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. 2014 Aug 11;225(4):377–389. doi: 10.1111/joa.12224

Morphology and fibre-type distribution in the tongue of the Pogona vitticeps lizard (Iguania, Agamidae)

Leïla-Nastasia Zghikh 1, Emilie Vangysel 2, Denis Nonclercq 1, Alexandre Legrand 3, Bernard Blairon 3, Cécile Berri 4, Thierry Bordeau 4, Christophe Rémy 5, Carmen Burtéa 2, Stéphane J Montuelle 6,7, Vincent Bels 8
PMCID: PMC4174021  PMID: 25109482

Abstract

Agamid lizards use tongue prehension for capturing all types of prey. The purpose of this study was to investigate the functional relationship between tongue structure, both surface and musculature, and function during prey capture in Pogona vitticeps. The lack of a detailed description of the distribution of fibre-types in the tongue muscles in some iguanian lizards has hindered the understanding of the functional morphology of the lizard tongue. Three methodological approaches were used to fill this gap. First, morphological analyses were performed (i) on the tongue surface through scanning electron microscopy, and (ii) on the lingual muscle by histological coloration and histochemistry to identify fibre-typing. Secondly, kinematics of prey capture was quantified by using high-speed video recordings to determine the movement capabilities of the tongue. Finally, electromyography (EMG) was used to identify the motor pattern tongue muscles during prey capture. Morphological and functional data were combined to discuss the functional morphology of the tongue in agamid lizards, in relation to their diet. During tongue protraction, M. genioglossus contracts 420 ± 96 ms before tongue–prey contact. Subsequently, Mm. verticalis and hyoglossus contract throughout tongue protraction and retraction. Significant differences are found between the timing of activity of the protractor muscles between omnivorous agamids (Pogona sp., this study) and insectivorous species (Agama sp.), despite similar tongue and jaw kinematics. The data confirm that specialisation toward a diet which includes more vegetal materials is associated with significant changes in tongue morphology and function. Histoenzymology demonstrates that protractor and retractor muscles differ in fibre composition. The proportion of fast glycolytic fibres is significantly higher in the M. hyoglossus (retractor muscle) than in the M. genioglossus (protractor muscle), and this difference is proposed to be associated with differences in the velocity of tongue protrusion and retraction (5 ± 5 and 40 ± 13 cm s−1, respectively), similar to Chamaeleonidae. This study provides a way to compare fibre-types and composition in all iguanian and scleroglossan lizards that use tongue prehension to catch prey.

Keywords: Agamidae, capture, electromyography, fibre typing, muscle, tongue

Introduction

In Squamates, it is acknowledged that the tongue is a highly versatile system performing a wide diversity of functions (Bels et al. 1994; Cooper, 1994; Herrel et al. 1998; Schwenk, 2000; Bels, 2003). While the phylogeny of Squamates is still subject to discussion (Estes et al. 1988; Vitt et al. 2003; Vidal & Hedges, 2005, 2009; Reilly & McBrayer, 2007; Lee, 2009; Pyron et al. 2013), the monophyly of the three main iguanian families (i.e. Pleurodonta, Agamidae and Chamaeleonidae) is accepted, based on molecular and/or morpho-functional data (Pyron et al. 2013). In Iguania, the tongue plays a key role, not only during prey capture (Schwenk & Throckmorton, 1989; Wainwright et al. 1991; Delheusy & Bels, 1992; Herrel et al. 1995; Meyers & Herrel, 2005; Schaerlaeken et al. 2007) but also in food transport and swallowing (Bels et al. 1994; Schwenk, 2000) and drinking (Schwenk & Greene, 1987; Sherbrooke, 1993; Wagemans et al. 1999), as well as for vomerolfaction (Herrel et al. 1998; Cooper, 2000) and social interaction (Jenssen, 1977; Carpenter, 1978). In other lizards (Scleroglossa sensu Estes et al. 1988), the tongue may also be used for capturing particular food items (Urbani & Bels, 1995; Smith et al. 1999; Reilly & McBrayer, 2007; Montuelle et al. 2009; Broeckhoven & Mouton, 2013).

In arguably all Squamates, the tongue is a highly mobile structure that is described as a muscular- hydrostat (Kier & Smith, 1985; Smith & Kier, 1989; Chiel et al. 1992). In addition to producing tongue movements, muscles act as a mechanical support during movement. This feature results from the fact that muscle cells contain an incompressible fluid which keeps them in constant volume deformation. Thus, tongue movements and deformations depend primarily on its muscular organisation. Tongue musculature comprises extrinsic (i.e. origin outside of the tongue) and intrinsic (i.e. origin and attachment both confined in the volume of the tongue) muscle fibres, which are detailed in Schwenk (2001).

Morphological (e.g. surface and musculature) and functional characteristics of tongue protrusion during food capture have been investigated from an evolutionary point of view (Schwenk, 2000; Iwasaki, 2002). For example, Schwenk & Bell (1988) suggest that tongue protrusion in some species of Agamidae (e.g. Phrynocephalus helioscopus) may represent a functional intermediate between lingual protrusion in Sphenodon and the rest of the Iguanian clade on one hand, and the highly derived lingual projection of Chamaeleonidae on the other hand. In Iguanids, Agamids (see tongue contact in Moloch horridus; Meyers & Herrel, 2005) and Chamaeleonids, as well as in Scleroglossan lizards that use tongue prehension, the foretongue makes contact with, and adheres to, the food (Bels et al. 1994; Schwenk, 2000; Reilly & McBrayer, 2007). As iguanian lizards, the integration of tongue morphology and function in agamid lizards is particularly interesting for several reasons. The main points of interest are the diversity in tongue morphology (Gnanamuthu, 1937; Smith, 1988; Herrel et al. 1998), as it is used for prey capture, chemoreception and drinking (Herrel et al. 1995, 1998; Schwenk, 2000; Schaerlaeken et al. 2007), the variability in diet (i.e. herbivorous, scavenger, insectivorous, omnivorous) and the mechanism of tongue function, as it is either considered to be similar to Sphenodon and other Iguanids (representing an ancestral state) or a cryptic intermediate between the ancestral state and the highly derived mode characteristic of Chamaeleonids (Schwenk & Bell, 1988). Tongue muscles and dorsal surface have been studied in some agamid species (Gnanamuthu, 1937; Smith, 1988; Herrel et al. 1995, 1998) but not in the omnivorous P. vitticeps.

