Morphology of the Femoral Glands in the Lizard Ameiva Ameiva (Teiidae) and Their Possible Role in Semiochemical Dispersion

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JOURNAL OF MORPHOLOGY 00:000–000 (2007)

Morphology of the Femoral Glands in the Lizard Ameiva ameiva (Teiidae) and Their Possible Role in Semiochemical Dispersion

Beatriz A. Imparato,1 Marta M. Antoniazzi,1 Miguel T. Rodrigues,2 and Carlos Jared1,2*

1Laborato´rio de Biologia Celular, Instituto Butantan, CEP 05503-900, Saõ Paulo, Brazil 2Departamento de Zoologia, Instituto de Biocieˆncias, Universidade de Saõ Paulo, Saõ Paulo, Brazil

ABSTRACT Many lizards have epidermal glands in munication are poorly known. Most morphological the cloacal or femoral region with semiochemical func- papers on this subject deal with European, Asian, tion related to sexual behavior and/or territorial demar- Australian, and African species (Cole, 1966a,b; cation. Externally, these glands are recognized as a row Maderson, 1968, 1970, 1985; Maderson and Chiu, of pores, opening individually in the center of a modified 1970, 1981; Chiu and Maderson, 1975; Schaefer, scale. In many species the pores are used as systematic characters. They form a glandular cord or, in some spe- 1901, 1902; Cohn, 1904; van Wyk et al., 1992; Duj- cies, a row of glandular beads below the dermis, and are sebayeva, 1998). More recently, the morphology of connected to the exterior through the ducts, which con- pre-cloacal glands in an amphisbaenian (Amphis- tinuously liberate a solid secretion. Dead cells, desqua- baena alba) was reported and their role in the nat- mated from the secretory epithelium, constitute the ural history of this species was analyzed (Anto- secretion, known as ‘‘a secretion plug.’’ The present work niazzi et al., 1993, 1994; Jared et al., 1999). focusses on the morphology of the femoral glands of the Femoral glands are the most common semio- teiid lizard Ameiva ameiva, correlating it to the way in chemical sources in lizards (Jullien and Renous- which the secretion is deposited in the environment. The Lećuru, 1973) although some glandular scales or results here obtained are compared to those available ‘‘generation’’ glands (Maderson, 1967, 1968) have for other lizards and amphisbaenians. We observed that the diameter of the glandular pores did not show signifi- been described in cordylids in other regions of the cant differences between males and females. The glands body (van Wyk et al., 1992). Femoral glands are comprise germinative and secretory cells, which pass localized in one or two rows on the ventral side of through at least three stages of differentiation, during each thigh. The first morphological studies on fem- which an accumulation of cytoplasmic granules, with a oral glands date from the end of XIX century and glycoprotein content, occurs. The cells eventually die beginning of XX century (A´ braha´m, 1930), and and desquamate from the secretory epithelium, forming concerned primarily European species. The subject a secretory plug mostly constituted by juxtaposed non- was ignored by morphologists until the 1960s, when fragmented secretory cells. Because of the arrangement papers by Gabe and Saint-Girons (1965) and Cole of the rosette-like scales surrounding the femoral pores, (1966a,b) were published. Uva (1967), Gasc et al. we suggest that when the animal is in a resting position, with its femoral regions touching the ground, these (1970), Chiu and Maderson (1975) and Chauhan scales may be involved in the breakage of their respec- (1986) then reported additional data on other species. tive plugs, depositing tiny portions on the substrate. In Although semiochemical communication is very this manner, it seems that the method for signal disper- important in lizard biology, studies focusing on sion in this species involves specifically adapted struc- these aspects are rare (Mason, 1992; Cooper, 1994; tures and does not simply involve the chance breakage Cooper et al., 1996). However, many experiments of the plug, as the gland secretes it. Signal dispersion have demonstrated that several species are able to must also be intimately associated with the animal’s recognize chemical signals secreted by femoral movement within its territory. J. Morphol. 00:000–000, glands (Cooper and Vitt, 1984; Alberts, 1989, 1993; 2007. Ó 2007 Wiley-Liss, Inc. Lopez et al., 1998, Martin and Lopez, 2000). KEY WORDS: squamata; lizards; teiidae; femoral pores; The localization of glands in both lizards and epidermal glands; Ameiva ameiva; pheromone amphisbaenians suggests that signals are pas-

Among the Squamata, semiochemicals have an *Correspondence to: C. Jared, Instituto Butantan, Laborato´rio de important role in sexual behavior, defense, preda- Biologia Celular, Av. Vital Brasil 1500, CEP 05503-900, Sa˜o Paulo, tion, territorial marking, orientation, intraspeciﬁc Brazil. E-mail: [email protected] aggregation, and parental care (Halpern, 1992). Published online in Besides their importance in squamate biology, the Wiley InterScience (www.interscience.wiley.com) morphology of the structures related to semiocom- DOI: 10.1002/jmor.10473

