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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.
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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.
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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 paper||Other sourcesa|
|Geophis brachycephalus||Geophis nasalis||Geophis semidoliatus||Atractus reticulatus||Dipsas indica||Sibynomorphus mikanii|
|Infralabial gland divided in a lateral (il1) and a medial (il2) portion||Present||Present||Present||Absent||Present||Present|
|Muscle levator anguli oris distinct and well developed||Present||Present||Present||Present||Present||Present|
|Muscle adductor mandibulae externus medialis associated with the infralabial gland||Present||Present||Present||Absent||Absent||Absent|
|Lumen in the medial portion (il2) of the infralabial gland||Present||Present||Present||Absent||Absent||Absent|
|Vesicle-like structures in the medial portion (il2) of the infralabial gland||Present||Present||Present||Absent||Absent||Absent|
|Attachment of the muscle levator anguli oris on the tip of dentary||Absent||Absent||Absent||Absent||Present||Present|
|Attachment of the muscle levator anguli oris on the posterior portion of infralabial gland||Present||Present||Present||Present||Absent||Absent|
|Mandibular duct of the medial infralabial gland that opens in the epithelium of the mouth||Present||Present||Present||Absent||Present||Present|
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.