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Keywords:

  • dipsadidae;
  • goo-eaters;
  • adductor muscles;
  • lumen;
  • supralabial glands;
  • infralabial glands

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

Geophis belongs to the goo-eating dipsadine assemblage of snakes that are known to feed exclusively on earthworms, snails, and slugs. Although the unusual feeding strategies of the goo-eating dipsadines are well known (but poorly documented), little attention has been paid to their internal anatomy. Here, we describe a new and noteworthy morphological and histochemical condition of the infralabial glands in three species of Geophis (G. brachycephalus, G. nasalis and G. semidoliatus), all earthworm feeders. Their infralabial glands are constituted of two distinct parts: an anterolateral portion composed of mucous and seromucous cells that stretches from the tip of the dentary to the corner of the mouth, and a tubular posteromedial portion that is exclusively seromucous. The anterolateral portion receives fibers of the levator anguli oris muscle that attaches on its posterodorsal extremity while the posteromedial portion extends posteriorly to the corner of the mouth where it receives fibers of the adductor mandibulae externus medialis muscle. Furthermore, the posteromedial portion of the infralabial gland is constituted by large acini filled with secretion that is periodic acid-Schiff positive. These acini release their secretion directly into a large lumen located in the middle of the glandular portion. In the three species examined, the supralabial glands show a traditional configuration, being constituted of mucous and seromucous cells and retaining an enlarged part in its caudal region that resembles a Duvernoy's gland. The presence in Geophis of an expanded lumen in part of the infralabial gland that is compressed by an adjacent muscle suggests a more specialized role for the secretion produced by these glands that may not be related to envenomation but rather to prey transport and mucus control. J. Morphol. 275:87–99, 2014. © 2013 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

In snakes, the labial (infralabial and supralabial glands), venom, and Duvernoy's glands are among the best-known oral glands (Taub, 1966). Venom glands are present only in advanced snakes (Caenophidia) with a front-fanged venom delivery system (displayed by some Atractaspididae, all Elapidae and Viperidae), while Duvernoy's glands are present in a number of endoglyptodont colubroidean snakes (sensu Zaher et al., 2009) without a front-fanged system (Vidal, 2002; Kardong, 2002). Infralabial and supralabial glands seem to occur in all snakes that have been studied to date (Smith and Bellairs, 1947; Kochva, 1978; Underwood, 2002).

The usual classification of reptile oral glands is based on the types of secretion granules, and depends on the distinct histochemical composition of these granules (Gabe and Saint-Girons, 1969; Kochva, 1978). Although there is still uncertainty regarding the relationship between cell types recognized by histologists and their secretion, it is clear that mucous cells secrete mucins and that all venom glands contain serous cells of some type (Underwood, 1997).

Venom glands are generally surrounded by muscles that act as compressors during the bite (Haas, 1973), have a lumen (a large encapsulated reservoir where the secretion may be stored), and release venom through a single duct that connects directly with the fang, constituting a high-pressure system (Kardong and Lavin-Murcio, 1993; Jackson, 2003). The venom and Duvernoy's glands are considered homologous and a component of the venom-delivery system in snakes, which are usually constituted of serous cells and associated with toxin production (Taub, 1966; Kochva, 1987; Jackson, 2003; Fry et al., 2008). Infralabial and supralabial glands, on the other hand, lack any association with adjacent muscles and are composed of a row of small glands and their short, individual ducts, which are predominantly constituted of mucous cells with the function of producing mucous secretion mainly for lubrication (Kochva, 1978).

In Neotropical snakes of the subfamily Dipsadinae Bonaparte, 1838 particularly in “goo-eater” snakes, the Duvernoy's glands seem to be reduced or absent while infralabial glands are well developed and constituted predominantly of seromucous cells (Taub, 1967a; Fernandes, 1995; Oliveira et al., 2008). This fact is probably related to their highly specialized feeding behavior (Gans, 1972), which is mainly shown in dipsadine snakes that feed on snails (Savitzky, 1983; Sazima, 1989). Additionally, Zaher (1999) pointed out that snakes of the genus Geophis Wagler, 1830 show a posterior expansion of the infralabial glands that tends to be surrounded by fibers of the adductor mandibulae externus medialis pars posterior (AEM) muscle that probably act as a “compressor glandulae.” Similarly, the goo-eating genera Atractus, Sibynomorphus, Sibon, Dipsas, Ninia, and Adelphicos show distinct instances of muscle attachments into their often hypertrophied infralabial glands. Preliminary observations revealed a series of noteworthy specializations related to oral glands and head muscles of goo-eating snakes that stimulated a research program focused on the topic (Zaher, 1996, 1999; Antoniazzi et al., 2005; Oliveira et al., 2008).