Although the histology of tongue musculature has already been described in some agamid species, the fibre-type composition of each tongue muscle has not been studied for this family. To date, there has only been one study of lingual fibre-type composition focusing on Chameleonids (Herrel et al. 2001). The lack of detailed knowledge of the distribution of fibre-types in the tongue muscles limits the understanding of the functional morphology of the iguanian tongue. Indeed, the comparison of homologous protractor muscles in agamids and chameleons (Herrel et al. 2001) could open discussions about the evolution of the form and function of the iguanian tongue. Furthermore, Throckmorton & Saubert (1982) described the presence of three groups, in terms of distribution of fibre-types in the jaw muscles of Tupinambis nigropunctatus: homogeneous (one type of fibre), mixed (a mixture of three fibre-types: slow oxidative, fast oxidative-glycolytic and fast glycolytic) and compartmentalised (a mixture of three fibre-types with clear regional territories). These authors suggested that the presence of anatomically different regions within a muscle reveals functional differentiations. It is thus necessary to establish a relationship between the typological properties of the protractor and retractor muscles, and their effect on the tongue function.

Given that the tongue of P. vitticeps is used to catch all types of the food, the aim of this study is to investigate two morphological properties related to tongue function during prey capture. To achieve this, the papillary surface was analysed through scanning electron microscopy and the musculature was studied by histology and magnetic resonance imaging. Additionally, the fibre-type composition and distribution of the three tongue muscles was examined using histochemical procedures to identify the oxidative and glycolytic capacities of tongue fibres. Moreover, the function of these muscles in tongue movements (i.e. protractor vs. retractor) was assessed with electromyography. This data was compared with anatomical and functional data available for other lizards with different diets within the agamid phylogeny (Honda et al. 2000; Hugall et al. 2008).

Materials and methods

Animals

Eight male adult individuals (mean snout–vent length, SVL: 198 ± 26 mm) of the species P. vitticeps were used in the experiments. All the specimens were obtained from the Muséum d'Histoire Naturelle de Tournai, Belgium, and were maintained in the animal housing facility of the University of Mons (UMONS, Belgium). The animals were treated according to the guidelines specified by the Belgian Ministry of Trade and Agriculture and under the control of the UMONS ethical commission (agreement LA1500021). The lizards were placed individually in terraria (120 × 60 × 60 cm) and maintained in a controlled environment with 12 h of daylight and a temperature of 34 °C during the day and 22 °C at night. The animals were fed with live adult crickets (Acheta domesticus), mealworms (Tenebrio molitor), new-born mice and vegetables every 2 days and received water ad libitum. All the animals were euthanised by decapitation under the control of the UMONS Committee for Survey of Experimental Studies and Animal Welfare.

Scanning electron microscopy

For the scanning electron microscopy (SEM), the tongue of two P. vitticeps (SVL: 188 ± 18 mm) was removed and fixed overnight in a modified Karnovsky solution, rinsed in buffered, and then post-fixed in unbuffered, 1% osmium tetroxide at 36 °C for 120 min. To remove the mucus, the tongue was treated with 8 N HCl at 60 °C for 30 min (Beisser et al. 2004). The samples were dehydrated in graded ethanol and dried using the critical-point method, before being mounted on aluminium stubs, coated with gold in a sputter-coater and observed with a JEOL JSM-6100 scanning electron microscope.

Magnetic resonance imaging

For the magnetic resonance imaging (MRI), two individual P. vitticeps (SVL: 188.5 ± 16.2 mm) were used. To immobilise the subject inside the magnetic bore, each lizard was anaesthetised using an intramuscular injection of ketamine (200 mg kg−1 body mass, Ketalar®). The subject was considered anaesthetised when it lost the ability to push itself back on its feet after being turned on its back and responded weakly to tail pinching. MRI experiments were performed on a 300 MHz (7 T) Bruker Biospec imaging system (Bruker, Ettlingen, Germany) equipped with a Pharmascan horizontal magnet. Head images were acquired with a rapid-acquisition relaxation-enhanced (RARE) imaging protocol using the following parameters: TR/TE = 3000/48.7 ms, RARE factor = 4, NEX = 4, matrix = 256 × 256, FOV = 2.3 cm, slice thickness = 1 mm, 20 coronal slices, spatial resolution = 90 μm, TA = 12 min 48 s.

Histology

After euthanasia, the lower jaws of two P. vitticeps (SVL: 225.5 ± 36.1 mm) were fixed by immersion in Duboscq–Brazil fluid for 48 h and decalcified in 5% trichloroacetic acid for 24 h. The samples, after dehydration and paraffin-embedding, were cut in serial sections of 5-μm thicknesses on a Reichert Autocut 2040 microtome and placed on silane-coated glass slides. After rehydration, the sections were stained with PAS (periodic acid-Schiff reaction), haematoxylin, Orange G and Fast green to allow histologic examination.

Fibre-typing

Four tongues of P. vitticeps (SVL: 202 ± 34 mm) were frozen in isopentane that had been precooled with liquid nitrogen and stored at –80 °C. The samples were cut at 8 μm in a cryostat microtome (LEICA CM1950) at –21 °C, and placed on Superfrost® Plus slides (Thermo Scientific). After sectioning, the frozen sections were air-dried for 45–60 min and used for the histochemical identification of muscle fibre types.

