ecological indicator whereby epidermal and integumentary specializations may be eco- logically correlated.

Key words: , skin, , histochemistry, scales

The epidermis of reptiles produces two classes of INTRODUCTION keratins: soft, or alpha-keratins, and hard, or beta-keratins Reptiles are terrestrial that have become com- (O’Guin et al., 1987; Alibardi and Toni, 2006). The epidermis pletely independent of the aquatic environment; their skin is of Natrix piscator is complex in its organization and consists dry and has low permeability to water. All reptilian species of a series of small discrete units, with the scales contiguous have characteristically scaly skin, which is one of the main at the hinge region (Singh, 1989). Goslar (1958), in describ- features distinguishing them from other amniotes ( and ing the scale epidermis of Natrix natrix, gave a concise mammals). The reptilian integument protects the account of the carbohydrate histochemistry. Baberjee and from mechanical damage and dehydration (Maderson et al., Mittal (1978) reported mucous cells in the hinge epidermis, 1985; Matoltsy and Bereiter-Hahn, 1986). The different and described carbohydrates in cellular components of the scale patterns observed are a result of a long evolutionary epidermis of Natrix piscator. The first comparative micro- process that allowed each species to adapt to its specific scopic study of vertebrate integumentary development environment (Alibardi and Toni, 2006). included observations on embryos of Natrix sp., Anguis The epidermis is derived from the embryonic ectoderm, fragilis and Lacerta sp. (Kerbert, 1876), and described the while the dermis is derived from somatic mesoderm. The two-layered condition of the epidermis in early stages of rep- dermis may be closely or loosely attached to the fascia of tiles and birds. The report argued that this condition was deeper muscles (Hall, 1980; Noden, 1980). Both the epi- common to all vertebrates, quoting observations on fish dermis and the dermis may contain neural crest-derived pig- (Rienecke, 1869) and amphibians (Stricker, 1872). ment cells, although those seen in the epidermis are usually A scaled integument with dermal ossification was pres- only of the melanogenic type (Noden, 1980). ent in basic amniotes of the Carboniferous Period, which were by definition reptiles, about 340 million years ago * Corresponding author. Phone: +2-012-2928210; (Colbert et al., 2001; Pough et al., 2001). Skin morpho- Fax : +2-082-2328088; genesis and epidermal differentiation in therapsids and sau- E-mail: [email protected] ropsids probably began to diversify since the Upper Carbon- doi:10.2108/zsj.28.743 iferous, as suggested by anatomical and paleontological 744 R. E. Abo-Eleneen and A. A. Allam data (Maderson and Alibardi, 2000). Integument evolu- tion in reptiles was centred on variations of scale shape in relation to adaptation to their habitat, functional pur- poses (especially water-loss limitation), composition, and degree of keratinization (Sawyer and Knapp, 2003; Saw- yer et al., 2003; Wu et al., 2004). Scales protect the body of the snake, aid it in loco- motion, and allow moisture to be retained (Gans, 1974; Mullin, 1996). Broad variations of scale morphology, size, and overlap is found in modern reptiles, repre- sented by the numerous species of and snakes (Pough et al., 2001). These variations include the numer- ous variations in their superficial micro-ornamentation (Arnold, 2002). The present study is concerned with the histochem- ical characterization of carbohydrates, proteins in the scales, hinge epidermis of a water snake and a sandy snake as well as with the difference in the skin of two selected snakes. The first was Natrix tessellata, which is one of the most common snakes in the vicinity of the Nile Area (water) in Egypt. Its maximum length is 1.0–1.3 m. The colour may vary from greyish green to brownish or almost black, with dark spots on the back (Vlcek et al., 2010). The second was Cerastes vipera, which is small and stout having an average length of 35–55 cm. It has a broad, triangular head with small eyes set well forward and situated on the junction of the side and the top of the head. It is a sandy species (Mallow et al., 2003). MATERIALS AND METHODS In the present study, 20 specimens of Natrix tessellata (Hanas El Maiya) were captured from the Egyptian Nile Delta regions (water habitats), and 20 specimens of Cerastes vipera (Haiya Qarah) were collected from sandy areas in the Mediterranean coastal desert of Egypt.