Goo-eaters constitute a putative and speciose group of Dipsadinae snakes, whose species feed mostly on soft and viscous invertebrates (generally mollusks such as slugs and snails, and annelids; Cadle and Greene, 1993). Although monophyly of the group and relationships between genera of the dipsadine subfamily remain poorly elucidated, the genus Geophis seems to be more closely related to the goo-eating dipsadine genus Atractus (Pyron et al., 2011; Grazziotin et al., 2012), and thus represented a natural candidate for expanding our comparisons previously started with the similarly fossorial Atractus (Oliveira et al., 2008).

Geophis contains over 40 species distributed from northern Mexico to northwestern Colombia and northern Ecuador in South America, for which only a few species are well represented in museum collections (Downs, 1967; Wilson and Townsend, 2007). These are small, leaf-litter, semifossorial or fossorial snakes that feed mainly on earthworms, but also on leeches and slugs (Downs, 1967; Campbell and Murphy, 1977; Seib, 1985; Campbell et al., 1983; Savage and Watling, 2008). However, apart from several reports on stomach contents available in the literature (e.g., Seib, 1985), almost nothing is known of the feeding behavior of Geophis, except for their predilection to annelids.

This study is part of a series of publications that investigate the morphological and functional aspects of the oral glands and associated structures in the goo-eating dipsadine clade of Neotropical snakes (Zaher, 1996; Oliveira et al., 2008). Here, we describe the anatomical, histological and histochemical features of the labial glands and related musculature in three species of the genus Geophis [G. brachycephalus (Cope, 1871), G. nasalis (Cope, 1868), and G. semidoliatus (Duméril et al., 1854)] and compare them with the goo-eating Atractus reticulatus (Boulenger, 1885), Dipsas indica Laurenti, 1768 and Sibynomorphus mikanii (Schlegel, 1837) described in our latest contribution of the series (Oliveira et al., 2008). Both morphological and histochemical features of the infralabial glands of Geophis described herein suggest a specialized role that may be related more specifically to mucus control and prey transport rather than immobilization of their viscous prey.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

Muscular and Glandular Morphology

Specimens used in this study belong to the following collections: Museum of Natural History, University of Kansas, Lawrence (KU); National Museum of Natural History, Washington (USNM); Museum of Vertebrate Zoology, University of California, Berkeley (MVZ). We dissected the head muscles and glands of two individuals of G. brachycephalus (KU 35878; KU 63805), two of G. nasalis (MVZ 148181; MVZ 148267), and one of G. semidoliatus (USNM 224839) that were kindly made available for study. Our study was restricted to only three species due to the scarcity of representatives of most species of Geophis in scientific collections. Only a few specimens were thus made available for dissection and histological preparation. However, we believe our sample illustrated accurately the morphological condition of the labial glands and associated muscles in Geophis. All dissections were performed under a stereomicroscope Olympus SZX 12 equipped with a camera lucida.

McDowell (1972) and Groombridge (1979) provided important studies on the morphology of the palate, which also offered some information on the soft tissue anatomy of the floor of the mouth, and here we follow their terminology. Glandular terminology follows Taub (1966), Kochva (1978), and Underwood (2002). The terminology for the external mandibular adductor muscles is still in dispute among authors (see Rieppel, 1980; McDowell, 1986; Zaher, 1994a, 1994b). Here, we follow the terminology of Zaher (1994b,1996).

Histology and Histochemistry

All histological sections were performed on specimens belonging to scientific collections. Heads were skinned from the nostril to the neck and removed from the specimens at the level of the first cervical vertebra. Specimens and their head skin were thus returned to their jar in the collection. Although dissections were carefully performed, we consider that some of the more detailed anatomical aspects of the epithelium of the mouth may have been irreparably damaged. This seems to be the case for the openings of the main ducts belonging to the infralabial glands since these were not found in the available histological sections (see results below).

After removal of the skin and disarticulation of the neck, the entire heads were submitted to decalcification in 4.13% aqueous EDTA, pH 7.2, renewed every other 3 days, constantly stirred for 60 days. The decalcified heads were then divided sagittally into two halves, dehydrated in ethanol, embedded in paraffin, and submitted to serial sagittal or transversel sectioning. The sections (7 μm) were performed in a Microm HM 340 E microtome with disposable steel blades. The sections were submitted to hematoxylin-eosin (HE) staining, for the general study of the tissues, and to Mallory trichrome staining (Junqueira et al., 1979), for identification of the collagen and muscular fibers and epithelia.

Sections were still subjected to the following histochemical staining procedures (according to Bancroft and Stevens, 1996): periodic acid-Schiff (PAS), alcian blue pH 2.5, combined alcian blue (pH 2.5) and PAS (Pearse, 1985; Kiernan, 2001), and bromophenol blue (BB). PAS and alcian blue were applied for identification of neutral and acid mucosubstances, respectively, and BB for identification of proteins.

