2004

Asiatic Herpetological ResearchVol. 10, pp. 176-181

Histochemical Characterization of the Lingual Salivary Glands of theHouse Gecko, Ptyodactylus hasselquistii (Squamata: Gekkonidae)
BASHIR M. JARRAR AND NOORY T. TAIB
Zoology Department, College of Science, King Saud University, P.O.Box 2455, Riyadh 11451 Saudi Arabia.
Abstract. - Histochemical investigations of the lingual salivary glands of the house gecko, Ptyodactylus hasselquistii
have been conducted. The glands are comprised of mucous and mucoserous cells. Mucous cells secrete or elaborate
neutral mucosubstances, neuraminidase sensitive carboxylated mucins, hyaluronidase resistant sulfomucins, but are
devoid of proteins. The mucoserous cells secrete and elaborate neutral mucosubstances and glycoproteins but are
devoid of sialomucins and sulfomucins. The results are discussed in the context of the feeding habits and phylogenyof reptiles.
Key words. - Histochemistry, lingual, salivary glands, house gecko, Ptyodactylus hasselquistii, Gekkonidae.

Introduction
Histochemical studies on the lingual salivary glands ofvertebrates have mainly been concerned with mammals,
whereas little attention has been paid to the lingual salivary glands of non-mammalian vertebrates. Most studies on the lingual salivary glands of reptiles havefocused with morphological and histological aspects
while few histochemical studies have been carried onthese glands (Raynaud, 1961; Gabe and Saint-Girons,
1969; Lopes et al., 1982; Taib and Jarrar, 1985a; 1985b;
1985c; 1986; Taib, 1986, Asgah et al., 1990).
Nevertheles, the literature on the lingual secretions oflizards is rather scanty and their consitituents have yet
to be determined.
The present study is a detailed histochemical characterization of the lingual salivary glands of the house
gecko, Ptyodactylus hasselquistii.

Materials and Methods
Twenty adults of each male and female house gecko
Ptyodactylus hasselquistii were trapped from different
houses in Riyadh city, Saudi Arabia. They were killed byetherization and the whole tongue was removed fromeach animal and quickly immersed for 24 hrs in one of
the following fixatives: neutral buffered formalin,
Bouin's fluid and Gendre's fluid. They were then thoroughly washed in running water, processed for serial
sectioning at 4-5 µm thickness and the sections were
stained with haematoxylin-eosin or with Mallory
trichrome for histological examination, whereas the secretary cells of the glands were characterized by the criterion of Gabe and Saint-Girons (1969). Other sections
were used for the following histochemical reactions:© 2004 by Asiatic Herpetological Research

Neutral mucosubstances. - Periodic acid-Schiff (PAS)technique (Gurr, 1962), PAS after diastase digestion(McManus and Mowry, 1964), PAS after alpha-amylase
digestion (Luna, 1968), PAS after acetylation blockade(McManus and Cason, 1950), PAS after acetylationsaponification (Oxello et al.,1958), PAS after phenylhydrazine treatment (Spicer et al., 1967) and PAS after
treatment with chloroform and methanol.
Acid mucosubstances. - Alcian blue (AB) at pH 2.5,
1.0, and 0.4 (Mowry, 1956; Luna, 1968).
Distinction between acidic and neurtal mucosubstances. - AB (pH 2.5)-PAS (Mowry and Winkler, 1956)
and AB (pH 1.0)-PAS (Spicer et al., 1967).
Distinction between sulfomucins and sialomucins. Aldehyde fuchsion (AF) and AF-AB, pH 2.5 (Spicer andMeyer, 1960); weak (25ºC, 16 hr), mild (37ºC, 4hr) andstrong (60ºC h hr) methylation-saponification- AB (PH2.5) (Spicer, et al., 1967); toluidine blue (TB) buffered at
pH 1.7 and 3.4 (Landsmeer, 1951), critical electrolyte
concentration (CEC) technique for extinction ofalcianophilia at pH 5.6 in the presence of gradual concentation of Mg2+ (Scott and Dorling, 1965).
Enzyme digestion tests. - Diastase-PAS technique(McManus and Mowry, 1964); neuraminidase(Sialidase, Vibrio chlolerae, type V)-AB (pH 2.5)
(Spicer and Warren, 1960); hyaluronidase (testicular)AB (pH 2.5) (Spicer et al., 1967). Ribonuclease digestion (Love and Rabotli 1963); neuraminidase-TB (pH3.7), hyaluronidase-TB (pH 2.0) were employed. In
each case control sections were incubated for the samelength of time at the same temperature in buffer solutions without the enzyme.