Ó 2007 WILEY-LISS, INC. 2 B.A. IMPARATO ET AL. sively deposited in the environment during locomo- Histochemistry tion of the animals within their territory (Maderson, Historesin and paraffin sections were subjected to the follow- 1986; Jared et al., 1999). Among lizards possessing ing histochemical staining procedures (according to Bancroft femoral glands, signal dispersion involves the and Stevens, 1996): periodic acid-Schiff (PAS) and alcian blue breakage of small portions of the secretion plugs. pH 2.5, for identification of neutral and acidic mucosubstances, These remain on the substrate, releasing chemical respectively, and bromophenol blue, for identification of pro- signals for a period of time that must be related to teins. Sudan black B was used for identification of lipidic substances. both the natural history and environment of a par- ticular species (Alberts, 1993). Ameiva ameiva is a teiid lizard with a broad geo- Photomicrography graphic distribution in Central and South Ameri- Photographs were obtained by using a Leica DMLB micro- cas, from Panama´ to South Brazil and North Ar- scope equipped with a Leica MPS60 photographic system. gentina (Vanzolini et al., 1980; Colli, 1991). The aim of the present paper was the study of the morphology and some aspects of the histochemistry of Scanning Electron Microscopy the femoral glands of the teiid Ameiva ameiva, A strip of skin from the femoral region containing 5–7 femo- and the comparison of the results with those ral pores was removed from the animals, fixed in Karnovsky so- obtained for other lizards and amphibaenians. We lution (Karnovsky, 1965) for 24 h, post-fixed in 1% osmium te- hope to contribute to the integrative knowledge of troxide, incubated in 1% tannin, and postfixed once more in 1% osmium tetroxide (Mizuhira and Futaesaku, 1974; Simionescu the natural history of lizards, especially in relation and Simionescu, 1976). The material was dried by the critical to chemical communication. Our data suggest that point method, coated with gold, and examined in a JEOL SM in Ameiva ameiva the differentiated scales con- 6100 and in a JEOL JSM 5300, operating between 15 and 25 kV. taining the femoral pores and the secretion plugs are structures adapted to the dispersion of chemi- Transmission Electron Microscopy cal signal on the substrate and seem to be associated with the locomotion of these animals through- Fragments of the femoral glands (separated from the skin) of out their territory. about 1 mm3 were fixed in Karnovsky solution, pH 7.2 (Kar- novsky, 1965) for 24 h, post-fixed in 1% osmium tetroxide, con- trasted in 2% uranyl acetate, dehydrated in ethanol and embedded in epoxy resin. Ultrathin sections (60 lm) were obtained in MATERIALS AND METHODS a Sorvall MT 6000 ultramicrotome and were examined with a LEO 906E, operating at 80 kV. Animals

Adult individuals of Ameiva ameiva Linnaeus 1758 were collected at Palmas (State of Tocantins, Brazil) and maintained for Morphometrical Study 2 years in the vivarium of the Laboratory of Cellular Biology of Adult male (N ¼ 14) and female (N ¼ 14) specimens of the Instituto Butantan. They were housed in terraria contain- Ameiva ameiva from the zoological collection of Museu de Zoo- ing humid earth at the bottom with dry branches above and logia da Universidade de Saõ Paulo, all from the same popula- pieces of plastic pipe for shelter. They were daily exposed to tion, were studied. The number and the diameter of the pores sunlight during the morning period. Food was supplied weekly in the femoral region of males and females were quantified and and was composed of a variation of live items (beetles - Tenebrio possible differences in both sexes were analyzed by Student molitor, crickets - Gryllus sp, cockroaches - Pycnocellus surina- test. The diameters of the pores of three regions in the pore row mensis), fruits, eggs, and minced beef. (the two extreme regions, named P1 and P3, and the middle For the morphological studies, six animals were sacrificed region, named P2) were measured. Because data were normally with a intraperitoneal overdose of sodic thiopental just after distributed and had homogeneous variances, they were ana- being collected from nature. Samples of the skin of the femoral lyzed by ANOVA test. region containing the femoral glands were removed and pro- cessed as described below. Voucher specimens were deposited in the collection of the RESULTS Museum of Zoology, University of Saõ Paulo (MZUSP). General Anatomy and Histology of the Femoral Glands