In lizards, different protocols have been used previously to identify muscle fibres as slow oxidative (SO), fast oxidative-glycolytic (FOG) or fast glycolytic (FG) These protocols are based on histochemical activities of myosin adenosine triphosphatase (mATPase) with nicotinamide adenine dinucleotide dehydrogenase (NADH-dehydrogenase) or succinate dehydrogenase activity (SDH) (Gleeson et al. 1980; Putnam et al. 1980; Gleeson, 1983; Gleeson & Harrison, 1988; Mutungi, 1992; Bonine et al. 2005). The drawback of these techniques is that the detection of the various enzymes is carried out in separate sections. Recently, Moritz & Schilling (2013) developed an original protocol based on the combination of reactions to observe the three fibre types in the same histological preparation: FG fibres stained dark for alkali-stable mATPase, FOG fibres stained dark for NADH-dehydrogenase and alkali-stable mATPase, and SO fibres stained dark for NADH-dehydrogenase. In the present study, the detection of SDH and mATPase after acid pre-incubation activity was combined to identify SO (stained dark for SDH and mATPase after acid pre-incubation), FOG (stained dark for SDH) and FG fibres (Table 1).

Table 1.

Histochemical characteristics of muscle fibres identified in the lingual muscles of Pogona vitticeps

Type SDH mATPase (pH 4.1) Acidic combination
SO/tonic +++ +++ +++
FOG ++ − ++
FG + − +
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+++, Strong activity; ++, moderate activity; +, weak activity; −, no activity; SDH, succinate dehydrogenase; mATPase, myosin adenosine triphosphatase; SO, slow oxidative fibres; FOG, fast oxidative-glycolytic fibres; FG, fast glycolytic fibres.

Sections were treated as follows:

  1. Incubation at 37 °C for 40 min in a solution of succinate 50 mm, Nitroblue-tetrazolium 0.625 mm and Meldolablau 41.5 μm (pH 7.6).

  2. Rinsed in 0.9% NaCl.

  3. Fixation for 10 min in 4% formaldehyde in 9% NaCl.

  4. Rinsed in distilled water adjusted with NaHCO3 to pH 7.

  5. Acid pre-incubation for 10 min at pH 4.1.

  6. Rinsed in Tris buffer (pH 7.8).

  7. mATPase incubation at 37 °C for 60 min.

  8. Rinsed three times in 1% CaCl2 2H2O, once in 2% CoCl2 for 3 min, and then in distilled water.

  9. Visualisation in 1% (NH4)2S for 3 min.

  10. Rinsed in water for 20 min.

  11. Dehydrated and coverslipped with synthetic mount.

Morphometric analysis

The percentage of the reactive area occupied by each fibre type in the protractor (M. hyoglossus) and retractor (Mm. genioglossus and verticalis) muscles was analysed by morphometric analysis at low magnification (2.5×) after the interactive selection of the muscular surface. The procedure was based on a hardware system consisting of a Zeiss Axioplan microscope equipped with a ProgRes C10 plus colour camera (Jenoptik, Germany) connected to an IBM-compatible PC, and a software designed for morphometry and colour analysis (KS 400 IMAGING system, Carl Zeiss Vision GmbH, Munich, Germany). This image-analysing system distinguished the reactive areas based on differences in colour and contrast. For each muscle, the area occupied by each fibre-type was calculated as a percentage of the total area sampled. For each specimen, individual values were pooled for each muscle (Mm. hyoglossus, genioglossus and verticalis).

Electromyographic and video recordings

Seven male adult individuals (SVL: 237 ± 34 mm) were used to determine the pattern of tongue muscular activity and tongue kinematics. The animals were filmed in a 80 × 25 × 20 cm vivarium at 19 ± 0.9 °C, and live crickets (length: = 24.07 ± 1.36 mm) were offered 10 cm away from the tip of the snout of the lizards. Prior to the electromyography (EMG) recording, the animals were anaesthetised by an intravenous injection of propofol (10 mg kg−1, Diprivan® 1% AstraZeneca™) in the ventral caudal vein. Bipolar electrodes were prepared from 30-cm-long silver twisted (140 μm) and folded wire (Phymep, s.a.r.l, France) following published methods (Loeb & Gans, 1986). At the tip of the electrodes, 1 mm of the Teflon insulation was stripped to expose the wire. A pair of electrodes was inserted into the protractor muscles (Mm. genioglossus and verticalis) using hypodermic needles (0.6 mm), the retractor (M. hyoglossus) muscle and in the M. depressor mandibulae responsible for opening the jaw (Bels et al. 1994; Herrel & De Vree, 1999; Schwenk, 2000); a ground electrode was inserted into the vertically caudal muscle. The animals were allowed to recover for 24 h after the implantation of the electrodes. When fully recovered, the lizards were placed in the experimental vivarium and the electrodes were welded to a connector that was set on the animal's back.

Electromyography signals were processed using amplifiers (BMA-400, CWE, Ardmore, PA, USA). The raw signals were digitalised (NI-6341 E®, National Instrument Co, Austin, TX, USA) with a sampling rate of 15 kHz using Labview® software (National Instruments). The raw signal was subjected to a band pass-filter between 100 and 1000 Hertz, rectification and a moving average (time constant 100 ms; MA821®, CWE). The onset of activity was determined by the visual inspection of the earliest rise in EMG activity above baseline. A 200-ms-long baseline window was selected prior to any activity and the baseline amplitude during this baseline reference window was removed from the signal. For each muscle separately, the onset of EMG activity was identified on the integrated EMG signal and repetitive bursts were identified on the corresponding raw EMG signal. The onset of EMG activity was defined as the first motor unit potential having amplitude over 125% of the baseline reference amplitude.