Photographs were taken with a DFC425 digital camera in a Leica M205a stereoscopic microscope and a Leica M2500 microscope using Leica Application Suite software (Version 3.8). After image acquisition, measurements of the acinar diameters in labial glands were taken using the Analysis package from Leica Application software. Acinar measurements were taken from 30 acini on each gland in at least three different histological slides for each species.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

Supralabial Glands

General morphology

In all three species examined (G. brachycephalus, G. nasalis, G. semidoliatus), supralabial glands were located lateral to the maxilla from the rostral gland to the anterior edge of the muscle levator anguli oris (LAO; Fig. 1A–C). These glands were whitish, like the anterolateral portion of the infralabial glands, and their acini were barely visible during dissection. No muscle fibers were associated with the supralabial glands. In G. brachycephalus and G. nasalis, the anterior region of the supralabial glands was thin and elongated, whereas the posterior part was enlarged below the orbit (Fig. 2A–C).

image

Figure 1. Schematic drawing of the skinned heads of Geophis brachycephalus (A), Geophis nasalis (B), and Geophis semidoliatus (C), showing the location of the cephalic glands and head muscles. aem2, muscle adductor mandibulae externus medialis; aep, muscle adductor mandibulae externus profundus; aes, muscle adductor mandibulae externus superficialis; il1, anterolateral portion of the infralabial gland; ap.aes, aponeurosis of the adductor mandibulae externus superficialis; hg, harderian gland; lao, muscle levator anguli oris; ng, nasal gland; il2, posteromedial portion of the infralabial gland; qml, quadrato-maxillary ligament; sl, supralabial gland.

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image

Figure 2. (A–B) Geophis brachycephalus. A: Lateral view of the dissected head showing oral glands and superficial muscles. B: Lateral view with a higher magnification, showing both anterolateral (il1) and posteromedial (il2) portions of the infralabial gland; the muscle LAO attaches to the anterolateral portion while the muscle adductor mandibulae externus medialis (aem2) attaches to the posteromedial portion. C: Ventrolateral view of the head of Geophis nasalis stained with alcian blue and alizarin red, showing both anterolateral (il1) and posteromedial (il2) portions of the infralabial gland and the supralabial gland (sl) with an enlarged posterior part. d, dentary; f, frontal; hg, harderian gland; ncm, muscle neurocostomandibularis; ng, nasal gland; p, parietal; pfr, prefrontal; pg, muscle pterygoideus; qml, quadrato-maxillary ligament; sl, supralabial gland.

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Histology and histochemistry

In G. brachycephalus (Fig. 3H,I), the supralabial gland was constituted predominantly of seromucous cells, although some mucous cells were observed intermingled among the seromucous cells, being very similar to the anterolateral portion of the infralabial gland.

image

Figure 3. Geophis brachycephalus. A: Sagittal section of the head stained with HE, showing both anterolateral (il1) and posteromedial (il2) portions of the infralabial gland. B: Transverse section of the posterior region of the head stained with HE, showing the posteromedial portion (il2) embraced by the muscle adductor mandibulae externus medialis (aem2). C: Sagittal section of the mandibular region showing the anterolateral portion (il1) with its duct (d-il1) and the posteromedial portion (il2) with a lumen (lu) in its center; the arrows numbered as 1 and 2 point to the main ducts belonging to the anterolateral and posteromedial portions of the infralabial gland, respectively. D: Sagittal section with a higher magnification, showing acini opening into the duct (d-il1); the arrows point to the apical part of the cytoplasm of cells lining the ducts that reacted positively to alcian blue (pH 2.5). E: Details of the seromucous (sm) and mucous cells (m) in the anterolateral portion of the infralabial gland; F: Detail of the lumen (lu) of the posteromedial portion of the gland, full of secretion; G: Sagittal section with a higher magnification of the posteromedial section evidencing acini full of secretion and vesicles (v). (C-G) Sections stained by conjugated reaction of the methods alcian blue (pH 2.5) and PAS. H: Transverse section of the maxillary region stained with HE, showing supralabial gland (sl) constituted by seromucous (sm) and mucous cells (m) and its short duct opening in the oral cavity (oc). I: Detail of the seromucous (sm) and mucous cells (m) in the supralabial gland. cp, compound bone; dt, dentary teeth; mx, maxilla; om, oral mucosa.

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In G. nasalis, the secretory epithelium of the supralabial gland was composed predominantly of acini with a narrow lumen and composed by cells that reacted positively to both alcian blue (pH 2.5) and PAS, characterizing their mucous condition (Fig. 4I,J). Only a small part of the acini from the supralabial gland was constituted of seromucous cells that reacted negatively to alcian blue (pH 2.5), but were strongly positive to PAS. These seromucous acini were mostly concentrated in the dorsal region of the gland (Fig. 4I,K). The supralabial glands showed many short ducts that opened into the epithelium along the mouth.