Histology In Ameiva ameiva (Fig. 1), the ventral skin of the thighs, in both males and females, shows a sin- The skin containing the femoral glands was cut in strips of gle row of pores (Fig. 2), whose number varies 2 about 3 3 5mm using a razor blade and fixed in 4% parafor- from 17 to 23 in males and from 16 to 22 in maldehyde in PBS 0.1 M, pH 7.2 for 24 h. The material was dehydrated and embedded either in historesin (glycol methacry- females. Each pore opens in the center of a modi- late, Leica) or paraffin (at 608C). fied scale, resembling a rosette (Fig. 3). Because of For general study of the tissues, 2 lm historesin sections their prominence, the row of pore-bearing scales were stained with toluidine-fuchsin (Junqueira, 1995) and 5–7 stand out from the neighboring scales and, for this lm paraffin sections were stained with hematoxylin–eosin. reason, are easily recognized (Figs. 2, 3). Studying Picro-Sirius and N.M.C. trichrome stains (Junqueira et al., 1979) were used for the identification of collagen and muscular the skin from the dermal side reveals that each of fibers, and the Weigert’s resorcin-fuchsin stain (Bancroft and the pores corresponds to a gland. The row of Stevens, 1996), for the identification of elastic fibers. glands, superimposed on one another, forms a deli-

Journal of Morphology DOI 10.1002/jmor Figures 1–3 (legend on page 4)

Figures 4–6 (legend on page 4) 4 B.A. IMPARATO ET AL. cate yellowish cord (Fig. 4), which is juxtaposed to The secretion plug emerges from the pore in the the dermis by delicate connective tissue. When form of a rod, the tip of which is usually about the this tissue is removed, it is possible to observe that same level of the skin surface. While a smooth each one of the glands is branched and flat (Fig. cover (Fig. 7) envelops its lateral aspect, small 4). In sections of this region, the glandular bodies units corresponding to the dead cells, which are are cut in different planes and internal ductules desquamated from the holocrine epithelium, form appear among the lobules (Fig. 5). The main ducts its distal surface (Fig. 8). Most of these dead cells connecting the glandular bodies to the exterior are are intact and show no signs of abrasion from con- visible in sections cut transverse or longitudinal to tact with the substrate (Fig. 8). the axis of the femur. The ducts are always filled with a solid plug comprising a yellowish and brit- Light Microscopy of the tle secretion (Fig. 5). Secretory Epithelium A thin layer of connective tissue, which is supplied by many blood vessels, covers each of the Germinative cells and secretory cells (Fig. 9A–F) femoral glands. The connective tissue forms septa comprise the glands. To facilitate the morphologi- dividing the gland into lobules (Fig. 6). Each lobule cal description of the differentiation process, the comprises a holocrine epithelium that converges to secretory cells were divided into three subsequen- the ductules, which finally connect to the main tial stages (S1,S2, and S3), according to their duct. structural characteristics. The main duct is lined by a stratified epithe- The germinative cells (G) are localized at the pe- lium, which is continuous with the skin epidermis riphery of the lobules. They are elongated and (Fig. 5). The holocrine secretion that fills the duct have voluminous nuclei (Fig. 9A). The cells in the comprises small units with an affinity for toluidine first stage of differentiation (S1) have the same blue (in toluidine blue-fuchsin staining) (Fig. 6) or localization as the germinative cells but are gener- for eosin (in hematoxylin–eosin staining). ally more spherical. Their nuclei are also spherical and voluminous and usually have one or two con- Scanning Electron Microscopy of the spicuous nucleoli. During the first stage, the cyto- Glandular Pore Region plasm is lightly stained and does not possess secretion granules (Fig. 9A). The scale containing the glandular pore com- The second stage of differentiation (S2) is charac- prise three or four plates arranged like the petals terized by cells with a larger volume and observed of a flower (Fig. 7). One of the plates is always in different phases of the secretory process. These larger and more prominent when compared to the phases can be identified by the increase in the others (Fig. 7, insert). Each one of the scale plates, number of secretory granules and were named P1, specially the larger one, shows a fine arched inter- P2, and P3.InP1, the cells are usually spherical nal border, which contacts the plug (Fig. 7). and the cytoplasm has a number of discrete granules (Fig. 9B). In P2, small granules that are sometimes metachromatic (Fig. 9B) characterize the Figs. 1–3 Fig. 1. A male specimen. Fig. 2. Ventral side of cells. In the last phase (P ), the granules occupy a the thighs showing the pair of femoral pore rows (arrows). Fig. 3 3. Macrophotography of the femoral pores. The glandular ducts large portion of the cytoplasm, are larger and open in the center of a row of modified scales (arrows) resem- spherical, but are heterogeneous in size and bling rosettes. The duct lumina are filled by the secretion plugs in their tinctorial properties (Fig. 9C). Irregular (*). granules containing heterogeneous secretion (Fig. 9D) which usually forms a half-stained pattern Figs. 4–6 Fig. 4. Dermal side of the skin in the region of the femoral pores. The femoral glands are packed close together within the granule distinguish late P3. In the forming a delicate yellowish cord. The arrows point to some most differentiated cells, the irregular nuclei tend branched glandular bodies, while the arrowheads indicate the to be displaced to the periphery, and are smaller glandular ducts. Fig. 5. Histological section of the skin in the in relation to the total cell volume. The cells region of the femoral pores. Part of the glandular cord is visible beneath the dermis, with two glandular bodies sectioned in dif- remain spherical. ferent planes, longitudinal (G1) and transversal (G2), and two In the third and last stage (S3), the secretory main ducts transversally sectioned (D1 and D2), full of secre- cells, already close to the duct, assume a flat form tion. The whole is enveloped in a delicate connective tissue and the cytoplasm is full of irregular granules with (arrowheads). The asterisks indicate internal ductules in the heterogeneous secretion. The peripheral nuclei are distal portion of one of the glands. One main duct (D3) opens through the epidermis (Ep). De, dermis. Glycol methacrylate, pycnotic (Fig. 9E). At the end of this stage, the cells toluidine-fuchsin stain. Fig. 6. Magnification of part of Figure 5, desquamate from the secretory epithelium, enter focusing on a transverse section of gland body. It is divided into the duct, and become part of the solid plug. lobules separated by loose connective tissue (ct). The lobules are During the differentiation process, cells of a dif- constituted by germinative cells (arrowheads), in the periphery, and secretory cells in different stages of differentiation (*). ferent type may be seen among the secretory cells. Arrows, blood vessels; d, ductule. Glycol methacrylate, tolui- They contain dense inclusions suggesting clusters dine-fuchsin stain. of secretion granules lying inside a large vacuole,

