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Mucous droplets with multiple membranes in the accessory submandibular glands of long-winged bats.

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THE ANATOMICAL RECORD 240:178-188 (1994)
Mucous Droplets With Multiple Membranes in the Accessory
Submandibular Glands of Long-Winged Bats
Department of Oral Biology, School of Dentistry, Case Western Reserve University,
Cleveland, Ohio (B.T.);Department of Biological Sciences, Illinois State University,
Normal, Illinois (C.J.P.); Department of Anatomy, Schools of Dentistry and Medicine, West
Virginia University, Morgantown, West Virginia (C.A.P.)
Background: Certain species of bats possess two sets of
submandibular glands, namely, principal and accessory. The ultrastructure and histochemistry of the accessory submandibular gland was examined in three species of long-winged bats.
Methods: Specimens of Miniopterus schreibersi and M. magnator were
live-trapped in Thailand, and of M. inflatus were live-trapped in Kenya. For
electron microscopy, accessory submandibular lands were initially fixed in
triple aldehyde-DMSO, postfixed in osmium tetroxide, and embedded in
Epon-Maraglas. A portion of the glands collected in Thailand (M.
schreibersi and M. magnator) was fixed in buffered formalin and embedded in paraffin. Sections of the latter material were subjected to a battery
of histochemical tests for glycoconjugates.
Results: Although in all three species the accessory submandibular
glands have normal histological structure, the glands in two, M. schreibersi
and M. magnator, were distinguished by possessing mucous droplets of
unusual morphology. These droplets, whose identity as mucous was confirmed by histochemical tests for glygoconjugates, are delimited by manifold membranes: up to 10 in M. schreibersi and fewer, but still multiple, in
M. magnator. In both species, the entire array of surface membranes may
fold inward in the fashion of mitochondria1 cristae, forming packets of
membranes, many of which have the spurious appearance of floating free
in the droplet matrix. These multipartite limiting membranes appear to
originate simply by Golgi saccules and moderately large, flattened Golgi
vesicles repeatedly wrapping themselves around the surface of nascent
mucous droplets. During exocytosis, the outermost membrane of each mucous droplet contacts the luminal membrane, this barrier ruptures, then
the remainder of the dropletmultiple membranes and matrix-ither
into the lumen or are cast out in toto. In either case, a great deal of membrane phospholipid is added to the saliva. This salivary lipid may permit
these bats to consume insects that normally are able to repel predators with
chemical defenses that make them unpalatable. The third species that we
studied, M. inflatus, has mucous droplets of normal appearance, i.e., they
have only one limiting membrane.
Conclusions: The varying structure of mucous secretory products among
the species of Miniopterus provides important clues as to the evolution of
this genus as well as to the evolution of secretory cells in general.
0 1994 Wiley-Liss, Inc.
Key words: Accessory submandibular gland, Salivary gland, Mucous
droplets, Golgi apparatus, Secretion, Exocytosis, Bats
Early electron microscopic studies on exocrine
glands, especially the exocrine pancreas, led to the erFoneous notion, still widely held, that secretory granules are homogeneous bodies that lack obvious substructure. With the introduction of aldehyde fixatives,
it has become apparent that structureless secretory
Received February 17, 1994; accepted May 2, 1994.
Address reprint requests to Dr. Carleton J. Phillips, Department of
Biological Sciences, Felmley Hall Room 206, Illinois State University,
Normal, IL 61761-6901
granules are the exception and that the vast majority
of exocrine cell types produce secretory granules with
morphological patterning. This is nowhere more evident than in the case of mammalian salivary glands;
these organs have been examined in a variety of species and a seemingly unending spectrum of granule
morphology has been uncovered (Phillips et al., 1987,
1993; Phillips and Tandler, 1987; Tandler et al., 1990;
Tandler and Phillips, 1993a). The designs often are
characteristic for each species and, indeed, might even
be used to trace phylogenetic relationships or to distinguish between species that otherwise are morphologically similar (Phillips et al., 1987).