The subject was filmed at 250 Hz with a high-speed video camera (SANYO) in the lateral view. Only unmistakably lateral sequences were analysed. At the end of the recording session, a still frame of a 5 × 5 cm grid was recorded for scaling. To synchronise the electromyographic and kinematic data, a signal generator triggered a light-emitting diode visible on the video camera, and the signal output was recorded simultaneously on the EMG signals. Mean (± SD) data were used to summarise the pattern of onset and offset of the protractor and retractor muscles relative to the tongue contact on the prey (time 0) during the capture cycle.

Kinematic analysis

From the recorded video sequences, the tongue protraction and retraction velocities were calculated. The frame during which the tongue began to move between the jaws was considered the start time of tongue protraction (it was not possible to determine tongue movement within the buccal cavity). The frame during which the contact between the tongue and the prey occurred was identified. The time between the start of tongue protraction and the maximum protraction was considered to represent the tongue protraction duration. The difference between the tongue position at the start of protraction and the tongue position at tongue–prey contact was considered to be the tongue protraction distance. This data was used to approximate the average velocity of the tongue during protraction as the ratio of the protraction distance over the tongue protraction duration. Similarly, for tongue retraction, the frame during which the jaw closed on the prey was identified as the end of tongue retraction. The tongue retraction duration was calculated between the tongue–prey contact and the end of tongue retraction. The tongue retraction distance was calculated as the difference between tongue position at tongue–prey contact and the tongue position when the tongue stopped moving within the oral cavity. The ratio between the retraction distance and time was used to approximate the average velocity of tongue retraction. Note that for distance measurements, the point of the tongue that made contact with the prey was tracked.

Statistical analysis

The statistical analysis was performed using minitab® Release 14.20 for Windows (Minitab, State College, PA, USA). First, Ryan-joiner tests (similar to Shapiro–Wilk tests) were used to check the normality of the data. For comparisons of the fibre-type percentages (SO, FOG and FG) in each of the three muscles (Mm. hyoglossus, genioglossus or verticalis) a Kruskal–Wallis test was used. A post-hoc Mann–Whitney U-test was performed for pair-wise comparisons. Comparisons of one fibre-type (SO, FOG or FG) between the three muscles (Mm. hyoglossus, genioglossus and verticalis) were made with nonparametric analyses of variance. The velocities of the tongue protrusion and retraction were compared with the Mann–Whitney U-test. The significance level for all of the tests was set at P < 0.05.

Results

Tongue surface

Based on the terminology of Rabinowitz & Tandler (1986) and Herrel et al. (1998), the tongue of P. vitticeps is divided into: the tip, foretongue and hindtongue (Fig. 1A). The tip of the tongue is slightly bifurcated and devoid of papilla on the front area, whereas its posterior region is characterised by cylindriform papillae (Fig. 1B). The foretongue is characterised by plumose papillae (Fig. 1C) that merge posteriorly to form densely packed plumose papillae on the hindtongue (Fig. 1D). The tongue of P. vitticeps is thick and fleshy (Figs 2 and 3). Figure 2 presents an MRI image of the head of a P. vitticeps in dorsal view showing that the tongue occupies almost the whole of the oral cavity. Long reticular papillae of columnar serous cells are present on the surface of the foretongue (Fig. 3A) and are covered by a simple cylindrical epithelium. These reticular papillae are elongated across the foretongue but shorten and thicken posteriorly (i.e. toward the hindtongue). The lingual epithelium is not keratinised except very lightly on the tip of the tongue, forming the ventral pallet. The tip of the tongue presents a pavimentous stratified epithelium. The foretongue is also characterised by tubular serous-secreting glands (S) characterised by PAS-negative secretory cells, with a spherical nucleus (Fig. 3C). Tubuloalveolar mucous-secreting glands (M), characterised by PAS-positive cells revealing the presence of mucopolysaccharide (Gabe & Saint Girons, 1969), are concentrated at the hindtongue (Fig. 3B). These tubuloalveolar mucous-secreting glands are localised on the lamina propria and are capped by a non-keratinised stratified squamous pavimentous epithelium. The ‘frenulum’ (F; Fig. 3A), which attaches the tongue to the floor of the mouth, is covered by a stratified pavimentous festooned epithelium. Small mucous-secreting glands are confined to this epithelium.

Fig. 1.

Fig. 1

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Scanning electron micrographs of the tongue tip, foretongue and hindtongue in Pogona vitticeps. (A) Schematic illustration of the tongue showing the subdivision into three regions. 1: tongue tip, 2: foretongue and 3: hindtongue. The tongue tip (B) presents cylindriform papillae in this posterior area. Note the minor bifurcation (arrow) of the tongue (B) and the presence of numerous elevated plumose papillae (pl) in the foretongue (C) and less developed papillae in the hindtongue (D).

Fig. 2.

Fig. 2

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A MRI transverse plane view of Pogona vitticeps head showing the large occupation of the tongue in the oral cavity. E, eye; Ep, entoglossal process; Gl, M. genioglossus lateralis; Gm, M. genioglossus medialis; H, M. hyoglossus; V, M. verticalis.

Fig. 3.

Fig. 3

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(A) Parasagittal section through the lower jaw and the tongue of Pogona vitticeps showing the intrinsic and extrinsic muscles and elements of hyoid. Ep: entoglossal process; F, frenulum; Gl, M. genioglossus lateralis; Gm, M. genioglossus medialis; H, M. hyoglossus; L, M. lateralis; Md, mandible; T, M. transversalis; V, M. verticalis. (B) Tubular mucous-secreting glands (M) are concentrated at the hindtongue. (C) Serous-secreting glands (S) are located exclusively in the foretongue.