Journal of Morphology DOI 10.1002/jmor FEMORAL GLANDS IN THE LIZARD AMEIVA AMEIVA 5

Figs. 7 and 8. Fig. 7. The femoral pore, showing the cylindrical secretory plug (P), with its smooth cover (Co), emerging through a rosette of scales. The rosette is composed of a larger plate (**) and three small plates (*). This arrangement is best observed in the insert. The arrows point to the arched internal border of the larger plate. SEM. Fig. 8. The plug’s surface (P), at higher magni- ﬁcation, where many dead secretory cells (*) may be observed. The arrangement of the cells, which are juxtaposed to one another, gives an idea of the friability of this structure. SEM. and occupy a considerable part of the cell cyto- tive to bromophenol blue, although the granules plasm (Fig. 9C,F). are not all positive to the same intensity. Alcian Blue at different pHs (1.0, 2.5, and 3.1) and Sudan Black were negative for all glandular Histochemical Observations structures. of the Femoral Glands The secretory cells showed a strong and homoge- Light Microscopy of the Rosette neous PAS positivity (before and after amylase treatment) either in the secretion granules or in The skin of Ameiva ameiva comprises a corniﬁed the plug. Similarly, granules and plug were posi- epidermis and a dermis essentially constituted by

Journal of Morphology DOI 10.1002/jmor Figure 9

Figures 10–13 FEMORAL GLANDS IN THE LIZARD AMEIVA AMEIVA 7 dense regular connective tissue in a basket arrange- in other epithelial tissue, the cell membranes show ment with intermingled blood vessels. many interdigitations and desmosomes. The cyto- Longitudinal sections of femoral skin show the gen- plasm of germinative cells is quite electron lucent eral structure of the modified scales (rosettes) con- and is rich in mitochondria, granular and agranu- taining the pores, which may also be seen in trans- lar endoplasmic reticulum, free ribosomes, and verse section (Figs. 10–13). Picro Sirius staining Golgi apparatus (Fig. 14B). Bundles of intermedi- showed that the dermal connective tissue is basically ate filaments (tonofilaments) occur through the formed by collagen fibers, which stain bright red. cytoplasm or linked to desmosomes (Fig. 14A,B). N.M.C. trichrome staining revealed the characteristic The spherical nuclei with one to three nucleoli arrangement of dermal collagen fibers (which appear occupy a large volume of the cell and present pre- in deep blue color) within the rosette. The dermis dominantly loose chromatin (Fig. 14A). comprises an exceptionally well-developed deep layer The secretory cells in the first stage of differen- and a poorly developed superficial layer, immediately tiation (S1) can be distinguished from the germina- below the epidermis (Figs. 10 and 11). Immediately tive cells by their more spherical shape, and espe- around the duct the collagen fibers are thinner and cially by the small and scarce secretion granules are circularly arranged (Fig. 11). Between the deep in the cytoplasm (Fig. 14C). These granules are dermis and the circular fibers, there is zone of loose spherical and homogeneous. Similar to the germi- and less organized connective tissue well irrigated by native cells, the cytoplasm is still quite electron blood vessels (Fig. 11). lucent and tonofilaments and desmosomes are When Weigert’s Fucsine-Resorcine is applied to the abundant. An increase in the granular endoplas- sections, delicate purple elastic fibers are revealed mic reticulum (GER) and Golgi apparatus occurs among the collagen fibers, mainly in the well developed (Fig. 14D). The nuclei is spherical, with predomi- deep layer in the form of a net (Fig. 12) and following a nantly loose chromatin, and is less voluminous circular arrangement around the duct (Fig. 13). within the cell when compared to the situation in germinative cells (Fig. 14C). Transmission Electron Microscopy of the The second stage (S2) is characterized by nuclei Femoral Glands with irregular