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2004

Figure 1. Lingual glands of P. hasselquistii after staining
with haematoxylin-eosin. Note that the mucous cells of
the lingual glands are located in the papillar space of thepapillae. x950.

Figure 2. Lingual glands of P. hasselquistii after staining
with PAS. The reactivity of the glands confirms the presence of the neutral mucosubstances. x950.

Figure 3. Lingual glands of P. hasselquistii after staining
with AB (2.5), confirming the presence of sialomucins
and sulfomucins. x950.

Figure 4. Lingual glands of P. hasselquistii after staining
with AB (1.0)-PAS. The bluish purple color indicates thepresence of neutral and sulfated mucosubstances simultaneously. x950.

Figure 5. Lingual glands of P. hasselquistii after staining
with CEC at 0.3M Mg++., confirming that the mucosubstances produced by the glands contain carboxyl andsulfated groups. x950.

Figure 6. Lingual glands of P. hasselquistii after staining
with MBPB, indicating the protein contents of the glands
secretion. x700.

2004

Asiatic Herpetological ResearchProteins. - Mercuric bromophenol blue method (Mazia
et al., 1953); ninhydrin-Schiff (Yasuma and Itchikawa,1953), mercuric-bromophenol blue (MBPB) and PAS
after trypsin digestion (Pearse, 1972).
Photographs. - Photographs were taken with a 35mm
Zeiss Ikon camera on Kodacolor NR 100 film.

Results
The lingual salivary glands of the house gecko,
Ptyodactylus hasselquistii occupy the papillar invagination of the posterior two-thirds of the dorsal surface
together with the lateral sides of the tongue. The anterior part of the tongue is devoid of any glandular structure
and covered by keratinized squamous epithelium. These
glands are made of mucous cells located in the inner
papillar space of the filiform papillae (Fig. 1) together
with simple tubular structures made of mucoserous cells
seen at the most posterior part of the dorsum. The
mucous cells have an alveolar cytoplasm and flattened,basally located nuclei with clear apical ends resting on adelicate basement membrane.
As summarized in Table 1, the mucous cells of thelingual glands of P. hasselquistii exhibited strong PAS
reactivity (Fig. 2) which was neither labile to alphaamylase nor to saliva digestion but completely lost byphenylhydrazane treatment. However, this reactivity
was completely blocked by acetylation and was partly
restored by deacetylation-PAS sequential techniques.
They showed marked alcianophilia at both pH 2.5 (Fig.
3) and 1.0 but to lesser extent at pH 0.4. They also reacted with both PAS and AB and stained bluish purple withAB (2.5)- PAS and AB (1.0) PAS (Fig. 4). These glands
also reacted with AF as well as with AF-AB (2.5) and
AF-AB (1.0). The alcianophilia of the glands was partly lost at pH 2.5 with acid hydrolysis and weak methylation and there after restored by saponificiation techniques. They demonstrated alcianophilia with the CEC
techniques at 0.1M, 0.2M, and to some extent at 0.3M
Mg2+ (Fig. 5) and showed metachromasia at pH 3.4 and1.7 but reacted negatively to all protein detection tests.
The mucoserous cells of the glands showed PAS
reaction, exhibited no alcianophilia at pH 2.5 and 1.0
and were orthochromatic at pH 3.4 and 1.7 but reacted
positively to all protein detection tests (Fig. 6). No sexual dimorphism was observed in the lingual secretion of
the species under study.