The tongue contains skeletal striated muscles arranged in well-defined fascicles (Fig. 3A). The tongue musculature can be divided into extrinsic and intrinsic muscles, as described in detail by Gnanamuthu (1937) and Smith (1988). The extrinsic musculature (Fig. 3A) includes Mm. genioglossus and hyoglossus. The M. hyoglossus constitutes the main body of the tongue and is composed of longitudinal fibres originating from the first ceratobranchial and extending anteriorly. The M. genioglossus has three parts: the M. genioglossus medialis, which forms the median ventral surface of the tongue; the M. genioglossus lateralis, which forms the lateral edge of the tongue; and the M. genioglossus internus, which separates the two portions of the M. hyoglossus in the anterior region of the tongue.

The intrinsic musculature includes three muscles: Mm. verticalis, longitudinalis and transversalis. In P. vitticeps, M. verticalis is composed of a ring of fibres that surround the entoglossal process of the hyobranchial apparatus (ring muscle sensu Smith 1988; Fig. 3A). The M. longitudinalis is composed of longitudinal fibres that run beneath the tongue surface for most of the length of the tongue. Some longitudinalis fibres turn dorsally in the connective axis of papillae. M. transversalis is composed of transversal fibres located between the M. longitudinalis fibres.

Histochemistry

In the tongue of P. vitticeps three different skeletal muscle fibre-types are found: slow oxidative (SO), fast oxidative-glycolytic (FOG) and fast glycolytic (FG) fibres (Fig. 4). The retractor (hyoglossus) muscle is characterised by a greater percentage of FG than FOG and SO fibres (Fig. 5A). Indeed, more than 50% of these muscle fibres are FG fibres (Table 2). However, this muscle is distinctly compartmentalised. Figure 6 shows an area on the dorsal side of the tongue under the papillae. This area is composed exclusively of FG fibres, whereas oxidative fibres (FOG and SO) are mixed with glycolytic fibres in the basal region and the edge of M. hyoglossus that is in contact with the M. verticalis. The protractor muscle (M. genioglossus) is composed of oxidative fibres (SO and FOG), which are distributed randomly. This muscle shows a greater percentage of fibres characterised by a histochemical profile corresponding to SO fibres than FOG fibres. In contrast, FG fibres are absent (Fig. 5B). Similar to M. genioglossus, M. verticalis is mainly composed of SO fibres, but it shows a low proportion of FG fibres (Table 2). In both muscles, significant pair-wise comparisons (Mann–Whitney U-tests) show that the proportion of SO fibres is significantly greater than of FOG fibres and FG fibres (Fig. 5C). Moreover, in M. verticalis, the proportion of FOG fibres is significantly higher than that of FG fibres. The muscle fibres in the Mm. genioglossus and verticalis are mixed, in contrast to the highly compartmentalised M. hyoglossus.

Fig. 4.

Fig. 4

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Histochemical labelling on serial sections of the M. hyoglossus in Pogona vitticeps stained for the activities of (A) succinate dehydrogenase activity (SDH) and (B) a combination of histochemical reactions (see Material and methods). SO, slow oxidative fibres; FOG, fast oxidative-glycolytic fibres and FG, fast glycolytic fibres.

Fig. 5.

Fig. 5

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Boxplots showing the percentage of different fibre types in the Mm. hyoglossus (A), genioglossus (B) and verticalis (C). Box plot values consist of the median (line), first and third quartiles (upper and lower edges of box). *Significant difference (P < 0.05). SO, slow oxidative fibres; FOG, fast oxidative-glycolytic fibres and FG, fast glycolytic fibres. n = 4.

Table 2.

Percentage of SO, FOG and FG fibres in the protractor and retractor muscles of Pogona vitticeps

% SO fibres % FOG fibres % FG fibres
Muscle Mean SD Mean SD Mean SD
M. hyoglossus 22 6 21 3 57 4
M. genioglossus 83 9 17 9 0 0
M. verticalis 60 8 31 12 9 7
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SD, Standard deviation.

Fig. 6.

Fig. 6

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Right hyoglossus muscle of Pogona vitticeps stained in order to detect succinate dehydrogenase activity. The start is located in the dorsal region rich in FG fibres. By contrast, FOG and SO fibres dominate in the lower and lateral parts of the ring muscle.

The proportion of SO fibres is significantly higher for the M. genioglossus than for the Mm. hyoglossus and verticalis (Fig. 7A). No significant difference is observed in the proportion of FOG fibres between the three muscles (Fig. 7B). Finally, the proportion of FG fibres is significantly greater in M. hyoglossus than in both Mm. genioglossus and verticalis (Fig. 7C).

Fig. 7.

Fig. 7

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Boxplots showing the percentage of slow oxidative fibres (A), fast oxidative-glycolytic fibres (B) and fast glycolytic fibres (C) in the Mm. hyoglossus, genioglossus and verticalis. Box plot values consist of the median (line), first and third quartiles (upper and lower edges of box). *Significant difference (P < 0.05). n = 4.

Electromyography

After detecting the prey, P. vitticeps positions its head and lunges towards the prey as previously observed in other agamids. Overall, the head, jaw and tongue movements are similar to previous descriptions (Schwenk & Throckmorton, 1989; Kraklau, 1991; Herrel et al. 1995; Meyers & Herrel, 2005; Schaerlaeken et al. 2007). After slowly approaching the prey, the subject stops and the jaw starts to open. The lizard is always in the typical ‘head-up’ posture (Montuelle et al. 2008) before moving the trophic system toward the prey. As soon as the lizard lunges at the prey, the tongue is protracted between the tips of the jaws. The dorsal surface of the tongue is presented in front of the prey by an anterior curling movement of the foretongue under the tapered tip of the entoglossal process (Smith, 1988; Schwenk, 2000). The tongue protracts continuously and touches the prey. The mean duration of tongue protrusion is 327 ± 155 ms. The mean duration of tongue–prey contact before the beginning of the retraction is 6 ± 3 ms. Then, the tongue retracts with the prey and recovers its initial position while the jaw is closing, as previously described for other Agamid species (Schwenk & Throckmorton, 1989; Kraklau, 1991; Herrel et al. 1995; Meyers & Herrel, 2005; Schaerlaeken et al. 2007). The mean duration of tongue retraction is 109 ± 25 ms.