shapes, an increase in cytoplasmic volume and in the number of the secretion gran- At the ultrastructural level, the germinative ules. It is possible to distinguish at least three cells are polygonal or spindle-shaped (Fig. 14A). As phases in this stage: (P1) In the initial phase, cells have spherical, homogeneous granules of different Fig. 9. Histological sections at high large magnification sizes occupying part of the cytoplasm (Fig. 14E). showing the internal arrangement of the glandular lobules. Lamellae of the granular endoplasmic reticum are Gland cells are seen in different stages of differentiation. Ger- quite developed and dilated and Golgi cisternae minative cells (G) lie at the periphery (A–C, E and F). Secre- were often seen (Fig. 14F). (P2) The cytoplasm of tory cells in the first stage of differentiation (S1) have no granules (A, D, and F). Secretory cells in the second stage of differ- the cells contains many large spherical granules, entiation, divided in three subsequent phases (P1,P2 eP3), in some of them with heterogeneous content (half which the granules are secreted, increase in number, and electron dense, half electron lucent). The GER is undergoes maturation (B–D). Secretory cells in the third stage well developed and is still dilated (Fig. 15A). Other of differentiation (S3), in which they acquire a flat form, present evident signals of decline and finally desquamate from the se- organelles are not so visible and, as the number of cretory epithelium (E). Cells with dense inclusions (*), observed granules increases, nuclei are more electron dense among the other cells (C, D, and F). Arrowheads point to pyc- and are pushed towards the cell periphery. (P3)In notic nuclei. Glycol methacrylate, toluidine-fuchsin stain. the last phase of S2, the cytoplasm is replete with secretion granules with heterogeneous content Figs. 10–13. Fig. 10. Histological section through a rosette- shaped scale containing a femoral pore transversally sectioned. (Fig. 15B). The nuclei often appear already The arrangement of the dermis in the scale is better observed shrunk, are electron dense and have an irregular in Figure 11, which corresponds to a high magnification of the shape. While most organelles are barely observed area indicated by the rectangle. The dermis comprises a thin among the granules, it seems that there is an superficial layer immediately below the epidermis (Ep), a well- developed deep layer (dt) mainly formed by collagen fibers, and increase in the number of lipidic inclusions, with a circular layer (ct), also mainly formed by collagen fibers, medium electron density (Fig. 15B). around the duct. Between the deep layer and the circular layer In the last stage of differentiation (S3), the cells there are areas mainly composed of loose and less organized show evident signs of cytoplasmic disorganization connective tissue (lt) with many blood vessels (arrows). P, secre- and acquire a flatter form (Fig. 15C). The granules tion plug; *, duct epithelium. Paraffin. N.M.C. trichrome stain. Fig. 11. Magnified image of the area indicated by the rectangle occupy practically the whole volume of the cells. in Figure 10. Fig. 12. Details of a histological section through The nuclei are pycnotic or absent. There is an one rosette-shaped scale containing a femoral pore transversally increase in the number of lipidic inclusions (Fig. sectioned. The section was stained to demonstrate the delicate 15C). The cells in this stage are beginning to des- elastic fibers (arrows) which form a disorganized net among the collagen fibers in the deep layer. Ep, epidermis; *, duct epithe- quamate from the secretory epithelium, or are al- lium. Paraffin. Weigert’s resorcine-fuchsin. Fig. 13. The circular ready desquamated and lie loose in the duct, form- arrangement of collagen fibers around the duct. ing the secretion plug. Ultrastructure of the plug

Journal of Morphology DOI 10.1002/jmor 8 B.A. IMPARATO ET AL.

