Mucous droplets with multiple membranes in the accessory submandibular glands of long-winged bats.код для вставкиСкачать
THE ANATOMICAL RECORD 240:178-188 (1994) Mucous Droplets With Multiple Membranes in the Accessory Submandibular Glands of Long-Winged Bats BERNARD TANDLER, CARLETON J. PHILLIPS, AND CARLIN A. PINKSTAFF 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.) ABSTRACT 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 flow 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 c 1994 WILEY LISS, INC 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 MUCOUS DROPLETS WITH MULTIPLE MEMBRANES IN BATS 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 literature. 179 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. Histochemistry 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 MATERIALS AND METHODS 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 180 B. TANDLER ET AL. 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 MUCOUS DROPLETS WITH MUL,TIPLE MEMBRANES I N BATS TABLE 1. Glycoconjugate histochemistry of the accessory submandibular gland of Miniooterus schreibersi' Stain PAS PAPS AB, pH 2.5 AB-PAS HID HID-PA HID-AB PAPD Mucous tubules Strongly PAS + Moderately PAS + Moderately AB + Strongly PAS +; Some AB + droplets Weakly HID + Moderately HID + Moderately HID + ; Some AB + droplets Moderatelv PAPD + Serious demilunes +(?) - '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- 181 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. DISCUSSION 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 182 B. TANDLER ET AL. 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 MUCOUS DROPLETS WITH M U L T I P L E MEMBRANES I N BATS 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- 183 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 184 B. TANDLER E T AL. 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. MUCOUS DROPLETS WITH MULTIPLE MEMBRANES IN BATS 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. 185 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. 186 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, 1992). 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 MUCOUS DROPLETS WITH MULTIPLE MEMBRANES IN BATS 187 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 LITERATURE CITED 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. Bjorkman, 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 615. 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 K. Spencer, ed. Academic Press, San Diego, pp. 213-242. 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 645. salivary phospholipids enable M . schreibersi and M . Karnovsky, M.J. 1965 A formaldehyde-glutaraldehyde fixative of magnator to eat species of insects that otherwise would high osmolality for use in electron microscopy. J. Cell Biol., 27: be unpalatable. All species of Miniopterus are “insec137a-138a (abstract). tivorous,” but the details of their diets are unknown. In Kingdon, J . 1974 East African Mammals. An Atlas of Evolution in Africa. Vol. 11, Part A (Insectiuores and Bats). University of Chigeneral terms, they are thought to eat high-flying, cago Press, Chicago, pp. 307-311. small-sized, hard-bodied beetles, a s well as soft-bodied Lillie, R.D., and H.M. Fullmer 1976 Histopathologic Technic and insects (Kingdon, 1974; Nowack, 1991). Although hunPractical Histochemistry, 4th Edition. McGraw Hill, New York, pp. 629-630. dreds of species of bats are insectivorous, this does not G. 1961a Advantages of a phosphate buffer for OsO, solumean that it necessarily is a simple matter for them to Millonig, tions in fixation. J. Appl. Physics, 32.1636 (abstract). use insects as a nutrient resource. Indeed, many flying Millonig, G. 1961b A modified procedure for lead staining of thin insects have chemical defenses against predation: desections. J. Biophys. Biochem. Cytol., 22.736-739. fenses can consist of a mixture of phenols, quinones, Mowry, R.W. 1963 The special value of methods that color both acidic and vicinal hydroxyl groups in the histochemical study of mucins. and sequestered plant allochemicals (Jones et al., With revised directions for the colloidal iron stain, the use of 1988).Phospholipid-rich saliva might counteract insect alcian blue G8X and their combinations with the periodic acidchemical defenses by selectively blocking taste bud Schiff reaction. Ann. N. Y. Acad. Sci., 106t402-423. transduction or by producing a physical barrier to pro- Munger, B.L. 1964 Histochemical studies on seromucous and mucoussecreting cells of human salivary glands. Am. J. Anat., 125.411tect the bats’ digestive tract, or both. If this is so, bat 429. species in which phospholipids are added to the saliva Nagato, T., B. Tandler, and C.J. Phillips 1984 Unusual smooth endocould well have a n advantage by being able to feed on plasmic reticulum in submandibular acinar cells of the male round-eared bat, Tonatia syluicola. J. Ultrastruct. Res., 87.275insects that cannot be used as food by bat species that 284. lack salivary phospholipids. R.M. 1991 Walker’s Mammals of the World. Vol. I, 5th EdiSupport for the hypothesis that salivary lipids might Nowak, tion. The Johns Hopkins University Press, Baltimore, pp. 366serve a protective role in the digestive tract comes from 368. observations on several species of frog-eating bats in Orci, L., M. Amherdt, R. Montesano, P. Vassalli, and P. Perrelet 1981 Topology of morphologically detectable protein and cholesterol in the genera Trachops and Megaderma. Anurans can be membranes of