image

Figure 4. Geophis nasalis. A: Sagittal section of the head stained with HE, showing both anterolateral (il1) and posteromedial (il2) portions of the infralabial gland and their associated muscles. B: Detail of the muscle adductor mandibulae externus medialis (aem2) attached to the posteromedial portion (il2). C: Sagittal section stained with HE, showing contrast between anterolateral (il1) and posteromedial (il2) portions. D: Sagittal section stained by a reaction of bromofenol blue, showing seromucous cells in both portions of the infralabial gland. E: Sagittal section stained by conjugated reaction of alcian blue (pH 2.5) and PAS, showing mucous cells only in the anterolateral portion of the gland (il1). F: Sagittal section stained with HE, showing a duct (d-il1) distending into the anterolateral portion of the infralabial gland (il1). G: Transverse section of the posterior region of the infralabial gland stained with HE, showing a duct (d-il1) of the anterolateral portion (il1) of the infralabial gland opening under an infralabial scale, and illustrating the relationship between both anterolateral (il1) and posteromedial (il2) portions and between the anterolateral portion and the muscle LAO. H: Transverse section stained with HE, showing the relationship between the posteromedial portion (il2) and the muscle adductor mandibulae externus medialis (aem2). I: Sagittal section stained with a conjugated reaction of alcian blue (pH 2.5) and PAS, showing the supralabial gland (sl) and the anterolateral portion (il1) of the infralabial gland. J: Sagittal section stained with alcian blue (pH 2.5), showing the presence of acid mucous in mucous cells of the supralabial gland (sl). K: Sagittal section stained with PAS, showing a strong positive reaction in seromucous (sm) and mucous cells (m) and demonstrating the presence of neutral mucous.

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In G. semidoliatus, the supralabial gland was constituted by both mucous and seromucous cells arranged in acini (Fig. 5D). The seromucous cells were polygonal, with basal and rounded nuclei and stained more intensively with HE than mucous cells (Fig. 5D). The granules in the cytoplasm of the seromucous cells were rounded and reacted positively to PAS (Fig. 5E). Mucous cells were also rounded and their cytoplasm stained weakly with HE (Fig. 5D). The granules in the cytoplasm of the mucous cells were reacted strongly to alcian blue (pH 2.5) and were smaller than those of seromucous cells (Fig. 5E). Histochemical results were summarized in the Table 1.

image

Figure 5. Geophis semidoliatus. A: Sagittal section of the mandibular region showing the posteromedial portion (il2) with a lumen (lu) and the association between the gland and muscle adductor mandibulae externus medialis (aem2); section stained by a conjugated reaction of alcian blue (pH 2.5) and PAS methods. B: Sagittal section of the posteromedial portion (il2) of the gland stained with HE and shown in a higher magnification, evidencing acini full of secretion and vesicles (v). C: Sagittal section of the posteromedial portion of the gland stained by a conjugated reaction using alcian blue (pH 2.5) and PAS methods and shown in a higher magnification, showing acini full of secretion and vesicles (v). D: Sagittal section of the supralabial gland stained with HE, showing its general structure. E: Sagittal section of the supralabial gland showing seromucous cells positive to PAS and mucous cells with intensive reaction to alcian blue; stained by a conjugated reaction using alcian blue (pH 2.5) and PAS methods.

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Table 1. Summarized histochemical results of labial glands in three species of Geophis
    Infralabial glands
 Supralabial glandsil1il2
 PASAABBPASAABBPASAABB
  1. Abbreviations: AA, alcian blue; BB, bromophenol blue; il1, anterolateral portion of the infralabial gland; il2, posteromedial portion of the infralabial gland; PAS, periodic acid-Schiff

  2. a

    cells concentrated in the central region of the gland.

  3. b

    cells concentrated in the peripheral region of the gland.

  4. (+) positive to the reaction;

  5. (-) negative to the reaction;

Geophis brachycephalus++a+b++a+b++
Geophis nasalis++a+b++a+b++
Geophis semidoliatus++a+b++a+b++
ClassificationMucous/seromucousMucous/seromucousSeromucous

Infralabial Glands

General morphology

In all three species examined, infralabial glands had distinct anterolateral and posteromedial parts that were tightly associated but distinguished, during dissection, by color and acinar size (Figs. 1A–C, 2A–C, and 6). The anterolateral portion of the infralabial gland (il1) extended along the internal lip margin, just under the infralabial scales, from the tip of the dentary bone to the level of the corner of the mouth (Fig. 1A–C). A series of small ducts extending from this portion of the gland opened into the mouth along the lip located medially to the infralabial scales. Additionally to the series of small ducts, the il1 also presented a single large, anteroposteriorly directed duct that opened in the anteriormost region of the mouth, at the level of the tip of the dentary. In preserved specimens, the il1 was whitish and showed small acini that were difficult to observe during dissection (Fig. 2B,C). The posteromedial portion of the infralabial gland (il2) extended ventrally to the il1 as a striped and elongated structure along the anterior region of the mouth. Just before the level of the corner of the mouth, the il2 enlarged while extending caudally until approaching or reaching the anterior edge of the muscle adductor mandibulae externus superficialis (AES; Fig. 1A–C). The il2 showed a darker color, with larger, more globular lobules than those of the il1, being more visible during dissections (Fig. 2B,C). The il2 of the gland lacked the small ducts connected to the mouth, typical of an infralabial gland, its secretion being discharged through a single, large duct that was medially positioned and anteroposteriorly oriented.

image

Figure 6. Schematic drawing of the infralabial gland in Geophis, representing major morphological structures. aem2, muscle adductor mandibulae externus medialis; d-il1, duct of the anterolateral portion of the infralabial gland; d-il2, duct of the posteromedial portion of the infralabial gland; il1, anterolateral portion of the infralabial gland; il2, posteromedial portion of the infralabial gland; lao, muscle levator anguli oris; lu, lumen; oc, oral cavity; v, vesicles.