Figure 14

Journal of Morphology DOI 10.1002/jmor FEMORAL GLANDS IN THE LIZARD AMEIVA AMEIVA 9 shows that it comprises electron-dense polyhedral variations (Gasc et al., 1970; Rastogi, 1975). Some units, measuring about 1.3 lm in the larger axis. of these differences may be related to the way epi- A few secretory cells, mainly in the first stages dermal glands are used for chemical communica- of differentiation, contain dense inclusions in their tion within each group. cytoplasm (Fig. 15D). Rosette-like differentiated scales are not exclu- sive of teiids but can also be found in other lizard families such as iguanids and lacertids (Cole, Morphometrical Study 1966a; Blasco, 1975). Our histological observations The number of pores in males and females did not on Ameiva ameiva indicate that this structure pos- show differences (meanmales ¼ 20.27, meanfemales ¼ sibly derives from the fragmentation of one single 19.67, N ¼ 28, t ¼ 0.0159). The comparison of pore scale (Fig. 5). diameters among regions P1, P2,andP3 did not show Femoral pores have been used as systematic differences in males (meanP1 ¼ 0.51 mm, meanP2 ¼ characters for a long time (Linnaeus, 1758; 0.57 mm, meanP3 ¼ 0.49 mm, F ¼ 3.15, N ¼ 42) as Dume´ril and Bibron, 1834). They are also used to well as in females (meanP1 ¼ 0.34 mm, meanP2 ¼ define sexual dimorphism (Cole, 1966a). In the 0.36 mm, meanP3 ¼ 0.35 mm, F ¼ 0.52, N ¼ 42). case of captive Ameiva ameiva we have not The comparison of pore diameters between males observed any significant circannual modification in and females for regions P1, P2,andP3 also did not the external morphology of femoral glands show differences (tP1 ¼ 3.63E08, tP2 ¼ 1.93E07, although the animals remained sexually active, tP3 ¼ 3.25E05). with copulations being frequently observed. Our morphometrical results indicated that in Ameiva ameiva no sexual dimorphism can be observed in DISCUSSION relation to the number or to the diameter of pores. Epidermal glands in pre-anal or femoral regions This apparent similarity in the glands of both are wellknown in several lizard families (Jullien sexes could indicate that they may be used by all and Renous-Lećuru, 1973; Rastogi, 1975) although individuals for territorial demarcation. However, they can be found in other body regions (Simon, differences in the biochemical composition of gland 1983; Dujsebayeva, 1998). Many behavioral and secretion between males and females cannot be biochemical studies conducted in recent decades discarded. have demonstrated the role of these glands in lizard In relation to their internal anatomy, femoral chemical communication (Pough et al., 2001). There glands of Ameiva ameiva correspond to the model is a considerable external variation of epidermal proposed by Gabe and Saint Girons (1965) for liz- glands in relation to their distribution in the body ards in general. In this model, the glands are flat (Kluge, 1967a,b; Dujsebayeva, 1998), the shape of and branched, with an internal ramification of the scales containing the pores (Blasco, 1975; van ductules connected to a main duct, which leads to Wyk et al., 1992), and the coloration of the secre- the secretion to the exterior. The solid secretion tion plug (Cole, 1966b; Blasco, 1975). Internally plug is generally the same color of the gland in these glands can also present anatomical and color which it is produced (Cole, 1966b). In Ameiva ameiva, femoral glands and secretion plugs all have the same yellowish coloration. Fig. 14. The cells constituting the femoral gland lobules. Femoral glands in Ameiva ameiva, as in other (TEM). A: Polygonal germinative cell with a medium electronlu- squamates, are holocrine. The extrusion of the cent cytoplasm and a voluminous nucleus (N), with loose chro- solid secretion plug through the pore is due to the matin and a well developed nucleolus (n). The cell is connected pressure exerted by the distal proliferation and to the neighboring cells by many desmosomes (arrows). B: Higher magnification of a germinative cell. The cytoplasm is differentiation of the secretory cells within tubules rich in tonofilaments (arrows), mitochondria (m), agranular (Antoniazzi et al., 1993). This process, however, endoplasmic reticulum (*), small vesicles (arrowheads) and free does not explain the lateral aspect of the plug, visi- ribosomes spread, among the other organelles. C: First stage of ble by scanning electron microscopy (Figs. 7, 8). differentiation S1 of a secretory cell. As in the germinative cell, the cytoplasm is medium electronlucent and the nucleus (N), This seems to be a structure formed by the accu- with a prominent nucleolus (n), occupies a large volume of the mulation of desquamated epithelial layers since, cell. The first secretion granules (arrows) lie close to both the according to Maderson (1972), the gland duct epi- granular endoplasmic reticulum (GER) and Golgi apparatus thelia do not show cyclic changes of the type that (Go). n, nucleolus. D: Higher magnification of rectangle marked are associated with periodic shedding. in (C), showing details of the Golgi apparatus (Go), surrounded by many small vesicles (arrowheads), and of the GER. G, secre- The ultrastructure of the germinative cells is tion granule. E: First phase P1 of the second stage of differen- typical of epidermal cells, showing large numbers tiation. The total cell volume is enlarged and the number of se- of tonofilaments and desmosomes. These cells cretory granules (G) has increased. Arrow, bundle of tonofila- reveal an intense metabolic activity, which is mor- ments; *, Golgi apparatus. F: Enlarged area of a cell in P1. The secretion granules are spherical with dense, homogeneous con- phologically evidenced by the cytoplasm rich in dif- tent. Go, Golgi apparatus; GRE, granular endoplasmic reticu- ferent organelles. The observation of histological lum. and ultrastructural differences among the secre-

Journal of Morphology DOI 10.1002/jmor 10 B.A. IMPARATO ET AL.