Figure 8 summarises the pattern of muscle activities during tongue movements, in relation to the jaw opening muscle (M. depressor mandibulae). The M. genioglossus is the first muscle to be active in producing tongue protrusion/protraction and this activity ends shortly after tongue–prey contact. On average, M. genioglossus contraction starts 420 ± 96 ms before the time of tongue–prey contact. M. verticalis is active during tongue protrusion and stops 2 ± 9 ms after tongue–prey contact. M. hyoglossus is active during tongue protrusion/protraction from 133 ± 55 ms before tongue–prey contact and remains active throughout tongue retraction. The activity of these muscles produces an anterior displacement of the tongue toward the prey. The velocity of tongue protrusion/protraction is 5 ± 5 cm s−1. The mean velocity of tongue retraction was calculated as 40 ± 13 cm s−1 and is significantly higher than the protrusion velocity (W = 28; P = 0.0022). Tongue movements occur synchronously with jaw cycles and the M. depressor mandibulae is active during jaw opening, as demonstrated for other iguanian lizards (Herrel et al. 1995; Schwenk, 2000).

Fig. 8.

Fig. 8

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Time course of EMG activity in the Mm. depressor mandibulae, hyoglossus, genioglossus and verticalis during prey capture. Onset and offset of EMG activity are calculated with respect to the tongue–prey contact (time = 0 ms) and expressed in mean ± SE.

Discussion

Morphology of the tongue

It was observed that the tongue of P. vitticeps occupies a large portion of the oral cavity (Fig. 2). The tongue surface presents papillae, except for the anterior part of the tip of the tongue (Fig. 1). Two types of glandular cells were observed in the surface papillae: PAS-positive and PAS-negative cells. Serous glands (PAS-negative) were located on the tip of the tongue and the foretongue, whereas mucous glands (PAS-positive) were located in the hindtongue, as previously described for other agamid lizards by Smith (1988). The collected data also show that the tongue of P. vitticeps is covered by reticular papillae, in accordance with previous reports (Schwenk, 1988; Smith, 1988).

The scanning electron microscopy of the tongue surface of P. vitticeps (Fig. 1) showed that the tongue tip is slightly bifurcated, similarly to other agamids, Stellagama (Plocederma) stellio and Uromastyx acanthinurus (Herrel et al. 1998). These authors also studied the surface of the tongue of these two species and as with these lizards, the surface of the anterior part of the tongue tip of P. vitticeps is smooth, whereas the posterior part of the tongue tip presents cylindriform papillae. The foretongue of both species studied by Herrel et al. (1998) differs markedly. In U. acanthinurus, the foretongue is divided into a medial part composed of densely packed triangular papillae with extensive microstructure, and a lateral part with rectangular papillae on its lateral part. The foretongue of S. stellio is characterised by plumose papillae, as observed for P. vitticeps (Fig. 1). The hindtongues of S. stellio and P. vitticeps are covered by densely packed plumose papillae; however, that of U. acanthinurus is covered by densely packed cylindriform papillae. Consequently, U. acanthinurus is herbivorous (Smith, 1988; Herrel et al. 1999), whereas S. stellio is insectivorous (Herrel et al. 1999) and P. vitticeps is omnivorous (Cooper, 2000; Schaerlaeken et al. 2007). Dietary specialisation is proposed to have effect on tongue morphology at two levels. First, on the surface of the foretongue, which is in contact with food during feeding (Schwenk, 2000), and secondly, on the papillae of the hindtongue. Furthermore, tongue bifurcation is more prominent in U. acanthinurus than in S. stellio and P. vitticeps.

Moreover, as described by Smith (1988) and reviewed by Herrel et al. (1998), the M. verticalis does not surround the entoglossal process in U. acanthinurus. In P. vitticeps, the M. verticalis completely surrounds the lingual process, which is the main morphological feature of the intrinsic musculature of agamid lizards (Smith, 1988). Therefore, positional and histological features of this muscle are similar in species exploiting arthropods as food resources (e.g. Agama agama, Trapelus mutabilis, Paralaudakia caucasia; Smith 1988). In contrast, the extrinsic musculature is similar across all the other agamid lizards that have been studied to date, except Leiolepis sp. (Smith, 1988).

The phylogeny of Agamidae has been investigated on morphological and molecular clues and remains rather controversial (Moody, 1980; Witten, 1982; Joger, 1991; Honda et al. 2000; Hugall et al. 2008). In previous comparative analysis, muscular clues were discussed by Smith (1988) in relation to the morphological evolution of agamids (Moody, 1980). Present morphological and functional data can be discussed in light of the phylogeny of Agamidae inferred from mitochondrial DNA sequences (Honda et al. 2000; Hugall et al. 2008). Using three analyses (neighbour-joining, maximum-likelihood and maximum parsimony) based on molecular data and a most-parsimonious tree generated from morphological data provided by Moody (1980), Honda et al. (2000) propose that P. vitticeps and S. stellio belong to different groups with a common ancestor, and Phrynocephalus sp. studied by Schwenk & Bell (1988) shares a common ancestor with Agama. The herbivorous Uromastyx aegyptia shares a common ancestor with Leiolepis (Honda et al. 2000). It could be suggested that the ancestral mechanism of tongue protraction during prey capture in agamid lizards is based on the contraction of the extrinsic M. genioglossus and the intrinsic M. verticalis (Herrel et al. 1995; this study). This implies that lingual translation could be considered an evolutionary intermediate between ‘typical lingual prehension’ and tongue projection in Chamaeleonidae, as suggested by Schwenk & Bell (1988). Functional data recorded by EMG for P. vitticeps and S. stellio support this hypothesis because the two main protractor tongue muscles (Mm. genioglossus and verticalis) act simultaneously during tongue protrusion, although their contraction does not start at the same time relative to tongue–prey contact. The contraction of M. hyoglossus simultaneously with the protractor muscles is also observed in all agamids studied to date (Herrel et al. 1995; the present study), suggesting a similar mechanism in tongue deformation and protrusion in agamids whose diet includes evasive preys (e.g. arthropods). The herbivorous Uromastyx sp., which is suggested to share a common ancestor with Pogona, does not have a M. verticalis that completely surrounds the entoglossal process as demonstrated by Smith (1988) and the M. genioglossus could play the key role in tongue protrusion.