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Histology and histochemistry

In all species studied, both portions of the infralabial gland were wrapped in a thin layer of connective tissue where muscle fibers from the LAO and AEM (sensu Zaher, 1994b) were inserted (Figs. 3A,B, 4A,B, 5A). The il1 exhibited narrower acini than the il2 (Figs. 3A, 4A, Table 2). The basic differences among infralabial glands of the three species were mainly related to the number and disposition of mucous and seromucous cells, as summarized in the Table 1.

Table 2. Acinar diameters (in μm) present in labial glands belonging to three species of Geophis
    Infralabial glands
Supralabial glandsil1il2
RangeMeanSDRangeMeanSDRangeMeanSD
  1. Abbreviations: il1, anterolateral portion of the infralabial gland; il2, posteromedial portion of the infralabial gland; SD, standard deviation.

  2. Values correspond to thirty acinar diameters for each gland.

Geophis brachycephalus40.70–96.3762.4512.6638.13–89.9362.9811.5675.06–159.83113.4623.51
Geophis nasalis39.69–87.4464.0612.0249.18–114.7776.2917.0444.35–104.0578.2913.9
Geophis semidoliatus48.22–84.8164.119.9031.78–60.0349.546.8368.64–152.9696.9222.74

In G. brachycephalus, most acini from the il1 were constituted of seromucous cells, positive to PAS and negative to alcian blue (pH 2.5; Fig. 3C). The mucous acini (alcian blue positive) were mostly concentrated in the central region of the gland (Table 1) and were observed delivering secretion into a single duct that extends longitudinally along the entire il1 (Fig. 3C,D). Both seromucous and mucous cells were polygonal, with basal and rounded nuclei. In seromucous cells, the secretory granules were larger than those from mucous cells and stained only with PAS, while in mucous cells the small secretory granules were intensely stained with alcian blue (pH 2.5) and PAS (Fig. 3E). The duct was covered with cells similar to the seromucous cells from acini, although with a more columnar shape, and only the apical region of these cells stained positively for alcian blue (pH 2.5; Fig. 3D). The il2 consisted of acini with an enlarged lumen that contained large amounts of secretions (Fig. 3C,G). The internal spaces of the acini forming the il2 were significantly wider than the spaces present in the acini of the il1 (Table 2). Cells lining the acini in the il2 showed basal and rounded nuclei, with a smaller cytoplasm than the one present in seromucous cells of the il1 (Fig. 3F,G). The acini from the former region converged toward the middle of the gland, opening directly into an enlarged lumen (Fig. 3C,F). The lumen was covered with seromucous cells, with basal and rounded nuclei and a cytoplasm that was found to be lower than the one observed in the cells lining the acini (Fig. 3F). The secretion found in the central lumen of the gland and in the acini of the il2 was PAS-positive, and contained large numbers of vesicle-like structures (Fig. 3G). These structures showed a broad range in size, from very small (smaller than the nucleus of the cells) to large structures (equivalent in size to a few seromucous cells). The largest structures were mostly concentrated close to the epithelium of the acini. There was also a gradient of PAS reaction, with smaller vesicles being positive to the method, while the larger ones were frequently negative (Fig. 3G).

In G. nasalis, the il1 was constituted by acini made up of mucous cells that stained to HE and positive to alcian blue (pH 2.5), and seromucous cells strongly positive to HE and bromofenol blue (Fig. 4C–E). The seromucous cells were restricted to the peripheral region of the gland (Fig. 4D, Table 1). As in G. brachycephalus, the il1 also presented a large duct that extended along its medial surface, reaching the anterior region of the mouth at the level of the anterior tip of the dentary. According to the available histological sections, the duct opened anterolaterally to the tip of the dentary (Fig. 4F), although we failed to find the opening through a thorough inspection of the region under the microscope. The duct was constituted of mucous cells, with columnar cytoplasm and flattened and basal nuclei (Fig. 4F). In addition to this main duct, we also observed a series of short ducts that were mainly arranged perpendicularly to the gland and opened between the infralabial scales and the oral epithelium. More posteriorly, at the level of insertion of the LAO into the gland, the ducts surrounded the muscle bundle to reach the oral epithelium (Fig. 4G). The secretion produced by the cells that formed the il2 was discharged directly into the lumen formed in the central region of the gland (Fig. 4A,B). Cells from this glandular portion showed basal and rounded nuclei and rounded granules in their cytoplasm. These granules were seromucous in nature, reacting strongly to HE and to bromofenol blue, but negatively to alcian blue (pH 2.5; Fig. 4C–E). Both il1 and il2 were positive to PAS, although the il1 showed a stronger positive reaction than the il2.