Histological assay The skin of the specimens were directly fixed in 10% neu- tral formalin, then washed and dehydrated in ascending grades of ethyl alcohol, cleared in xylene, and embedded in paraffin. Serial 5 μm sections were cut and stained with Ehrlich’s haema- toxylin and eosin (Mallory, 1944). Periodic acid/Schiff’s (PAS) Fig. 1. (A–J) Light micrograph of vertical sections through skin. (A) Skin of method was used for polysaccharide staining (McManus, 1946). Natrix tessellata showing the slight overlapping scales separated by hinge In PAS, an acetylation blocks the hydroxyl groups from forming regions (H). Outer (OSS) and inner (ISS) scale surfaces are clearly differenti- acetyl esters, and a deacetylation hydrolyzes the acetyl esters ated (H.E., X 100). (B) Higher magnification of (A) showing that epidermis (E) and unblocks the reactive hydroxyl groups. To differentiate neu- and dermis (De) are clearly distinct. Melanocytes (M) and collagenous fibres tral mucopolysaccharides from the acid mucopolysaccharides, (Cf) are present in the dermis (H.E., X 400). (C) Skin of Cerastes vipera PAS/AB staining (Mowry, 1956) was employed. Bromophenol showing the high overlapping scales. The scales are separated by hinge blue was used for staining proteins (Mazia et al., 1953) to show regions (H) (H.E., X 100). (D) Higher magnification of (C) showing the epider- hardness. These sections were examined and photographed mis (E), dermis (De), collagenous fibres (Cf) and rare melanocytes (M) in the dermis (H.E., X 400) (E) Skin of Natrix tessellata showing accumulation of using a Leitz microscope. PAS positive material in the epidermis (E), the dermis (De) and the muscles (Mu) (PAS., X 100). (F) Skin of Cerastes vipera showing large amounts of Scanning electron microscopy (SEM) PAS positive material in the epidermis (E) and the dermis (De), the muscles Natrix tessellata and Cerastes vipera skins were fixed in (Mu) show a moderate amount (PAS., X 100). (G) Skin of Natrix tessellata 5% glutaraldehyde. The skins were then washed in 0.1 M caco- showing large amounts of PAS and Alcian blue stained material (mixed poly- dylate buffer and post-fixed in a solution of 1% osmium tetroxide saccharides) in the epidermis (E), the dermis (De) and the muscles (PAS- ° at 37 C for two hours. This procedure was followed by dehydra- Alcian, X 100). (H) Skin of Cerastes vipera showing accumulation of a mix- tion, critical point drying, and platinum-palladium ion-sputtering. ture of acid and neutral polysaccharides (magenta colour) in the epidermis The specimens were then examined under a scanning electron (E), the dermis (De) and the muscles (PAS-Alcian, X 100). (I) Skin of Natrix microscope (Jeol, JSM-5400LV). tessellata showing high levels of protein-containing materials in the epidermis (E) and the muscles (Mu) while the dermis (De) appears moderately stained Transmission electron microscopy (TEM) (Bromophenol blue, X 100). (J) Skin of Cerastes vipera showing high levels The skins were cut into one mm3 pieces and immediately of protein-containing materials in the epidermis (E) and the muscles (Mu), fixed in fresh 3% glutaraldehyde-formaldehyde at 4°C for 18−24 while the dermis (De) appears weakly stained (Bromophenol blue, X 100). Snake Skin Morphology 745 hours (pH 7.4) and then post-fixed in isotonic 1% osmium tetroxide tessellata showed a moderate amount of protein, in Cer- for one hour at 4°C. Serial dehydration in ethyl alcohol was carried astes vipera, the dermis appeared weakly stained. out in the following order: 50% alcohol 30 minutes, 70% alcohol 15 minutes (twice), 80% alcohol 15 minutes, 90% alcohol 15 minutes, Scanning electron microscopic studies absolute alcohol 30 minutes (twice). The specimens were then In Natrix tessellata, the skin was characterized by biden- passed twice through propylene oxide solution for 10 minutes. tate tips (Fig. 2A–D), while in Cerastes vipera the skin was The specimens were embedded in spur resin as follows: First, specimens were immersed in propylene oxide, then in 1:1 propyl- characterized by with the ventral surface clad ene oxide resin mixture 1 hour, then in 1:3 propylene oxide resin having larger tubercles (Fig. 2E–H). mixture overnight, then immersed in fresh pure resin at room tem- perature overnight. The next day, specimens were transferred to Ultrastructure electron microscopic studies capsules containing fresh resin and placed in an oven at 60°C for In both Natrix tessellata (Fig. 3A–C) and Cerastes one day to ensure polymerisation of the blocks. Semithin sections vipera (Fig. 3D–F), the epidermis exhibited numerous (1 μm) were cut from the blocks using an Ultracut Reichert-Jung microvilli. The thicker epidermis was mostly filled with a ultramicrotome with the aid of glass knives. Sections were stained dense network of keratin filaments. The epidermis of Natrix with toluidine blue and examined by light microscope. To detect the area of interest, Ultrathin sections were then prepared using the ultramicrotome glass knives, stained with uranyl acetate and