The role of the tongue muscles has been studied in Agamidae with different diets (herbivorous, insectivorous and omnivorous). However, the action of the complex muscles of the hyoid apparatus are less understood in agamid lizards (Herrel et al. 1995). In agamid lizards, Schwenk & Bell (1988) suggest that these muscles could play a key role in the tongue movements, as in Tuatara. This evolutionary hypothesis on the morphological variations of the tongue musculature with respect to diet seems to be supported by the kinematic data available. The diet of Pogona sp., Phrynocephalus sp. and Agama sp. include various arthropods that could escape quickly. To capture such mobile prey, it is suggested that it might be advantageous to position the body and the head far from the prey, while the tongue is protruded to decrease the risk of being detected by the prey, and to increase the speed of tongue protrusion to avoid prey escape. Another hypothesis is that the protractor tongue muscles allow the tongue to be maintained in a protruded position outside of the jaws for a longer time, which may be advantageous when the predator waits for the mobile prey to stop moving. Alternatively, a slower tongue protrusion is not detrimental when eating vegetables. Indeed, the duration of tongue protrusion in the herbivorous Uromastyx sp. is longer than in the other agamids studied (410 ± 139 ms; Schwenk & Throckmorton, 1989), being 120 ms in S. stellio (Herrel et al. 1995), approximately 190 ms in Phrynocephalus (Schwenk & Throckmorton, 1989) and 326 ± 155 ms in P. vitticeps eating on crickets, although tongue protrusion duration varies depending on prey type. Moreover, maximum tongue protrusion distance, which is calculated as the percentage of skull distance, is similar in the agamid species studied, taking up about 40% of skull length. In contrast, maximum tongue protrusion distance in Uromastyx sp. is only 20% of the length of the skull. Finally, in Uromastyx sp., the absence of M. verticalis that surrounds the entoglossal process, produces force against it, which moves the foretongue very rapidly toward the prey, no disadvantage when feeding on plant materials. Therefore, it is suggested that an omnivorous diet that includes evasive prey such as arthropods can be associated with a mechanism of tongue protrusion similar to the one of lizards characterised by an insectivorous diet, and that specialisation for a herbivorous diet can be associated with an evolutionary simplification of tongue morphology and function (e.g. loss of complex tongue intrinsic musculature, slower tongue protrusion, shorter tongue protrusion distance). Such evolutionary simplifications within feeding systems and mechanisms have been reported in various organisms (Hofstee & Pernet, 2011).

Histochemistry

In this study, the classification system of mammalian fibres established by Peter et al. (1972) for fast fibres was followed: fast oxidative-glycolytic (FOG) and fast glycolytic (FG) fibres, respectively. Gleeson et al. (1980) suggest that these fibres appear functionally similar in lizards and mammals, in terms of contraction time and fatigue resistance, and this classification has been used for lizards in various studies (Bonine et al. 2001, 2005). Regarding slow fibres, the histochemical and innervation study of Mutungi (1990) in the iliofibular muscle of Varanus exanthematicus, and the mechanical studies of Proske & Vaughan (1968) suggest that lizards have two types of slow fibres: slow oxidative (SO) and tonic fibres. SO, FOG and FG fibres belong to the twitch class of skeletal striated muscle fibres in vertebrates. Muscles rich in SO fibres are slow but highly resistant to fatigue, contrary to muscles rich in FG fibre, which are rapid and very sensitive to fatigue. Intermediately, muscles rich in FOG fibres are rapid and resistant to fatigue (Guthe, 1981). The second main class is tonic fibres, which have a slow contraction that can be maintained for prolonged periods with little energy expenditure (Rosen et al. 2004). Tonic fibres are found in locomotor muscles of lampreys, elasmobranchs, amphibians, reptiles and birds, and in the extraocular and ear muscles of some mammals (Putnam et al. 1980). In previous studies (Guthe, 1981; Gleeson, 1983; Morgan & Proske, 1984), only FG, FOG and tonic fibres were determined because no correlation was established between histochemistry and fibre ultrastructure or innervation, or contractile function (Mutungi, 1990). However, Gleeson et al. (1980) classified fibres into FOG, FG and tonic based on contractile properties, but they suggested that 30–50% of the tonic fibres may represent a mammalian-like SO group. In this study, SO fibres were not distinguished from tonic fibres, meaning that they were all classified as SO because no correlation was established with innervation or contractile function.