The infralabial gland of G. semidoliatus was very similar to the gland of G. brachycephalus. Seromucous cells with basal and rounded nuclei and granules that reacted positively to PAS constituted the il1. In this glandular portion, acini were formed exclusively by mucous cells (alcian blue positive) or by both mucous and seromucous cells. The il2 was constituted of acini with enlarged spaces that were totally filled with secretion (Fig. 5A–C). Within this glandular portion, we observed the presence of an extended lumen occupying the entire central region, and directly receiving the discharges from the acini (Fig. 5A). The granules in the cytoplasm of the cells were rounded and reacted positively to PAS (Fig. 5C). Several rounded vesicle-like structures were observed in the middle of the secretion in the large internal spaces of the acini and in the large lumen of the gland (Fig. 5A–C). The largest vesicular structures were mostly concentrated close to the epithelium of the acini (Fig. 5B) and reacted variably to PAS (Fig. 5C).

Muscles Associated with the Infralabial Glands

The muscle LAO was a thin, triangular sheet of muscle that originated behind the eyes, from the postorbital and the dorsolateral ridge of the parietal. The LAO was well developed and clearly distinct from the AES, from its origin to its insertion site. Fibers of this muscle were always directed dorsoventrally, running laterally to the harderian gland and converging ventrally to form a fusiform bundle at the level of the corner of the mouth that attached to the dorsal and posterior surfaces of the posterior third of the il1 (Figs. 1A–C, 2B). More anterolateral fibers of the LAO also attached to the rictal fold in the three species studied. The site of origin of the LAO varied in the three species studied, being more restricted (less developed dorsally) in G. nasalis. In G. brachycephalus, the posterior half of the LAO originated below the fibers of the AES while the anterior half was visible in lateral view. The LAO of G. brachycephalus appeared to form two slightly distinct bundles on its more ventrolateral region, with the posterior half attaching on the posterodorsal wall of the il1 while fibers of the anterior portion attached to the rictal fold. In G. nasalis, the majority of fibers of the LAO originated anteriorly to the AES, with the exception of the most posterior ones that were disposed laterally to the anteriormost part of the AES. All fibers converged ventrally and were attached to the rictal fold and posterodorsal surface of the il1. In G. semidoliatus, the LAO extended as a lateral bundle that overlapped the AES posteriorly, covering the latter almost completely. The fibers converged ventrally to form a narrow bundle at the level of the rictal fold, attaching to the latter and to the posterodoral surface of the il1.

The infralabial gland was also involved by the fibers of the AEM. The AEM originated along the anterolateral edge of the quadrate, from the anterodorsal corner of the bone to the lateral epicondyle, inserting on the whole lateral surface of the compound bone, from the level of the articular to the anterior surangular foramen. The more lateral fibers of the AEM that were just adjacent to the extended posterior part of il2 were tightly attached through their fascia to the wall of the gland, in a bipennate arrangement that suggested a function as compressors of the gland (Fig. 2B,C).

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

Although Taub (1967a, 1967b) pointed out that snake infralabial glands are generally mucous, he also acknowledged the serous nature of these glands in dipsadines, suggesting that they might be viewed as analogous to the Duvernoy's glands of other “Colubridae.” Several other authors also recorded seromucous cells in infralabial glands of a diverse number of colubroidean snakes (Gabe and Saint-Girons, 1969; Kochva, 1978; Baccari et al., 2002). In dipsadine snakes, they have been most evident, particularly in the infralabial glands of goo-eating snakes, as exemplified by Atractus reticulatus, Dipsas indica, and Sibynomorphus mikanii (Laporta-Ferreira and Salomão, 1991; Oliveira et al., 2008).

Our results indicate that the infralabial glands of at least three species of Geophis show a new and noteworthy specialization that was previously unreported among snakes. In addition to being predominantly composed of seromucous cells, these glands are divided in two independent, anterolateral (il1) and posteromedial (il2) portions that are both anatomically and histochemically distinct from each other (Fig. 6). While the il1 is constituted of both mucous and seromucous cells, the il2 is constituted exclusively of seromucous cells (Table 1). The il1 and il2 receives fibers of the LAO and AEM, respectively, acting independently as compressors of each portion of the gland. Each portion also shows a single large duct that runs longitudinally along its central region. Both ducts seem to open independently around the anterior region of the mouth, anterolaterally to the tip of the dentary (Fig. 3C). The large single duct of the il1 is not responsible for discharging all the secretion produced by the acini of that portion, since a fraction of that secretion is discharged through a series of smaller, typical infralabial ducts disposed along the lip of the mouth. On the other hand, secretion from the acini composing the il2 seems to be stored inside a large lumen formed along the posterior region of its single large duct, and is discharged only through that duct since no other openings were found in the gland. In that sense, the two portions of the infralabial gland seem to represent morpho-functionally independent units that are compressed by distinct muscular bundles (LAO and AEM, respectively) and discharge their secretions separately.

