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THE ANATOMICAL RECORD 251:114–121 (1998)
Ultrastructure of the Binary Parotid
Glands in the Free-tailed Bat,
Tadarida thersites.
I. Principal Parotid Gland
TOSHIKAZU NAGATO,1* BERNARD TANDLER,2
AND CARLETON J. PHILLIPS3
1Second Department of Oral Anatomy, Fukuoka Dental College, Fukuoka 814-0193, Japan
2Department of Oral Anatomy II, Kyushu Dental College, Kitakyushu 803-8580, Japan
3Department of Biological Sciences, Illinois State University, Normal, Illinois 61790-4120
ABSTRACT
Background: Many species of bats have two sets of submandibular
glands, principal and accessory. The accessory gland may resemble the
principal one but more often shows wide morphological divergence. The
free-tailed bat, Tadarida thersites, is very unusual in that it has two sets of
parotid glands rather than binary submandibular glands. We studied the
ultrastructure of the principal parotid gland to establish a baseline for
comparison with the accessory parotid.
Methods: Two specimens of adult free-tailed bats, one male and one
female, were live-trapped in western Kenya. Parotid glands were fixed for
electron microscopy using a protocol expressly designed for field fixation and
then embedded by conventional means.
Results: Histologically, the principal parotid is a typical serous gland. The
secretory granules of the endpiece cells have an unusual substructure in that
they contain variable numbers of lucent halos and one or several spherules.
Intercalated duct cells contain a significant number of dense, serous-like
granules. Striated ducts have the usual basal configuration of mitochondria
and folded plasma membranes, but the supranuclear cytoplasm contains
many small, dense granules, so that these ducts resemble the granular
convoluted tubules found in the submandibular glands of many families of
rodents. The apices of the duct cells have a peculiar contour—the luminal
surfaces obliquely invaginate into the apical cytoplasm, so that in thin section
the luminal membranes appear to be underlaid by a layer of vacuoles.
Conclusion: Although the principal parotid gland of the free-tailed bat
shows some distinctive, species-specific ultrastructural features, it basically
is similar to the parotid gland in two other molossid bats, Tadarida
brasiliensis and Molossus molossus. The distinctive features in the principal
parotid gland of T. thersites might relate to its feeding on hard-bodied insects
and perhaps to the production of lysozyme. Anat. Rec. 251:114–121,
1998. r 1998 Wiley-Liss, Inc.
Key words: principal parotid gland; salivary glands; granular ducts;
chiropterans; bats; Tadarida thersites
In his comprehensive review of chiropteran salivary
glands, Robin (1881) noted the presence in some species of
two sets of submandibular salivary glands, which he
labeled principal and accessory. His designations were
based solely on gross anatomical considerations. In our
large-scale, comparative ultrastructural study of bat salir 1998 WILEY-LISS, INC.
*Correspondence to: Dr. Toshikazu Nagato, 2nd Department of
Oral Anatomy, Fukuoka Dental College, 2-15-1 Tamura, Sawaraku, Fukuoka 814-0193, Japan
Received 23 December 1997; Accepted 20 January 1998
PRINCIPAL PAROTID GLAND IN TADARIDA
vary glands, we have come across such binary salivary
glands in a number of species. Rather than using gross
anatomical criteria as the basis for labeling, we have based
identification of principal and accessory glands entirely on
microscopic features (Tandler et al., 1996a). The usual
situation is that one of the sister glands is a close likeness
of conventional glands (this we designate as principal),
whereas the second gland may exhibit morphological—
and sometimes histochemical—differences from typical
glands (this we designate as accessory).
Although binary submandibular glands are not uncommon either in bats or in other kinds of mammals, accessory
parotid glands are extremely rare. In fact, the only species
in which they are reported to occur are humans (reviewed
by Toh et al., 1993) and several other primates (Huntington, 1913); rats, in which they are called anterior buccal
glands (Redman, 1972); sheep (Rodriguez-Veiga and
Zúñiga, 1985); and possibly laboratory mice (Domon and
Shiga, 1986). We have found that the free-tailed bat,
Tadarida thersites, also has an accessory parotid gland.
Both principal and accessory glands have a number of
unusual features, although the principal one is closer in
structure to conventional chiropteran parotid glands. The
ultrastructure of the principal parotid gland is detailed in
the present report; that of the accessory parotid gland is
described in the accompanying article (Tandler et al., 1998).
MATERIALS AND METHODS
Two adult specimens (one of each sex) of the free-tailed
bat, T. thersites, were collected by mist-netting in the
Kakamega Forest of western Kenya. Voucher specimens
(CJP 4889 and 4790) were deposited in the research
collection of the Carnegie Museum of Natural History,
Pittsburgh, Pennsylvania. The bats were killed in the field
with T-61 Euthanasia Solution; their major salivary glands
were immediately extirpated, immersed in fixative, and
minced. The initial fixative was cacodylate-buffered, halfstrength Karnovsky’s (1965) solution in 2.5% dimethyl
sulfoxide and contained 0.01 M LiOH and 0.01 M CaCl2.