Fig. 15. The cells constituting the femoral gland lobules. (TEM). A: Second phase P2 of the second stage of differentiation of a secretory cell. The secretion granules (G) increase in volume and in number and the contents of some of them (arrows) are not as homogeneous as phase 1 (F and G). The GER remains well developed. N, nucleus. B: Third phase P3 of the second stage of differentiation. The cytoplasm shows the first signs of disorganization although the main organelles, such as the GER are still visible. The contents of the secretion granules (G) are heterogeneous, sometimes half electrondense and half electronlucent (arrows). Lipid inclusions lie in the cytoplasm among the granules (arrowheads). N, nucleus. C: Third stage of differentiation S3 of the secretory cells. The cell is slightly flattened and the nucleus (N) lies in the periphery, shrinks as it becomes electron-dense and irregular. The cytoplasm is quite disorganized and the heterogeneous secretion granules, together with lipid inclusions (arrowheads) occupy most of it. D: Dense inclusions (I) resembling clusters of secreted material are quite common in the cytoplasm of many secretory cells, mainly in the first stages of differentiation. tory cells made it possible to distinguish at least presence of large inclusions that are apparently three different stages of differentiation. In the the accumulation of secretory material in the form third stage, small inclusions with medium electron of vesicles whose destiny in the cells we have not density seem to be lipidic in nature. Furthermore, being able yet to determine. All these differentia- we often observed in all differentiation stages the tion processes resemble what has been observed in

Journal of Morphology DOI 10.1002/jmor FEMORAL GLANDS IN THE LIZARD AMEIVA AMEIVA 11 the epidermal glands of other lizards (Cole, 1966a; Differently from amphisbaenians, in lizards it Gasc et al., 1970; Maderson, 1972) and amphisbae- has already been observed that distal portions of nians (Antoniazzi et al., 1993, 1994; Jared et al., 1999). femoral gland secretion plugs are fractured and In relation to the chemical nature of Ameiva deposited on the substrate, where they remain in ameiva femoral gland secretion, our histochemical the form of small secretion blocks (Alberts, 1993). and ultrastructural data indicate that it consists of According to our observations, that seems also to neutral mucosubstances and proteins, probably be valid in Ameiva ameiva. forming glycoproteins. The same results were Scanning electron microscopy showed that the obtained for other lizards (Gabe and Saint Girons, secretion plugs in this species are composed of 1965; Cole, 1966b; Uva, 1967; Gasc et al., 1970; dead cells that desquamate from the femoral gland Rastogi, 1975; Alberts, 1993) and amphisbaenians secretory epithelium. The fact that most of the (Antoniazzi et al., 1993, 1994). Despite the nega- cells seen on the plug’s surface are intact suggests tive result obtained with the use of Sudan Black B that, rather unlike amphisbaenians, they are not for detection of lipids, at the ultrastructural level, abraded by the substrate. Rather, the microscopi- in advanced stages of cell differentiation, we have cal appearance of the surface suggests that the observed small inclusions in the secretory cell cyto- frail plug is probably fractured, falling in small plasm that seem to be lipidic in nature. Lipids are pieces on the substrate. On the other hand, the common among substances involved in chemical epithelial cover may function to maintain the cells’ communication. (Alberts, 1990; Alberts et al., 1992; cohesion as a plug, favoring the prolongation of Halpern, 1992). Alberts (1993), working with the signal effect by preventing rapid secretion volatili- chemistry of pheromones in Dipsosaurus dorsalis, zation. Unlikely, in Amphisbaena alba, the surface discussed environmental influence on the composi- of the secretion plug shows a different arrange- tion of the femoral gland secretion of this iguanid. ment, in the manner of a cigar, with interspersed In Iguana iguana and Dipsosaurus dorsalis, the layers of keratinocytes and secretory cells desqua- low concentration of lipids in their glandular secre- mated from the glandular epithelium. In this case, tion could only be detected by gas chromatography the arrangement of the plug possibly helps in the (Alberts, 1990; Weldon et al., 1990). Thus, gradual fragmentation of the secretion during the although undetectable by histochemical techni- trail formation, modulating friction through the ques, small amounts of lipid might be present in presence of the interspersed keratinocyte layers teiid femoral gland secretions. (Jared et al., 1999). In Ameiva ameiva, on the The ventral localization of the epidermal glands other hand, we have observed that femoral pores on the body of lizards and amphisbaenians sug- only touch the substrate when the animal rests gests that their secretions are passively deposited the internal side of its thighs on the ground. In a in the environment (Maderson, 1986). Recent resting position, it probably presses the row of papers have demonstrated that in fact many spe- femoral pores against the substrate. The frail na- cies of lizards respond to secretions derived from ture of the secretion, resulting from the juxtaposi- these glands (Cooper and Vitt, 1986; Cooper et al., tion of the dead secretion cells, without being 1994; Cooper et al., 1996). However, the method interspersed with keratinocytes (as is the case in used by lizards for depositing chemical signals in amphisbaenians) may favor transverse fracturing the environment has not been studied so far. In of the plug and the deposition of secretion tiny por- amphisbaenians, (Antoniazzi et al., 1993, 1994), tions in the environment. This process may be studying the morphology of the pre-cloacal glands facilitated by the rosette-like shape and histologi- of Amphisbaena alba, demonstrated that, as a cal arrangement of the pore-bearing scales. result of the position and arrangement of these Microscopic examination of the row of femoral glands, the secretion plugs, perpendicular to the pores in Ameiva ameiva shows that some plugs body, make direct contact with the tunnel walls are more prominent than others relative to the where this species lives. These authors suggest skin surface. These differences in plug lengths are that, during locomotion, the plugs are naturally an indication of the size of the tiny pieces of secre- abraded and secretion fragments are deposited on tion deposited on the substrate. The plug deposi- the substrate, forming a trail. In fact, Jared et al. tion system may function each time the animal (1999) experimentally demonstrated the formation rests relaxing its legs on the ground. Then, in this of a secretion trail during locomotion of A. alba. species, the method for dispersing the pheromone Scanning electron microscopic examination, both seems not to consist of the chance breakage of the of the trail and of the abraded surface of the plug, plug but is much more complex. It involves both showed that the fragments of the trail correspond morphological characteristics of the rosettes and to the secretion granules of the secretory cells. The the structural and mechanical characteristics of trail then may constitute an extensive area for vol- the plug itself. These seem to be adaptations for atilization of chemical signals, which may be an ef- making dispersion more efficient through the con- ficient way for intraspecific (or interspecific) com- stant deposition on the substrate of tiny portions munication within tunnels. of secretion every time the animal rests. Although