Secretory granules contain a mixture of proteins,
both enzymatic and nonenzymatic, and glycoconjugates, lipids, certain vitamins, and electrolytes (Tandler and Phillips, 1993a). These granule components
sort themselves out according to chemical affinities
and interactions, electrical charge, and molecular configuration to yield particular designs based on the relative electron densities of these constituents. It seems
likely that these designs represent segregation of macromolecules or groups of macromolecules, a t least to
judge from experiments in which immunocytochemical
labeling has been used (Takano et al., 1993). Regardless of its substructure, each granule is delimited by a
single membrane, presumably of Golgi origin (Jamieson and Palade, 1967).
In the course of a large-scale study of the comparative ultrastructure of bat salivary glands, we have
encountered in two species of long-winged bats of the
genus Miniopterus mucous droplets of unique morphology. These droplets are delimited by a multiplicity of
membranes, some of which extend into the droplet matrix in formations akin to mitochondria1 cristae. The
apparent uniqueness of these secretory droplets is underscored by the fact that we have not encountered
similar structures in more than 40 genera of bats examined by us (Tandler e t al., 1990) a s well as by their
non-occurrence in the salivary glands of all other species of mammals for which data are available in the
mium tetroxide in the same buffer (Millonig, 1961a).
The specimens again were washed, this time in distilled water, and soaked overnight in acid-stabilized
0.25% uranyl acetate (Tandler, 1990). Rinsing with distilled water was followed by dehydration in ethanol,
passage through propylene oxide, and embedment in
Epon-Maraglas (Tandler and Walter, 1977). Thin sections were serially stained with acidified methanolic
uranyl acetate (Tandler, 1990) and lead tartrate (Millonig, 1961b) and were examined in a Siemens 101
electron microscope. Semithin sections were stained
with toluidine blue (Bjorkman, 1962) and examined in
a Zeiss Ultraphot.
For histochemical study, a portion of each tissue
specimen was fixed in 10% formalin in 0.5 M cacodylate buffer containing 0.1 M sucrose. This mixture also
contained 1%cetylpyridinium. Tissues were embedded
in paraffin and sectioned at 6 pm. Selected histochemical methods for the demonstration of glycoconjugates
were applied to the paraffin sections. The presence of
vicinal hydroxyl groups in glycoproteins was shown by
the periodic acid-Schiff (PAS) method (Mowry, 1963).
Control slides were treated with porcine pancreatic
a-amylase before PAS staining in order to determine
glycogen concentration (Lillie and Fullmer, 1976). Sections were pretreated by acetic acid-aniline condensation before the application of the PAS reaction in a n
effort to determine whether or not reactive aldehyde
groups from the fixative contributed to PAS staining
(Pearse, 1968). The periodic acid-phenylhydrazineSchiff (PAPS) method was used to show periodate reactive sialoglycoproteins (Reid et al., 1984; Spicer,
1961). Alcian blue (AB) a t pH 2.5 was used to demonstrate acidic glycoproteins (Mowry, 1963). Simultaneous staining of both PAS-positive and alcianophilic
glycoconjugates was accomplished by a combined
alcian blue-periodic acid-Schiff (AB-PAS) method
(Mowry, 1963). Selected diamine methods also were
used in a n attempt to further differentiate glycoprotein
types (Spicer et al., 1967). High iron diamine staining
preceded by periodic acid oxidation (HID-PA) was used
a s a general neutral glycoprotein stain, and periodic
Electron Microscopy
acid-p-diamine (PAPD) staining was used to show neuSpecimens of male Miniopterus schreibersi and M. tral glycoproteins that are thought to contain fucose,
magnator were live-trapped in Thailand. Salivary mannose, or galactose. A high iron diamine (HID)
glands were extirpated from anesthetized bats and method for sulfated glycoprotein and a combined high
fixed by immersion in triple aldehyde-DMSO (Kalt and iron diamine-alcian blue (HID-AB) method for the siTandler, 1971). Specimens of both male and female M. multaneous demonstration of sulfated and non-sulinflatus were live-trapped in Kenya; their salivary fated acidic glycoproteins also were used.
glands were immersion-fixed in a modification of KarRESULTS
novsky’s (1965) fixative that consisted of 2% formaldeLike many other bats, Miniopterus schreibersi, M.
hyde freshly titrated from paraformaldehyde, 3% glutaraldehyde, 2.5% DMSO, and traces of lithium and magnator, and M. inflatus possess two pairs of submancalcium, all in 0.4 M sodium cacodylate buffer contain- dibular glands: principal and accessory (Robin, 1881).