Discussion
The lingual salivary glands present great diversity inmorphology amongst the various groups of reptiles.
These glands are entirely absent from Varanidae,

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Amphisbaenia, Ophidia and some species of Chelonia
such as Chelonia mydas (Kochva, 1978). On the other
hand, these glands are simple consisting of three different types of goblet cells in most species of Testudinidae(Nalvade and Varute, 1976; Taib and Jarrar, 1984).
Some lizard possess mainly goblet cells together withsimple tubular glandular structures in their tongues(Nalvade and Varute, 1976; Shevliuk, 1976; Taib andJarrar 1985 b; 1985c and 1986), while others have moredeveloped lingual salivary glands as seen in some
Agamidae, Iguanidae, Gekkonidae, Anguidae andChamaleonidae (Gabe and Saint-Girons, 1969; Kochva,
1978; Asgah et al., 1990). On the bases of the results ofthe present study and in view of the criterion of Gab andSaint-Girons (1969), the lingual salivary glands of
Ptyodactylus hasselquistii are made of unicellularmucous goblet cells lining the dorsal epithelium of the
tongue with mucoserous simple tubular glandular apparatus at the base of the tongue. The structure of the lingual salivary glands of P. hasselquistii is different fromthose of Tupinambis teguixin, Agama blandfordi,
Uromastyx microlepis, Acanthodactylus schmidti and
Scincus mitranus, which have only mucous cells in their
lingual glands (Lopes et al, 1974; Taib and Jarrar,
1985b; 1985c; 1986; Taib, 1986). According to Gabe
and Saint-Girons (1969), the lingual glands are mucous
in Gekkonidae, mucoserous on Sphenodontidae,
Anguidae and Pygopodidae, but seromucous in
Chamaleonidae and serous in some speices of Iguanidae
and Agamidae. The grading from non-glandular tongues
through unicellular with or without simple tubular glandular structure to only simple tubular and to then tubulo-alveolar ones may reflect developmental stages
towards the definitive lingual glands of higher vertebrates (Shevliuk, 1976; Kochva, 1978).
A tentative interpretation of the types of mucosubstances in the lingual glands of P. hasselquistii can bemade from the results of the different histochemical
reactions used in the present investigation and from theclassification of mucosubstances proposed by Mowry
and Winkler, 1956; Spicer and Meyer, 1960; Scott andDorlling, 1965; Pearse, 1972). Neutral mucosubstances
are PAS positive, diastase resistant, as well as unstainable by cationic dyes. Acetylation produces derivativesof primary and secondary amines which prevent 1, 2
glycol groups, from reacting with PAS indicating thepresence of neutral mucosubstances or sialic acid, separately or simultaneously. Alcian blue is generally considered as being specific for identifying acid mucosubstances where alcianophilia at pH 2.5 and 1.0 is specificfor sialomucins and sulformucins respectively (Mowry
and Winkler, 1956). In the combined aldehyde fuchsinalcian blue sequential techniques, sulfomucins stain purple blue and sialomucins blue (Spicer and Meyer, 1960).

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Table 1. The histochemical reactions in the lingual salivary glands of Ptyodactylus hasselquistii.
Results

Histochemical reaction

PAS
Diastase digestion -PAS
Acetylation-deacetylation-PAS
Phenylhdrazine-PAS
AB (pH 0.4)
AB (pH 1.0)
AB (pH 2,5)
AB (pH 1.0) -PAS
AB (pH 2.5)-PASAF
AF- (AB pH 1.0)
AF- (AB pH 2.5)
Acid hydrolysis- AB (pH 2.5).
W. methylation - AB (2.5)
W. methylation-saponification - AB (pH 2.5)
M. methylation-AB (pH 2.5)
M. methylation-saponification -AB (pH 2.5)
S. methylation-AB (pH 2.5)
S. methylation-saponification- AB (pH 2.5)
TB (pH 1.7)
TB (pH 3.4)
CEC (AB, 0.1 M)
CEC (AB, 0.2 M)
CEC (AB, 0.3 M)
CEC (AB, 0.5 M)
Neuraminidase-AB (pH 2.5)
Hyaluronidase-AB (pH 2.5)
Ninhydrin-Schiff
Hg- bromophenol blue
Chloramine T-Schiff
Trypsin digestion-PAS(Chloroform + methanol)-PAS