Oliveira et al. (2008) described the presence of several morphological and histological features in the infralabial glands and some adjacent muscles of D. indica, S. mikanii, and A. reticulatus that are strikingly similar to the condition described in Geophis (Table 3). Dipsas indica, S. mikanii, and A. reticulatus and the three species of Geophis share the presence of seromucous infralabial glands and a well developed and distinct muscle LAO, and lack Duvernoy's glands. Further, in Geophis and A. reticulatus, LAO attaches to the posteromedial region of the il1. Similarly to Geophis, we also observed in Dipsas and Sibynomorphus the presence of an infralabial gland divided in two distinct portions (il1 and il2), with an enlarged il2 showing a large central duct and a il1 corresponding to a thin stretch of glandular tissue (Zaher, 1996; Oliveira et al., 2010). Atractus reticulatus differs from the three other goo-eating genera by retaining a single infralabial gland, without any trace of an il2. However, the topographic position of the single infralabial gland of Atractus and the presence of a series of small ducts opening in along the lip and below the infralabial scales supports the hypothesis that it is homologous to the il1 of Geophis, Dipsas, and Sibynomorphus. Geophis, Dipsas, and Sibynomorphus also show a large median duct of the il2 that opens in the epithelium of the floor of the mouth, without any close functional connection with the series of dentary teeth.

Table 3. Comparison of morphological features among dipsadine species examined
 This paperOther sourcesa
Geophis brachycephalusGeophis nasalisGeophis semidoliatusAtractus reticulatusDipsas indicaSibynomorphus mikanii
  1. a

    Fernandes (1995), Zaher (1996, 1999), Oliveira et al. (2008, 2010).

Infralabial gland divided in a lateral (il1) and a medial (il2) portionPresentPresentPresentAbsentPresentPresent
Muscle levator anguli oris distinct and well developedPresentPresentPresentPresentPresentPresent
Muscle adductor mandibulae externus medialis associated with the infralabial glandPresentPresentPresentAbsentAbsentAbsent
Lumen in the medial portion (il2) of the infralabial glandPresentPresentPresentAbsentAbsentAbsent
Vesicle-like structures in the medial portion (il2) of the infralabial glandPresentPresentPresentAbsentAbsentAbsent
Attachment of the muscle levator anguli oris on the tip of dentaryAbsentAbsentAbsentAbsentPresentPresent
Attachment of the muscle levator anguli oris on the posterior portion of infralabial glandPresentPresentPresentPresentAbsentAbsent
Mandibular duct of the medial infralabial gland that opens in the epithelium of the mouthPresentPresentPresentAbsentPresentPresent
Duvernoy's glandAbsentAbsentAbsentAbsentAbsentAbsent

As in Dipsas, Sibynomorphus, and Atractus, the il1 in the three species of Geophis analyzed has a more conventional cellular condition, being of a mixed glandular type (Gabe and Saint Girons, 1969; Baccari et al., 2002; Oliveira et al., 2008). Indeed, mucous cells primarily constitute the il1 of G. nasalis, while seromucous cells are restricted to the peripheral areas. Conversely, the il1 of G. brachycephalus and G. semidoliatus are constituted predominantly of seromucous cells, while mucous cells are restricted to the central area of the gland, just around the main duct.

In addition to the differences in the distribution of cellular types between the two portions of the infralabial gland of Geophis, there are also differences in the composition of their glandular acini. In all three species of Geophis, the il2 shows larger acini than the il1 (Table 2). The secretion present in the acini of the il2 shows distinct vesicle-like structures of unknown function. These vesicles are absent from the secretion of the acini of the il1, suggesting that they are specific of the il2. The lack of vesicles and small difference in diameter between the acini of il1 and il2 in G. nasalis (Table 2) are probably due to the fact that, in this species, the acini of the il2 were practically empty at the moment of their fixation.

Similar microvesicles have already been reported inside the secretory cells of venom glands and freshly extracted venom from Crotalus durissus terrificus (Carneiro et al., 2007). We confirmed the presence of vesicles in Geophis only inside the acini and lumen of the gland, never inside the cells. Vesicles in Geophis show a wide range in size and are not surrounded by membranes, while microvesicles in C. d. durissus are uniform in size and surrounded by membranes (Carneiro et al., 2007). The presence of vesicles in the lumen of the gland indicates that they are carried along with the secretion from the gland. However, like microvesicles, their function is still unknown. The large acini and vesicles, typical of the il2 of Geophis, are lacking in D. indica, S. mikanii, and A. reticulatus, suggesting that these structures are uniquely derived in Geophis, among goo-eating snakes (Table 3).