lead citrate and examined with a Joel CX 100 transmission electron microscope operated at an accelerating voltage of 60 KV. RESULTS Histological and histochemical studies The epidermis embodies stratum germinati- vum whose cells are responsible for the production of the different epidermal layers. The outermost layer of the epidermis, the oberhautchen, is the site of epidermal sculpturing and is the location of the spinules of the epidermis. In Natrix tessellata, the scales are slightly overlapping, whereas in Cerastes vipera, they are highly overlapping and each scale has an outer and an inner scale sur- face. The inner scale surface overlaps the outer surface of the next posterior scale. The two scales are joined through the hinge region (Fig. 1A, B). Among the specimens of each species studied, the epidermis of Natrix tessellata was thicker than the epidermis of Cerastes vipera. The connective tissue of the dermis can be divided into two layers, outer loose dermis and inner dense dermis (Fig. 1C, D). In Natrix tessellata (Fig. 1A, C), melano- cytes were densely distributed on the outer scale surface and collagen bundles could be seen. In Cerastes vipera (Fig. 1B, D), also melanocytes and collagenous fibres were distributed throughout the dermis. The application of PAS reaction on the skin of Natrix tessellata and Cerastes vipera (Fig. 1E, F) revealed a high polysaccharide content in the epi- dermis and the dermis. However, when the mus- cles of the studied species were compared, Natrix tessellata muscles were heavily loaded with poly- saccharides while in Cerastes vipera the muscles were moderately loaded with polysaccharides. The application of PAS-Alcian blue revealed a strong magenta colour indicating the presence of neutral Fig. 2. (A–H) Scanning electron micrograph of trunk skin scales. (A) Skin of the of Natrix tessellata showing scales with bidentate tips (Bt) (Scale bar, 100 and acidic mucin in the epidermis, the dermis, and μm). (B) Magnified portion of A (Scale bar, 10 μm). (C) Skin of Natrix tessellata the muscles of both studied species (Fig. 1G, H). illustrating bidentate tips (Bt) of scales (Scale bar, 100 μm). (D) Higher magnifi- Bromophenol blue staining of the skin of Natrix cation of (C) (Scale bar, 10 μm). (E) Skin of Cerastes vipera showing keeled tessellata and Cerastes vipera was used to reveal scales (Ks) (Scale bar, 100 μm). (F) Magnified portion of E (Scale bar, 10 μm). the distribution of protein in the epidermis and the (G) skin of Cerastes vipera illustrating ventral tubercles (Vt) (Scale bar, 100 muscles (Fig. 1I, J). While the dermis of Natrix μm). (H) Higher magnification of G (Scale bar, 10 μm). 746 R. E. Abo-Eleneen and A. A. Allam

of the skin of two snakes living in two different habitats. Natrix tessellata (Family: Colubridae), is never found far from water and is often found rest- ing or crawling on the bottom of streams or irriga- tion canals. The skin of Natrix tessellata was smooth, thin, and moist. In contrast, Cerastes vipera strictly inhabits sandy desert, particularly sand dune areas. The skin of both snakes was characterized by the presence of the scales and consisted of two distinct principle layers: an outer epidermis and an inner dermis. The morphology of many organisms seems to be related to the envi- ronment they live in. Nonetheless, many snakes are so similar in their morphological patterns that it becomes quite difficult to distinguish any adap- tive divergence that may exist. Many authors sug- gest that the micro-ornamentations on the scales of reptiles have important functional value (Rocha- Barbosa and Moraes e Silva, 2009). The characteristic scaled skin of reptiles is one of the main features that distinguishes them from the other amniotes, birds and mammals. The different scale patterns observed in Natrix tessellata and Cerastes vipera resulted from a long evolutionary history that allowed each spe- cies to adapt to its specific environment (Aubret et al., 2004 and Rocha-Barbosa and Moraes e Silva, 2009). The scales in both are overlapped and sep- arated by flexible hinge regions that facilitate body motility as mentioned by Spearman and Riley (1969). Areas of the hinge regions that are responsible for the facility of movement of the body regions are covered by elastic connective tissue (Spearman, 1969). Mittal and Singh (1987a, b) explained that the ventral surface elasticity and stretching are required for snakes to be able to move freely. Scanning electron microscopy recorded bidentate scale tips in Natrix tessellata, whereas in Cerastes vipera, the scales were Fig. 3. (A–F) Transmission electron micrograph. (A) Superficial layer of outer keeled. The types of scales were characteristic to scale surface of Natrix tessellata showing epidermis (E), dermis (De), superficial most reptiles (Abdeen et al., 2008b). microvilli (Mv), and dense fibrous material (Scale bar, 1 μm). (B) Epidermis layer The dermis in Natrix tessellata is thicker than of Natrix tessellata showing corneous layer (C), desmosomes (arrows), lipid the