Journal of Morphology DOI 10.1002/jmor 12 B.A. IMPARATO ET AL. having its own peculiarities, this method of signal Cooper WE Jr, Vitt LJ. 1984. Conspecific odor detection by the dispersion in A. ameiva, as occurs in amphisbae- male broad-headed skink, Eumeces laticeps: Effects the sex and site of odor source and of male reproductive conditions. J nians, is intimately dependent on the locomotion Exp Zool 230:199–209. of the animal within its territory. Cooper WE Jr, Vitt LJ. 1986. Lizards pheromones: Behavioral responses and adaptative significance in skinks of the genus Eumeces. In: Durvall D, Mu¨ ller-Schwarze D, Silverstein RM, ACKNOWLEDGMENTS editors. Chemical Signals in Vertebrates: Ecology, Evolution and Comparative Biology, Vol 4. New York: Plenum. pp 323– The authors are thankful to Dr. Ida S. Sano- 340. Martins and Dr. Jose´ Roberto M.C. Silva for the Cooper WE Jr, Lo´pez P, Salvador A. 1994. Pheromone detection use of the photomicroscopes. Thanks to Tatiana L. by an amphisbaenian. Anim Behav 47:1401–1411. Fernandes, Laurinda A. Soares, and Maria Helena Cooper WE Jr, van Wyk JH, Mouton PFN. 1996. Pheromonal detection and sex discrimination of conspecific substrate depos- Ferreira for their kind technical assistance, to its by the rock-dwelling cordilid lizard. Copeia 1996:839–845. Dr. Hana Suzuki and Leonardo de Oliveira for Dujsebayeva TN. 1998. The histology of callous scales of the their critical revision and to Alberto Barbosa de males of Asian rock agamas, Laudakia caucasia and Lauda- Carvalho for the help with the color figures. Thanks kia himalayana (Reptilia; Agamidae). Russ J Herpetol 5:160– are also due to Brazilian National Research Council 164. Dume´ril AMC, Bibron G. 1834. Erpe´tologie geńe´rale ou histoire CNPq, FAPESP, and Fundaçaõ Butantan. Specimens naturelle comple`te des reptiles. Vol 1, Paris: Roret. were collected under IBAMA permits Procs. n8 Gabe M, Saint-Girons H. 1965. 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Journal of Morphology DOI 10.1002/jmor