In contrast to fibre distribution in the locomotor muscles of lizards, the tongue has been neglected. Only Herrel et al. (2001) investigated the histochemical properties of the tongue in the lizard Trioceros (Chameleo) melleri. Here, the focus was on the composition of the extrinsic muscles: retractor (M. hyoglossus) and protractor (M. genioglossus) muscles, and the intrinsic verticalis muscle that plays a major role in tongue protrusion (Smith, 1988). M. verticalis forming a ring in some Agamidae is considered homologous to the central ‘accelerator’ muscle in Chamaeleonidae (Smith, 1988). Many authors consider tongue projection of Agamidae as the ancestral state of the ballistic projection in Chamaeleonidae (Schwenk & Bell, 1988; Herrel et al. 1995). In P. vitticeps, the protractor muscle (M. genioglossus) and the M. verticalis mostly comprise SO fibres, whereas the retractor muscle (M. hyoglossus) mostly comprises FG fibres (Fig. 5). These results are supported by the study of tongue protrusion and retraction during feeding in this species where it was found that tongue retraction is significantly quicker than tongue protrusion (40 ± 13 and 5 ± 5 cm s−1, respectively).

None of the tongue muscles is characterised by a homogeneous fibre-type composition. The protractor muscles contain a mixture of the three fibre-types, whereas the retractor muscle (M. hyoglossus) shows clear regionalisation (Fig. 6). FG fibres occupy a limited area in the dorsal side under the papillae and it is suggested that this region of the muscle plays a role in the deformation of the tongue before, and at, tongue–prey contact to increase the contact surface. Indeed, the predominance of FG fibres is consistent with a rapid and intense lingual deformation during tongue protrusion and tongue–prey contact.

Fibre composition in the tongue of P. vitticeps is different from that in T. melleri (Herrel et al. 2001). In T. melleri, the Mm. hyoglossus and genioglossus posterior are composed of FG fibres, and the Mm. genioglossus anterior and accelerator of FOG fibres. Moreover, no SO/tonic fibres were observed. M. verticalis plays a key role in tongue protrusion/projection during feeding in agamids and chamaeleonids. In Chamaeleo calyptratus, the peak velocity of tongue projection varies from 3.4 to 4.4 m s−1 depending on temperature (Anderson & Deban, 2010), and in Furcifer oustaleti, the maximum peak velocity of tongue projection varies between 4.9 and 5.9 m s−1 capturing prey at between 20 and 35 cm, respectively, between 29 and 31 °C (Wainwright et al. 1991). In Trioceros jacksonii, by dividing the tongue distance by the projection time presented in the kinematic profiles of Figures 3 and 4 in Wainwright & Bennett (1992), the average velocity of tongue projection can be estimated at between 1.5 and 2.6 m s−1. In P. vitticeps, tongue protrusion velocity is approximately 0.05 m s−1, which is about one-third of that in chameleons. This difference in tongue protrusion velocity can be explained by the difference in the percentage of fast fibres, although an energy-storage-and-release mechanism may play a key role in Chamaeleonidae (De Groot & Van Leeuwen, 2004). Regarding the retractor muscle (M. hyoglossus), the percentage of FG fibres is higher in T. melleri (90–100%; Herrel et al. 2001) than in P. vitticeps (56.5%). From the data available for Furcifer pardalis (Figure 5 in Herrel et al. 2000), the average velocity of tongue retraction can be estimated at 0.17 m s−1. In C. calyptratus, the peak velocity of tongue retraction varies from 0.8 to 1.9 m s−1 depending on temperature (Anderson & Deban, 2010). In F. oustaleti, Wainwright et al. (1991) measured a maximum tongue retraction velocity of between 2 and 3 m s−1 for similar distances between the predator and the prey at between 29 and 31 °C. In P. vitticeps the velocity is only 0.4 m s−1. In chameleons, the peak velocity of tongue retraction remains high and could be related to the high percentage of fast fibres. The question of tongue retraction from a functional and morphological perspective has yet to be investigated. From the kinematic data available (Wainwright et al. 1991; Wainwright & Bennett, 1992; Herrel et al. 2000) it seems that tongue retraction with the prey starts quickly and is then followed by a slower phase. This quick phase could be related to the high percentage of the fast fibres.

In conclusion, the data collected for the present study clearly indicate the basis of a functional and morphological relationship between the velocity of tongue protrusion and tongue fibre composition in the fast fibres found in agamids and chamaeleonids. However, a complete comparative analysis within iguanian and scleroglossan squamates that use tongue prehension has yet to be done. Except in the cordylid Ouroborus cataphractus (Broeckhoven & Mouton, 2013), all scleroglossan lizards use tongue prehension to capture prey with the dorsal surface of the foretongue (Urbani & Bels, 1995; Smith et al. 1999; Reilly & McBrayer, 2007; Montuelle et al. 2009), despite variability in tongue morphology (Gnanamuthu, 1937; Schwenk, 2000). The question of using a structure with highly different morphological features (muscular system and tongue surface) in a same function (e.g. prey prehension) has yet to be investigated in Squamates. Combining muscle morphology, including fibre-typing, with kinematics (i.e. velocity of tongue protrusion and retraction) and behaviour (i.e. prey selection) provides the opportunity to analyse the functional feature of the muscular hydrostat of the tongue.

Acknowledgments

The authors would like to thank Christophe Remy (Musée d'Histoire Naturelle de Tournai) for the provision of lizards, Bernard Blairon (UMONS, Laboratory of Physiology and Pharmacology) for his substantial assistance during the EMG recordings, Annik Maes (UMONS, Laboratory of Histology) for technical assistance and Paul Postiau (UMONS, Laboratory of Biology of Marine Organisms and Biomimetics) for his help with the Scanning Electron Microscope. Special thanks go to Dr Cécile Berri and Thierry Bordeau for their kind hospitality at the Unité de Recherches Avicoles of INRA, France, and for presenting the technique of histoenzymology. Thanks also go to the two anonymous reviewers for their comments and suggestions on a previous version of the manuscript. This work is part of the PhD project of Leïla-Nastasia, ZGHIKH. This study was supported by the FRIA-FNRS and the Muséum National d'Histoire Naturelle through the ATM ‘Formes possibles, Formes réalisées’.

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