Lumina are cavities used for the storage of large amounts of secretion produced by cells that can be released as one single, massive discharge. In snakes, lumina inside oral glands are known only in venom glands of viperids, elapids and in the genus Atractaspis, and in Duvernoy's glands of Dispholidus and Elapomorphus (Kochva, 1978; Salomão and Ferrarezzi, 1993). Venom glands of elapids show small lumina and the venom is stored mainly in the secretion granules inside the cells (Kochva, 1987). The large amount of secretion observed within the lumen of the infralabial glands, mainly in G. brachycephalus and G. nasalis, suggests that the lumen acts as a storage compartment for secretion in these snakes, a condition never reported before for any infralabial gland in snakes. Additionally, the secretory epithelium was relatively high and the presence of granules of secretion inside the cells indicate that part of the secretion may be stored inside the cells either, a condition that is analogous to the one present in venom glands of elapids. A lumen is also lacking in the il2 of Dipsas and Sibynomorphus (Table 3).

In snakes, oral glands are known to be associated with adjacent muscles that act as compressors of the gland. Among them are mainly the venom glands of vipers, elapids, and atractaspidids, and the Duvernoy's glands in a few colubroidean genera (e.g., Dispholidus, Mehelya, and Brachyophis; Kochva and Wollberg, 1970; Underwood and Kochva, 1993). On the other hand, labial glands in snakes are only rarely associated with adjacent muscles (Haas, 1973; Zaher, 1999). Snakes of the genus Geophis share with Enulius and Enuliophis a posterior expansion of the infralabial gland that tends to be embraced by the more lateral fibers of the AEM (Zaher, 1999: 34). We confirm Zaher's (1999) observation in the three species of Geophis examined, this condition being unique among snakes so far. The general morphology of the infralabial glands and their relationship with adjacent muscles are quite uniform among the three species of Geophis. However, the general pattern of these glands was distinct from the pattern found in the infralabial glands of Dipsas, Sibynomorphus, and, especially, Atractus, a genus closely related to Geophis and whose species also feed on earthworms (Table 3; Antoniazzi et al., 2005; Oliveira et al., 2008; Grazziotin et al., 2012).

Supralabial glands, on the other hand, are poorly developed in Geophis, which also seems to lack Duvernoy's or venom glands. Although considered homologous, the venom and Duvernoy's glands present a series of morphological and functional differences between them (Kardong, 2002). The synonymization of the terms Duvernoy's glands with venom glands was proposed by Fry et al. (2003), but the use of the term remains in dispute among authors (Weinstein et al., 2010, 2012; Fry et al., 2012). Taub (1967b) pointed out that Geophis multitorques has Duvernoy's glands with some mucous cells intermingled with the serous cells. Although G. brachycephalus and G. nasalis retain a posterior enlargement of the supralabial gland, our observations indicate that it cannot be considered a Duvernoy's gland because it is constituted by the same cell types present in the anterior part of the gland, being impossible to distinguish both parts from each other. Moreover, we could not verify the existence of a single duct in the posterior part nor an association between any duct with posterior maxillary teeth, which could indicate the presence of Duvernoy's gland. Our results agree with those previously described by others authors, which indicate that goo-eating dipsadine snakes lack Duvernoy's glands (Fernandes, 1995; Harvey et al., 2008; Oliveira et al., 2008).

This work emphasizes the wide morphological variation existing in labial glands of snakes. The presence of an expanded lumen within infralabial glands associated with the adjacent muscles, features never related before in an infralabial gland, suggest additional roles for the secretion of these glands, in addition to the already well-known lubrication role. The absence of Duvernoy's glands associated with extensive morphological variation in the infralabial glands may indicate that the soft and viscous invertebrates preyed on by these snakes impose functional demands on their feeding apparatus different from those typical of other nonviscous types of prey.

Although the presence of a lumen and an associated compressor muscle in the il2 may suggest a venomous condition of the infralabial gland in Geophis, this hypothesis is unlikely since the main glandular duct of the il2 is not functionally related to a specialized tooth but rather opens loosely on the epithelium of the floor of the mouth. Such unusual glandular complex is more likely to function as a highly specialized, protein-secreting system directed to the control of mucous secretion and assistance in the ingestion of their elongate, flexible and highly viscous preys. Similarly, the distinct il2 of Dipsas and Sibynomorphus also discharges its proteic secretion through a duct that opens on the epithelium of the mouth and may share the same adaptive purpose as the one hypothesized for Geophis.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

The authors would like to thank Elazar Kochva for his comments and help in interpretating the histological sections. Authors are deeply indebted to W. Ronald Heyer (USNM), Linda Trueb, Bill Duellman (KU), Jim McGuire and Carol L. Spencer (MVZ) for allowing the dissection and loan of specimens under their care, to Marta M. Antoniazzi and Carlos Jared for providing support and allowing the use of their histological facilities at the Laboratório de Biologia Celular of the Instituto Butantan, and Juan Camilo Arredondo for his help with the schematic drawing. The authors declare that no competing interests exist.

LITERATURE CITED

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED
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