ing 0.08 M sucrose. After a -24 hours sojourn in the The accessory glands in this group of bats are about
initial aldehyde mixture of whatever composition, the half the size of the principal glands and lie medial,
tissues were transferred to 0.05 M cacodylate buffer slightly inferior, and deep to the latter organs. Both
with 0.1 M sucrose for storage at ambient temperatures principal and accessory glands in these species are confor about 1 week. Once refrigeration became available, structed along conventional histological lines. They are
they were placed in cold, cacodylate-buffered 3% glu- mixed glands consisting largely of mucous tubules with
taraldehyde (Phillips, 1985). Back in the United serious demilunes, although the two gland types exStates, the tissue blocks were thoroughly washed in hibit histochemical differences. At the ultrastructural
phosphate-buffered sucrose and postfixed in 2% os- level, the demilune cells of the principal glands contain
Fig. 1. Light micrograph of an epoxy-embedded accessory submandibular gland of M . schreibersi. The
arrow indicates a transversely sectioned mucous tubule with dark granules. Cells in other endpieces that
have lighter or negatively stained granules are serous cells. Several striated ducts are present (bottom
center and right center). Toluidine blue. Bar, 10 pm.
Fig. 2. Serous granules in a demilune cell in the accessory submandibular gland of M . schreibersi.
These bipartite granules, which are identical to those in the other two species, consist of a dense outer
rim and a somewhat lighter punctate matrix. Bar, 1 pm.
a modest number of small, uniformly dense secretory
granules and the mucous tubules are laden with structureless, electron-lucent mucous droplets.
It is the accessory submandibular glands which are
the focus of this report. They are mixed glands consisting largely of mucous tubules with serious demilunes,
although in semithin epoxy sections these components
display a n anomolous staining pattern, i.e., the mucous
cells appear to have dense granules, whereas those in
the demilunes are only faintly stained (Fig. 1). The
definitive identification of these cell types rests on a
variety of histochemical tests (vide infra), and lends
credence to Pinkstaffs (1993) contention that morphology alone is a notoriously unreliable means by which to
classify salivary secretory cells. The results of a battery
of histochemical tests for glycoconjugates are given in
Table 1.The demilune cells, which are virtually devoid
of glycoconjugates, are quintessential serous cells regardless of which criteria are employed in their identification (Munger, 1964; Young and van Lennep, 1978;
Pinkstaff, 1993; Tandler and Phillips, 1993a). In contrast, the mucous tubules contain neutral glycoproteins, sialoglycoproteins, and sulfated glycoproteins.
The positive PAS-staining reaction indicates the presence of glycoproteins with vicinal hydroxyl groups. The
decrease in PAS staining in the PAPS-stained slides
indicates that a portion of the PAS-positive material is
neutral glycoprotein that remains unstained following
phenylhydrazine pretreatment. The residual PAS-positive material after such pretreatment probably is sialoglycoprotein (Reid et al., 1984). The moderate
HID-PA reaction, a s well as the moderate PAPD staining, shows the presence of neutral glycoprotein. Sulfated glycoproteins are revealed by the HID reaction.
Viewed in the electron microscope, the demilune
cells of the accessory submandibular gland appear to be
identical in all three species of Miniopterus. Their serious granules have a bipartite appearance-a moderately dense central matrix is surrounded by a denser
halo. A line of dense particles often is discernible a t the
junction of the two granule constituents (Fig. 2).
The mucous cells of the accessory glands are quite
similar in two of the species of Miniopterus, i.e., M .
schreibersi and M . magnator, but are different enough
so that the two can be distinguished from one another
on the basis of small variations in substructure of
secretory products. To deal with M . schreibersi first,
the mucous cells are pyramidal, bordering on a central
lumen and participating in the formation of intercellular canaliculi along their lateral borders. Hypolemmal
nerve terminals are abundant between adjacent cells.