After 20 hr in this mixture, tissues were washed and
stored in 0.5 M cacodylate buffer at ambient temperature.
Once refrigeration became available, tissue samples were
transferred to fresh 3% glutaraldehyde and stored at 4°C
(Phillips, 1985). In the laboratory, the parotid gland
samples were extensively washed in phosphate-buffered
sucrose and then postfixed for 2 hr in 2% osmium tetroxide
in the same buffer. After rinsing in distilled water, specimens were soaked overnight in acidified uranyl acetate
(Tandler, 1990). Another aqueous rinse was followed by
dehydration in ascending concentrations of ethanol, passage through propylene oxide, and embedment in Epon:
Maraglas (Tandler and Walter, 1977). Thin sections were
serially stained with acidified uranyl acetate (Tandler,
1990) and lead citrate (Venable and Coggeshall, 1965) and
examined in a JEOL 1200EX electron microscope (JEOL,
Tokyo, Japan).
RESULTS
At low magnification, the principal parotid gland of
T. thersites conforms in structure to conventional parotid
glands (Fig. 1). Secretory endpieces consist of wedgeshaped cells that contain numerous serous granules (classification based on ultrastructural morphology; cf. Young
and van Lennep, 1978; Tandler and Phillips, 1993) (Fig. 2).
115
The granules are rather large, measuring up to 1.4 µm in
diameter. Some contain a single (often several) eccentrically placed, dense spherule(s); these almost always are
encircled by a thin lucent halo. Small lucent haloes—some
unrelated to spherules, some containing material that
matches the spherules in density—may be scattered
throughout the granule matrix; these display a proclivity
for lining up against the inner aspect of the limiting
membrane (Fig. 3). Like other serous cells, those in the
principal parotid of T. thersites have a well-developed
rough endoplasmic reticulum (RER) and a prominent
Golgi apparatus. Lipid droplets tend to be abundant in
some cells, almost always in an infranuclear position.
The serous cells surround a more or less central lumen;
intracellular canaliculi originating from the endpiece lumen pervade the endpieces. The serous cells lack basal
folds. Very flat myoepithelial cells are present at the
endpiece periphery; their perikarya usually are located
near the endpiece-intercalated duct junction, and their
processes are joined to the adjacent serous cells by desmosomes.
Intercalated ducts consist of simple cuboidal epithelial
cells (Fig. 1) that often are edged by myoepithelial cell
processes (Fig. 4). Although almost every duct cell contains
a number of serous-type granules in its supranuclear
cytoplasm, some cells have more than others (Fig. 5).
These granules are smaller (,0.45 µm) than those in the
endpiece cells and have a homogeneously dense matrix. In
keeping with their secretory function, the duct cells have
an extensive Golgi apparatus. Where the Golgi saccules
are flat and parallel, a moderately dense material may be
present between adjacent saccules. Myoepithelial cell processes are particularly numerous on these ducts.
Consisting of pseudostratified epithelium, the major
intralobular ducts show a number of departures from
conventional striated ducts. Even at very low magnification (Fig. 6) it is apparent that although they exhibit basal
striations, they contain secretory granules in their apical
cytoplasm and that their luminal surface has an erose
contour. Figure 7 shows the overall cytology of these duct
cells—the infranuclear portion of the cell is typical in
structure, whereas the supranuclear cytoplasm is suffused
with granules. At higher magnification (Fig. 8), the basal
striations are revealed as consisting of highly plicated
plasma membranes interspersed with mitochondria.
The most striking feature of the tall cells is their content
of numerous serous-type granules (Figs. 6, 7, 9). These are
homogeneously dense and measure approximately 1.0 µm
in diameter. They largely are confined to a subluminal
zone; between the granules and the nucleus is a region
with a considerable amount of RER. A relatively small
Golgi apparatus sometimes caps the nucleus.
The luminal surface of the tall duct cells bears many
short, stubby microvilli. This surface is highly indented, so
that a section passing through an appropriate plane gives
the impression that the luminal plasmalemma is underFig. 1. (overleaf.) Survey electron micrograph of the principal parotid
gland of Tadarida thersites. The gland consists mainly of serous acini
composed of heavily granular cells. An intercalated duct is indicated by
the arrow; the light-appearing cell in its wall is a mononuclear wandering
cell. 31,150.
Fig. 2. (overleaf.) A transverse section through an entire endpiece
showing the general morphology of the serous cells and their abundance
of serous granules. 33,900.