dermis in Cerastes vipera. This difference is droplets (L), nuclei (N), and vacuoles (V) (Scale bar, 1 μm). (C) Desmosomes of appropriate to body size, in that the body of Natrix Natrix tessellata (arrow). Also visible, cytoplasm containing lipid droplets (L) and tessellata is of greater diameter than the body of vacuoles (V) (Scale bar, 1 μm). (D) Superficial layer of outer scale surface of Cerastes vipera. In both species, dermal collagen Cerastes vipera showing epidermis (E), dermis (De) and superficial microvilli (Mv) (Scale bar, 1 μm). (E) TEM showing large beta-filaments near the nucleus fibres occupy the dermis. Studies on the arrange- and melanosomes (arrowheads) in Cerastes vipera (Scale bar, 1 μm). (F) Skin of ment of dermal collagen fibres in snakes (Jayne, Cerastes vipera showing nucleus (N) and vacuoles (V) (Scale bar, 1 μm). 1988) and lizards (Mohammed, 1989) indicated that the fibres might facilitate lateral flexion and distension during swallowing. In addition, they are tessellata contained some mucus-like dense granules and useful for predicting skin stiffness or transmission of locomo- few coarse filaments, especially in the marginal region adja- tor forces. Jayne (1988) reported that the loading curves of cent to the microvilli surface. In both, the cytoplasm con- snakeskin collagen fibres most likely dominate the mechan- tained many pale vacuoles of lipid-like material, the nuclei ical behavior of the skin and are more flexible than in other were visible, and desmosomes were present. In Cerastes regions. Fat cells give the skin a moist feel. These observa- vipera melanosomes were concentrated along the central tions are similar to those recorded in geckos, core of the keratinocyte. Underwoodisaurus milii (Elkan, 1976) and Cyrtodactylus scaber (Mohammed, 1989). Some desert reptiles are able DISCUSSION use fat as a source of water, producing water as an end The present study revealed similar and distinct features product from metabolism of fat (Cloudsley-Thompson, Snake Skin Morphology 747

1971). The limitation of water and ion movement through the At the ultrastructural level, the epidermis of both species skin of snakes may be due to the richness of hydrophobic accumulated keratin filaments and showed alpha-keratiniza- lipids among the keratin network of the corneous layer of tion similar to that of avian (Matulionis, 1970; Sawyer and epidermis. Borg, 1979) and mammalian epidermis (Holbrook, 1991; Color change is known in both species studied. This Akiyama et al., 1999). Cytokeratins (alpha-keratins) are reflects the ability of snakes, particularly Cerastes vipera, to present in most epidermal layers, where they serve as change colors rapidly. Color change may be effected within cytoskeletal proteins for mechanical resistance to traction, minutes as a result of movement of melanosomes in the provide form to cells, or determine changes in shape of cells melanophore dendrites in response to the melanophore- (Fuchs and Weber, 1994; Coulombe and Omary, 2002). In stimulating hormone secreted by the pituitary gland (Sawyer snakes, the thickness of the corneum layer remains rela- et al., 1983). This is adaptation to desert habitats to help in tively thin in comparison to that of other reptiles, permitting the protection and catching prey where the snakes’ prey are movement and flexibility of the skin. The latter is achieved rare in desert. In other reptiles, pigmentation is lost, as in by the stretching and distension of hinge regions among lepidosaurian epidermis (Maderson, 1985) but crocodilians scales (Sawyer et al., 2000). and turtles retain their pattern of pigmentation, because it In conclusion, the present study confirmed that the mor- continues to be provided from the underlying pigment depo- phological and histological configurations of the skin of sition. Natrix tessellata and Cerastes vipera have a similar and dif- The present study revealed the presence of high levels ference characters, and also affected by to their particular of carbohydrates in the epidermis and dermis of both spe- habitats, aquatic or desert, respectively. cies. Alexander (1970) and Parakkal and Alexander (1972) reported similar results in lizards, turtles, and crocodiles. ACKNOWLEDGMENTS These structures have been regarded as mucous granules This project was supported by King Saud University, Deanship (Lavker, 1973). In fish epidermis, mucopolysaccharide of Scientific Research, College of Science Research Center. deposits have been observed in some cells undergoing ker- atinization, and these depositions are liberated into the inter- REFERENCES cellular space immediately before cells maturation (Mittal Abdeen A, Mostafa N, Abo-Eleneen R (2008a) Comparative histo- and Banerjee, 1974a, b; Mittal and Whitear, 1979). 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Int Mittal K, Whitear M (1979) Keratinization of fish skin with special ref- J Dev Biol 48: 249−270 erence to the catfish Bagarius bargarius. Cell Tissue Res 202: 213−223 (Received November 11, 2010 / Accepted March 7, 2011)