Mucous droplets of unusual morphology fill the supranuclear cytoplasm (Fig. 3). They are quite variable in
size, measuring up to 2 km in diameter. Instead of
being delimited by a single membrane, each droplet is
bounded by a series of wavy membranes, up to 10 in
TABLE 1. Glycoconjugate histochemistry of the
accessory submandibular gland of
Miniooterus schreibersi'
AB, pH 2.5
Mucous tubules
Strongly PAS +
Moderately PAS +
Moderately AB +
Strongly PAS +;
Some AB + droplets
Weakly HID +
Moderately HID +
Moderately HID + ;
Some AB + droplets
Moderatelv PAPD +
'PAS = periodic acid-Schiff; PAPS = periodic acid-phenylhydrazineSchiff; AB = alcian blue; HID = high iron diamine; HID-PA = high
iron diamine-periodic acid; HID-AB = high iron diamine-alcian blue;
PAPD = periodic acid-p-diamine.
number (Fig. 4). At some points along the droplet perimeter, the membranes are out of phase and make
contact with one another, whereas a t other points
where they are divergent, the space between adjacent
membranes is filled by material of low density. In
many of the droplets, the innermost limiting membrane is of marginally higher density than are its kindred membranes. The interior of the droplets consists
of a moderately dense matrix in which are randomly
disposed packets of membranes having the same configuration as the peripheral membranes. The outermost membrane of each of these internal packets is
slightly denser than are the inner ones. Fortuitous sections show that these packets of membranes represent
infoldings of the peripheral array of membranes, much
in the same fashion as cristae in mitochondria (Fig. 5).
Thus, the innermost peripheral membrane, which is of
higher density, is continuous with the outer membranes of the matrical packets, which membranes also
are dense.
The mucous cells of the accessory submandibular
gland in M . magnator have the same architecture as do
those in M . schreibersi, but their secretory droplets
generally are smaller and have fewer (though still multiple) peripheral and interior membranes. However,
scattered among the smaller droplets, which measure
about 0.5 bm in diameter, are some relatively large
ones that measure more than 2.5 pm. These large droplets have abundant interior packets of membranes; the
membranes in these formations alternate in fairly regular fashion with lucent spaces (Fig. 6). In some cells,
the smaller droplets contain amorphous densities.
When cut in precisely the correct plane, these densities
are seen to be square plates (Fig. 7) with a paracrystalline substructure. Such crystalloids consist of parallel linear densities with a periodicity of -10 nm.
The mode of formation of multimembraned secretory
droplets appears to be identical in M . schreibersi and
M . magnator. The earliest signs of incipient granules
are seen in relation to the Golgi apparatus (Fig. 8 ) ,
which is in the usual supranuclear position. Condensing vacuoles often are nestled in the theca of the
scyphate Golgi saccules; the vacuoles are characterized
by a n unusually dense membrane and a sparse fibril-
logranular matrix (Fig. 9). As the droplets mature,
they enlarge, become somewhat denser, and migrate to
the edge of the stacked Golgi saccules. At this point,
they begin to acquire supernumerary membranes. The
precise origin of these membranes is not entirely clear,
but it appears as though they are added to the external
surface of the prospective mucous droplets by the simple expedient of Golgi saccules wrapping themselves
around each droplet (Fig. 10). These excess membranes
are further supplemented by fusion with membranebound vacuoles, presumably also of Golgi origin (Fig.
11). As the droplets mature, the surface membranes
make incursions into the granule interior to form the
membranous packets.
The manner in which mucous droplets with extra
membranes are discharged from the secretory cells
readily can be reconstructed. Mature droplets in both
M . schreibersi and M . magnator move to the luminal
border of the cell and the outermost membrane of such
droplets fuses with the plasmalemma (Figs. 12 and 13).
At a slightly later stage, these fused membranes have
disappeared, and the droplets, still enshrouded by multiple membranes, sit in omega-shaped invaginations of
the cell surface that are lined by what originally was
the most external membrane of the droplets (Fig. 14);
the contents of the mucous droplet are still separated
from the lumen by the membranous array (Fig. 15). At
this point, the droplets with their mantle of membranes either are cast out in toto (Fig. 16) or these
membranes rupture, permitting the droplet contents to
ooze into the lumen (Fig. 171, leaving fragments of
membrane in the now vacated invaginations (Fig. 18).
The membrane lining a n individual invagination takes
on the properties of the plasmalemma and a second
mucous droplet fuses with it, the entire process repeating itself in a kind of chain exocytosis (Fig. 19).