MC

MSC

++,P
Nb
++,p
Cb
+B
+B
++, B
+,Bp
++,Bp
+,P
+,Bp
++,Bp
Pb
Pb
++,Bp
Cb
+,B
Cb
+,B
+
+
+
+
±
+B,pb
++B,Nb
Nb
Nb

++,P
Nb
++,p
Cb
+
+
+
-

Reactions: - negative; ± weak; +, moderately positive; ++, intensely positive; Cb, complete blockade; M,
mild; Pb, partial blockade; Nb, no blockade; S, strong; TB, toluidine blue; W, weak.
Colors: B, blue; Bp, bluish purple; P, pink.
Glands: MC, mucous cells; MSC, mucoserous cells.

Sialomucins can be identified by alcianophilia at pH 2.5
which is partially lost following acid hydrolysis andcompletely removed after neuraminidase digestion, but
neuroaminidase did not affect the staining of sulfatedmucosubstances. A loss of alcianophilia after
hyaluronidase digestion is due to the removal of
hyaluronic acid and chondroitin sulfates. Methylation
blocks subsequential stainig of simple mucosubstancesby esterification of carboxyl groups and complex sulfated mucosubstances desulphation. Subsequent treatment
with potassium hydroxide (saponification) after methylation will restore the staining of carboxyl groups (Druryet al., 1967). The mucosubstances that are stained at
0.1M MgCl2 in the CEC reaction, but not at 0.2M MgCl2
are believed to contain carboxyl group and no sulfate

groups. Sulfated mucosubstances, on the other hand,
stain strongly and selectively at 0.2M Mg2+ but lose
their alcianophilia at different levels with increasing
MgCl2 concentration (Spicer and Lillie, 1960). The lingual glands of the species under study resisted trypsin
digestion and the action of (chloroform-methanol)
which excludes the possibility of lipids and proteins.
Accordingly, the lingual salivary glands of of the house
gecko, Ptyodactylus hasselquistii contain neutral mucosubstances, sialadase labile carboxylated mucosubstances and hyaluronidase resistant sulfomucins and glycoproteins.
The lingual secretions of P. hasselquistii are different from those of some lizards such as Tupinambis
teguixin and Agama blandfordii which contain neutral

2004

Asiatic Herpetological Researchmucosbstances and sialomucins but no sulfomucius(Lopes et al., 1974; Taib and Jarrar, 1985c). They also
differ from the secretions of the lingual glands of
Uromastyx microlepis, Acanthodactylus schmiditi,
Scincus mitranus and Stenodactylus slevini which contain neutral mucosubstances, sialomucins and sulformucins but no glycoprotein (Taib and Jarrar, 1985b,
1986; Taib, 1986; Asgah et al., 1990). Neutral mucosubstances have been demonstrated in the secretions of allstudied reptiles that possess salivary glands while phylogenetically, the absence of sulfomucins in the lingual
glands would favour the concept that sialomucins secretive cells are more primitive than sulfated mucosubstances secretive ones. In addition, the heterogenous histochemical recactivity of the lingual glands might haveappeared in the evolutionary lines of reptiles to meet thedifferent changes in the feeding habits of various
species. Neutral mucins were present in the lingual
glands of almost all studied reptile species while sialomucins together with neutral mucosubastances wereidentified in the lingual glands of all insectivorous reptiles so far studied (Nalvade and Varute, 1972; Taib andJarrar, 1985c, 1986; Taib, 1986). The lingual glands of
all insectivorous and carnivorous reptiles studied thusfar
exhibited sulfomucins. More work is needed to elucidate
whether the lingual secretion diversity of reptiles imply
phylogenetic relationships or different feeding habits.

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