116
NAGATO ET AL.
Figs. 1 and 2.
PRINCIPAL PAROTID GLAND IN TADARIDA
117
Fig. 3. Higher magnification view of endpiece serous granules. The patterns exhibited by these granules
depends on just a few substructural elements: dense spherules of varying size, each of which is delimited by a
light halo, and haloes that encompass matrix material rather than spherules. The haloes tend to be aligned just
within the limiting membrane of the granules. 336,000.
laid by a series of lacunae (Fig. 9). Because the lining
membrane of these pseudolacunae is, in fact, the luminal
membrane, it also bears microvilli. These indentations
bear many V-shaped invaginations, indicating that they
are a preferred site for exocytosis of duct granules to take
place.
The sparse basal cells have a triangular shape, with the
triangle base resting on the basement membrane, to which
they are attached by hemidesmosomes. They also are
joined to their taller neighbors by desmosomes. Their few
mitochondria are considerably smaller than those in the
tall cells.
in the latter species, the principal parotid gland in
T. thersites has ‘‘conventional’’ secretory endpieces and as
is typical (but not uniformly so) of insectivorous bats, the
acinar cells can be described as serous (Phillips et al.,
1987, 1993).
Serous granules in the acinar cells of T. thersites add
another type of substructure to the already extensive
gallery of serous granule morphology (Phillips et al., 1987;
Tandler and Phillips, 1993). In addition to the spherules,
aliquots of matrix material are delimited by light halos.
DISCUSSION
Fig. 4. (overleaf.) Transverse section of an intercalated duct. The duct
cells contain a variable number of small, dense secretory granules. A
myoepithelial cell process (Mec) borders the duct on the right. The light
cells in the left half of the duct wall are mononuclear wandering cells that
have invaded the ductular epithelium. 36,500.
Fig. 5. (overleaf.) The apical cytoplasm of an intercalated duct cell that
is provided with many serous-type secretory granules. These granules
lack the substructure of their acinar cell counterparts. 328,800.
The molossid free-tailed bat, T. thersites, is a member of
the subgenus Mops, which is characterized by insectivorous bats specialized to feed on beetles (Freeman, 1979). In
general histology and ultrastructure, the principal parotid
gland of T. thersites is somewhat similar to a geographically wide-ranging New World species, T. brasiliensis. As
118
NAGATO ET AL.
Figs. 4 and 5.
PRINCIPAL PAROTID GLAND IN TADARIDA
119
Fig. 6. Survey electron micrograph of an obliquely sectioned striated duct. Even at this low magnification, it
is evident that the duct cells contain dense secretory granules and that their apical surfaces have a peculiar
configuration. 3780.
Fig. 7. Several striated duct cells whose supranuclear cytoplasm contains an abundance of dense
secretory granules. 33,700.
Although a similar pattern of halo-delineated matrix
material is present in the serous granules of the canine
parotid gland (Nagato and Tandler, 1986), those in the dog
do not have a propensity for alignment on the inner aspect
of the granule membrane. Secretory granules with multiple spherule-like inclusions are present in submandibular acinar cells of the nine-banded armadillo; each of these
small inclusions is enclosed by a relatively thick, lucent
halo (Ruby and Canning, 1978).
Serous granules in the principal parotid gland of
T. thersites have a radically different substructure from
those in the parotid gland in another beetle-eating molossid bat, Eumops glaucinus, that contain a single, large
spherule with a distinctive substructure. In contrast, the
serous granules in T. thersites bear some resemblance to
the great majority of those in the parotid gland of
T. brasiliensis; the latter granules have numerous small
spherules that are scattered throughout the granule matrix. In some but not all of these granules, the small
spherules are surrounded by a light halo. None of the granules
in T. brasiliensis contains large spherules (Tandler, unpublished observations). Occasional granules in T. brasiliensis
have an ornate substructure (Phillips et al., 1987).
Generally speaking, serous granules such as those in the
acinar cells of the principal parotid gland of T. thersites are
thought to be enzyme-rich, whereas seromucous granules
in frugiverous bats are considered to be low in enzyme
content (Phillips et al., 1993). It seems likely that lysozyme
is a component of the granules in T. thersites, because
immunohistochemical studies have revealed lysozymelike reactivity in the parotid gland acinar cell serous
granules in the related bat, T. brasiliensis (Phillips et al.,
1998)
Intercalated ducts in the principal parotid gland of
T. thersites are noteworthy because they are different from
those in T. brasiliensis. In thersites the duct cells typically
contain numerous electron-dense serous-type secretory
granules, whereas the homologous cells in brasiliensis
generally lack secretory granules (Tandler et al., 1998).