Histologically, the accessory submandibular glands
of M . inflatus match those of the other two species.
Ultrastructurally, however, the mucous droplets of M .
inflatus differ from those in M . schreibersi and M . rnagnator. The former have a mundane structure, with a
moderately dense, homogeneous matrix, and are delimited by a single membrane (Fig. 20). At no point in
their intracellular sojourn do these droplets acquire supernumerary membranes.
The most striking feature of the accessory submandibular salivary glands of Miniopterus schreibersi and
M . magnator is their content of mucous droplets with
multiple surface membranes. Although secretory products (whether mucous [Tandler, 19931 or serous [Tandler and Phillips, 1993a]), especially those in salivary
glands, display a large number of internal configurations resulting from the varied disposition and shape of
electron-dense and electron-lucent components, none
have ever been described in which the limiting membrane is anything but single.
The two most interesting questions relating to the
unusual mucous droplets in Miniopterus concern their
mode of formation and the manner in which their contents are liberated from the tubule cells. It is well established that the Golgi apparatus packages protein for
export. This process is carried out by accumulation of
secretory products in dilatations of the lateral borders
Fig. 3. Survey electron micrograph of a mucous cell in M . schreibersi. The apical cytoplasm contains
many mucous droplets in various stages of investment by membranes. Although superficially the fully
mature droplets resemble mitochondria, the latter organelles a r e easily identifiable by their smaller size,
elliptical shape, and high density (M = mitochondria). A hypolemmal nerve terminal with a varicosity
is indicated by the arrow. Bar, 1 pm.
of the Golgi saccules, followed by the separation of
these terminal dilatations to form condensing vacuoles
(Griffiths and Simons, 1986). Gradual accretion of additional secretory product coupled with loss of water
leads to concentration and densification of the vacuole
content so that the condensing vacuole takes on the
appearance and characteristics of a mature storage
granule. The limiting membrane of the secretory granules, thus, is a derivative of the Golgi membranes.
In Miniopterus, the condensing vacuoles have a
rather dense membrane, which retains its density
throughout the maturation process. For this reason, it
Fig. 4. A mature mucous droplet in M . schreibersi that is delimited
by multiple membranes. In addition, several packets of membranes
are present in the droplet matrix. Bar, 1 ym.
Fig. 5. A mucous droplet in M . schreibersi. I n this fortuitous section,
the multiple limiting membranes are seen to make an incursion into
the droplet matrix in much the same fashion as the inner mitochondrial membrane forms cristae. Sections that miss the connection of
the membrane packets with the exterior bounding membranes give
the spurious impression of sheaves of membranes floating free in the
droplet matrix. Bar, 1 pm.
is easy to determine that additional membranes, which
have normal density, are applied to the external surface of the maturing mucous droplet. Based on numer-
Fig. 6. A very large mucous droplet in M . magnator. In this species,
the number of limiting membranes, although multiple, is not so great
as in M . schreibersi, but there are more internal membrane formations. Bar, 1 pm.
Fig. 7. A mucous droplet in M . magnator that contains a square
crystalloid. Because of the angle of tilt of the crystalloid with respect
to the plane of section, the periodicity of the crystalloid is not discernible. Bar, 1 ym.
ous electron microscope images, we believe that the
bulk of the supernumerary membranes are added simply by trans Golgi saccules wrapping themselves
Fig. 8. The Golgi region of a mucous cell in M . schrezbersi showing
a series of prospective mucous droplets of varying degrees of maturity.
Only the droplet a t the right center has begun to acquire extra membranes. Bar, 1 pm.
Fig. 9. An incipient mucous droplet in M . mugnator. Note the relatively high density of its limiting membrane compared to the surrounding Golgi membranes. Bar, 1 pm.
Ftg. 10. An almost mature mucous droplet in M . schreibersi. A Golgi
saccule in the process of being added to the surface array of membranes on the droplet is indicated by the arrow. Bar, 1 pm.
Fig. 11. Two mucous droplets in M . schreibersz on which presumed
dilated Golgi saccules (flattened vacuoles?) apparently are being
added to the surface. Bar, 1 Fm.
Figs. 12-1 9. Reconstruction of the presumed sequence of exocytotic
events in Miniopterus. Figs. 12, 13, 16-19 from M . magnator. Bar, 1
pm. Figs. 14, 15 from M . schreibersi.