polypeptide-secreting cells. Philos. Trans. R. SOC. rebarbative prey because many species have integuLond. [Biol.], 296.47-54. mentary alkaloids and peptides that ordinarily should Palade, G.E. 1959 Functional changes in the structure of cell comporender them unpalatable. Certain species of tropical nents. In: Subcellular Particles. T. Hayashi, ed. Ronald Press, New York, pp. 64-83. bats that eat frogs exhibit highly modified follicular A.G.E. 1968 Histochemistry. Theoretical and Applied, Vol. 1, accessory submandibular glands (Phillips et al., 1987; Pearse, 3rd Edition. Little, Brown and Co., Boston, p. 707. Phillips and Tandler, 1994). The secretory cells in Phillips, C.J. 1985 Field fixation and storage of museum tissue colthese glands, at least those in Trachops cirrhosus lections suitable for electron microscopy. Acta Zool. Fennica. 170: 87-90. (Phillips and Tandler, 1985), add lipid to the saliva. Thus, salivary lipids appear to be correlated with the Phillips, C.J., and B. Tandler 1985 Unique salivary gland structure in two genera of tropical bats. Anat. Rec., 221:120A (abstract). ability to ingest animals that have chemical defenses Phillips, C.J., and B. Tandler 1987 Mammalian evolution a t the celagainst predation. lular level. Curr. Mammal., 2.1-66. ACKNOWLEDGMENTS This work was supported in part by NIDR grant DE 07648 (B.T., C.J.P.) and by funds from the West Virginia University Dental Corporation (C.A.P.).Thomas J. Slabe provided expert technical assistance. We Phillips, C.J., and B. Tandler 1994 Follicular architecture of the accessory submandibular gland in the African false vampire bat, Cardioderma cor. J. Mammal (In press). Phillips, C.J., T. Nagato, and B. Tandler 1987 Comparative ultrastructure and evolutionary patterns of acinar secretory product of parotid salivary glands in Neotropical bats. In: Studies in Neotropical Mammalogy: Essays in Honor of Philip Hershkovitz. 188 B. TANDLER ET AL B.D. Patterson and R.M. Timm, eds. Field Museum of Natural History, Chicago, pp. 213-229. Phillips, C.J., B. Tandler, and C.A. Pinkstaff 1987 Unique salivary glands in two genera of tropical microchiropteran bats: An example of evolutionary convergence in histology and histochemistry. J . Mammal., 68t235-242. Phillips, C.J., B. Tandler, and T. Nagato 1993 Evolutionary diversity of salivary gland acinar cells: a format for understanding molecular evolution. In: The Biology of the Salivary Glands. K. Dobrosielski-Vergona, ed. CRC Press, Boca Raton, pp. 41-80. Pinkstaff, C.A. 1993 Serous, seromucous and special serous cells in salivary glands. Microsc. Res. Tech., 26r21-31. Reid, P.E., W.L. Dunn, C.W. Ramey, E. Coret, L. Trueman, and M.G. Clay 1984 Histochemical studies of the mechanism of the periodic acid phenylhydrazine-Schiff (PAPS)procedure. Histochem. J . , 16; 641-649. Robin, H. 1881 Recherches anatomiques les mammiferes de 1’Ordre des Chiropteres. Ann. Sci. Nat. Zool., I2:1-180. Rothman, J.E., and L. Orci 1992 Molecular dissection of the secretory pathway. Nature, 355r409-415. Slomiany, B.L., V.L.N. Murty, and A. Slomiany 1985 Salivary lipids in health and disease. Prog. Lipid Res., 24r311-324. Specian, R.D., and M.R. Neutra 1980 Mechanism of rapid mucus secretion in globlet cells stimulated by acetylcholine. J . Cell Biol., 85t626-640. Spicer, S.S. 1961 The use of various cationic reagents in the histochemical differentiation of mucopolysaccharides. Am. J. Clin. Pathol., 36r393-407. Spicer, S.S., R.G. Horn, and T.J. Leppi 1967 Histochemistry of connective tissue mucopolysaccharides. In: The Connective Tissue. B.M. Wagner and D.E. Smith, eds. Williams and Wilkins, Baltimore, pp. 251-303. Takano, K., D. Malamud, A. Bennick, F. Oppenheim, and A.R. Hand 1993 Localization of salivary proteins in granules of human pa- rotid and submandibular gland. C.R.C. Rev. Oral Biol. Med., 4r399-405. Tanaka, Y., P. De Camilli, and J. Meldolesi 1980 Membrane interactions between secretion granules and plasmalemma in three exocrine glands. J. Cell Biol., 84:438-453. Tandler, B. 1990 Improved uranyl acetate staining for electron microscopy. J. Electron Microsc. Tech., 16:81-82. Tandler, B. 1993 Structure of mucous cells in salivary glands. Microsc. Res. Tech., 26:49-56. Tandler, B., and C.J. Phillips 1993a Structure of serous cells in salivary glands. Microscopy Res. Tech., 26r32-48. Tandler, B., and C.J. Phillips 1993b Giant mitochondria in the seromucous demilunar cells of the accessory submandibular gland of the long-haired fruit bat, Stenonycterus lanosus. Anat. Rec., 237; 157-162. Tandler, B., C.J. Phillips, and T. Nagato 1988 Parotid salivary gland ultrastructure in an omnivorous neotropical b a t Evolutionary diversity a t the cellular level. Zool. Scripta, 17r419-427. Tandler, B., C.J. Phillips, T. Nagato, and K. Toyoshima 1990 Comparative ultrastructure of chiropteran salivary glands. In Ultrastructure of the Extraparietal Glands of the Alimentary Tract. A. Riva and P. Motta, eds. Kluwer Academic Publishers, Boston, pp. 31-52. Tandler, B., and J.H. Poulsen 1976 Fusion of the envelope of mucous droplets with the luminal plasma membrane in acinar cells of the cat submandibular gland. J. Cell Biol., 68r775-781. Tandler, B., and R.J. Walter 1977 Epon-Maraglas embedment for electron microscopy. Stain Technol., 52t238-239. Venable, J.H., and R. Coggeshall 1965 A simplified lead citrate stain for use in electron microscopy. J. Cell Biol., 25 (No. 2, Pt. 11t407408. Young, J.A., and E.W. van Lennep 1978 The Morphology of Salivary Glands. Academic Press, London.