This particular interspecific difference is interesting because lysozyme-like immunoreactivity is associated with
parotid gland intercalated duct secretory granules in some
species of bats (Phillips et al., 1998). From comparative
studies, it appears that insectivorous bats that feed on
hard-bodied, chitinous insects such as beetles are far more
likely to have secretory intercalated ducts than are bats
that feed primarily on moths or other soft-bodied insects
(Tandler et al., 1998). Moreover, there appears to be a
correlation (observational rather than statistical) between
diet and the number and variety of parotid and submandibular gland cells that might produce lysozyme (Phillips
et al., 1998), Based on such data, we hypothesized that
120
NAGATO ET AL.
Fig. 8. The infranuclear portion of striated duct cells. Although these
cells clearly are involved in elaboration of an organic secretory product,
they possess the classical lineaments of striated ducts—basal compartments that are delineated by highly folded plasma membranes and that
contain rod-shaped, vertically oriented mitochondria. 315,200.
lysozyme might serve as a salivary chitinase in some
species (Phillips et al., 1998). Although both T. thersites
and T. brasiliensis eat beetles, large coleopterans compose
the bulk of the diet for thersites and less than half of the
diet for brasiliensis (Freeman, 1979).
Although the secretory endpieces of the principal parotid gland in T. thersites are typical in structure, their
striated ducts are modified. The tall cells are informed by
numerous secretory granules in their apical cytoplasm. In
keeping with their secretory function, many of the duct
cells have much more RER than is the usual case in these
gland components.
Although striated duct cells in many species may contain a modicum of secretory granules, these usually are
quite small (Tandler et al., 1990; Tandler, 1993). The only
other chiropteran salivary gland with numerous, fairly
sizeable secretory granules in the striated ducts is the
parotid gland of the vampire bat (Tandler et al., 1997). It
may not be fair to call the ducts in the vampire parotid
‘‘striated’’ because they have lost all traces of basal striations; however, by virtue of their position relative to
intercalated and excretory ducts, they certainly are homologous to striated ducts. These granular ducts in the vampire bat clearly are wholly given over to secretion. A
similar situation exists in both the parotid and submandibular glands of the slow loris, a primate (Tandler et al.,
1996). In these glands, like in the vampire bat parotid,
ducts homologous to striated ducts have lost their basal
striations and elaborate one or several giant (up to 11 µm
in diameter) secretory granules per constituent cell. Because the ducts in T. thersites still possess basal striations,
however reduced, it is highly likely that they still function
in ion transport despite their obvious commitment to
elaboration of organic products. In this respect, they
resemble the granular convoluted tubules of rodent submandibular glands (Tamarin and Sreebny, 1965).
In rodents, granular convoluted tubules lie between the
intercalated and striated ducts and are marked by the
presence of abundant serous type secretory granules in the
apical cytoplasm of their constituent cells. In some rodents, such as the laboratory mouse, these ducts are
sexually dimorphic. The structure and manifold functions
of granular convoluted tubules have been comprehensively
reviewed by Gresik (1994). Developmentally (and presumably evolutionarily), these ducts are direct lineal descendants of striated ducts (Gresik, 1994). As such, they retain
a reduced level of basal striations. Whole-cell patch clamp
studies of granular convoluted tubules from the submandibular gland of the male mouse have shown that these
ducts engage in Na1 conductance (Dinudom et al., 1993a);
this conductance is sensitive to amiloride (Dinudom et al.,
1993b). These data show that granular convoluted tubules
still retain the ability to transport electrolytes.
In contrast to rodent granular convoluted tubules, the
striated ducts of the submandibular gland of the European
hedgehog have a normal array of basal striations while
producing numerous secretory granules (Tandler and MacCallum, 1974). These granules must have an unusual
composition because they are better preserved by fixation
solely with osmium tetroxide than they are by initial
fixation in aldehyde mixtures followed by osmium. This
finding is contrary to all other secretory granules in any
salivary gland in any species.
The luminal surface of the granule-bearing duct cells in
T. thersites is quite irregular, appearing to have multiple
indentations. The only other species of bat in which we
have observed a comparable configuration of duct cell
apical surfaces is the mastiff bat, Eumops glaucinus,
which is a member of the same family (Molossidae) (Tandler, unpublished observations). In this respect, these surfaces resemble that of granular convoluted tubule cells in
the male mouse, which has been described as having
intracellular canaliculi with a modest number of microvilli
(Caramia, 1966). These canaliculi, which can penetrate
quite deeply into the granular convoluted tubule cells,
become much more evident after castration and show
images that strongly suggest that granule exocytosis takes
place at their lining membrane. Such canaliculi are absent
from granular convoluted tubule cells in the female mouse
(Caramia, 1966). In contrast to the mouse, surface indentations of duct cells were present in the female free-tailed
bat.
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