Fig. 12. A droplet has approached the luminal plasmalemma
Fig. 13. A droplet has fused with the luminal plasmalemma, which
has formed a blister.
Fig. 14. The luminal plasmalemma has ruptured, and possibly has
been avulsed. Bar, 1 pm.
Fig. 15. A higher magnification of the preceding illustration. The
gap in the luminal plasma membrane is evident. The contents of the
mucous droplet, however, still are separated from the lumen by several membranes. Bar, 0.1 IJ-m.
Fig. 16. The lumen of a mucous tubule contains several intact mucous droplets, each of which is delimited by a membrane.
Fig. 17. A mucous droplet whose limiting membranes have ruptured, permitting its contents to flow into the lumen.
Fig. 18. An omega-shaped invagination of the luminal surface representing a mucous droplet t h a t has completed exocytosis. The invagination still retains the membranous remnants of the droplet.
Fig. 19. A mucous droplet has fused with a n exocytotic invagination
in a type of chain exocytosis.
B. T A N D L E R E T AL.
Fig. 20. A mucous droplet in M . inflatus. Unlike the droplets in the
other two species, this one has a prosaic appearance, with only a
single surface membrane and no internal packets of membranes. Bar,
1 pm.
around the droplet. Trans Golgi saccules are readily
identifiable by cytochemical staining for thiamine pyrophosphatase (Hand, 1971) and positive staining of
the surface membranes of Miniopterus mucous droplets
for this enzyme would provide hard evidence that these
membranes indeed are of Golgi origin. Freeze-fracture
studies (Orci et al., 1981) of exocrine cells have shown
that from the point of formation of secretory granules
a t the trans face of the Golgi apparatus, the granule
membranes lose morphologically detectable protein
and gain morphologically detectable cholesterol. It
would be of interest to determine if the multiple membranes of Miniopterus undergo similar changes. Cytochemical and freeze-fracture studies are planned as
soon as additional specimens of these exotic bats become available. Finally, the mechanisms by which
multiple membranes are added to forming secretory
granules calls attention to the still unsettled problem
of intracellular trafficking from the Golgi apparatus to
other cytological components, including secretory products and the plasma membrane (Rothman and Orci,
The morphological mechanics of granule exocytosis
have been known since the original observation of this
phenomenon by Palade (1959). Secretory granules fuse
with the cell membrane to produce a five-layered
barrier (formed by fusion of their respective unit
membranes) between the granule contents and the cell
exterior. This five-layered barrier is reduced to a threelayered one by a mechanism that still is controversial,
the three-layered membrane is disrupted, and the
granule contents flow out of the cell, frequently into a
lumen; the former limiting membrane of the granule is
retained and incorporated into the cell surface, from
which it subsequently is retrieved by endocytosis (Am-
sterdam et al., 1969). Tandler and Poulsen (1976) presented evidence that during exocytosis of mucous droplets in the cat submandibular gland the conversion of
the five-layered barrier to a single membrane is accomplished by avulsion of the covering plasmalemma, a
process that should lead to the presence of phospholipids in the saliva. Biochemical analysis of human and
marmoset parotid and submandibular saliva has revealed that these fluids contain substantial quantities
of phospholipid (Slomiany et al., 1985). Tanaka e t al.
(19801, however, failed to find increased levels of phospholipid in rat parotid saliva after stimulation of protein discharge and concluded that the apparent loss of
plasma membrane during exocytosis is a fixation artefact. On the other hand, Specian and Neutra (1980)
have shown by morphological and stereological means
that, during stimulated secretion, intestinal goblet
cells shed apical membranes into the crypt lumen. In a t
least two species of Miniopterus, it is clear that, despite
the fact that the outermost membrane of each mucous
droplet remains behind as part of the plasmalemma, a
significant amount of membrane (and therefore of
phospholipid) is liberated into the saliva.
Taken out of context, the unique mucous droplets in
the accessory submandibular glands of M . schreibersi
and M . magnator may seem like novelties. However,
comparative ultrastructural data and other information about the biology of microchiropteran bats enable
us to consider the possible significance of the findings
reported here. In terms of mucous cells, there is the
question of the extent to which the secretory process is
conserved in nature. Insofar as bats are concerned,
there is the question of whether significant amounts of
salivary phospholipid might have a biological role in
the mouth or digestive tract either in combination with
salivary enzymes, or by itself.
Fundamentally, the secretory process appears to be
widely conserved; in fact, regulated eukaryotic secretory cells, ranging from yeast to many in mammals,
appear to share many basic molecular mechanisms
(Rothman and Orci, 1992). By the same token, there
can be considerable variation among secretory products exported by homologous salivary gland secretory
cells in different species (Ball, 1993). We previously
have shown that salivary gland acinar and duct cells
also can be highly divergent in terms of basic cytoarchitecture and mitochondria1 morphology and even can
exhibit alternative secretory pathways (Nagato et al.,
1984; Tandler e t al., 1988; Tandler and Phillips,
1993a,b). These various types of interspecific variation
in homologous cells take on significance when analyzed
on a comparative basis. For instance, if a phylogenetic
framework is used a s the basis for making comparisons, patterns of divergence in secretory cells emerge
and it is possible to correlate these patterns with speciation patterns that reflect adaptive radiation in diet
(Phillips et al., 1993). Perhaps the most striking examples have come from lineages of bats in which the diet
changed from carnivorylinsectivory to frugivory (Phillips et al., 1993).
In such a context, the accessory gland mucous cells in
the two species of Miniopterus represent a departure
from any previously described salivary gland secretory
cell. To fully appreciate the extent of this departure,
one need only to consider the fact that the mucous cells
in these two species differ from those in more than 200 thank Dr. Duane A. Schlitter for his cooperation and
species of bats whose salivary glands we have exam- assistance in the fieldwork phase of this study.
ined by electron microscopy (Tandler e t al., 1990). It
also is noteworthy that the three species of Miniopterus
differ from one another with respect to the structure of Amsterdam, A., I. Ohad, and M. Schramm 1969 Dynamic changes in
secretory products; usually such differences are found
the ultrastructure of the acinar cell of the rat parotid gland during the secretory cycle. J. Cell Biol., 41:753-773.
between genera or even families of bats rather than
Ball, W.D. 1993 Cell restricted secretory proteins a s markers of celamong species belonging to the same genus.
lular phenotype in salivary glands. In: Biology of the Salivary
The secretory apparatus in the accessory gland muGlands. K. Dobrosielski-Vergona, ed. CRC Press, Boca Raton, pp.
cous cells can be described as conservative in the Afri355-396.
N. 1962 Low magnification electron microscopy in histocan species, M . inflatus, and as derived in both Asian
logical work. Acta Morphol. Neerl. Scand., 4.344-348.
species, M. schreibersi and M. magnator. It is likely Griffiths,
G., and K. Simons 1986 The trans Golgi network: Sorting a t
that evolutionary modification in the secretory process
the exit site of the Golgi complex. Science, 234:438-443.
occurred during speciation and i t could be argued that Hand, A.R. 1971 Morphology and cytochemistry of the Golgi apparatus of rat salivary gland acinar cells. Am. J. Anat., I30:141-148.
the data imply a close relationship between the latter
J.D., and G.E. Palade 1967 Intracellular transport of secretwo species, with M. schreibersi being the most derived. Jamieson,
tory proteins in the pancreatic exocrine cell. 11. Transport to conComparative data for the other eight species in this
densing vacuoles and zymogen granules. J. Cell Biol., 34.597genus conceivably could reveal the entire evolutionary
Jones, C.G., D.W. Whitman, P.J. Silk, and M.S. Blum 1988 Diet
pathway that produced the modified mucous cells.
breadth and insect chemical defenses: A generalist grasshopper
The biological role(s) of phospholipid-rich saliva is
and general hypothesis. In: Chemical Mediation of Coevolution.
unknown, but information from other mammals and
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particularly from other species of bats can be used to Kalt, M.R., and B. Tandler 1971 A study of fixation of early amphibian embryos for electron microscopy. J. Ultrastruct. Res., 36t633construct hypotheses. Our working hypothesis is that
salivary phospholipids enable M . schreibersi and M . Karnovsky,
M.J. 1965 A formaldehyde-glutaraldehyde fixative of
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