THE AMERICAN JOURNAL OF ANATOMY 189:303-315 (1990)

Ultrastructure of the Principal and Accessory Submandibular Glands of the Common Vampire Bat BERNARD TANDLER, KUNIAKI TOYOSHIMA, AND CARLETON J. PHILLIPS Department of Oral Bcology (B.T., K.T.),School of Dentistry, Case Western Reserve University, Clevelnnd, Ohio 44106; and Department of Biology (C.J.P.I, Hofstra Uniuersity, Hempstead, New York 11550

ABSTRACT The principal and accessory submandibular glands of the common vampire bat, Desmodus rotundus, were examined by electron microscopy. The secretory endpieces of the principal gland consist of serous tubules capped at their blind ends by mucous acini. The substructure of the mucous droplets and of the serous granules varies according to the mode of speclmen preparation. With ferrocyanide-reduced osmium postfixation, the mucous droplets are moderately dense and homogeneous; the serous granules often have a polygonal outline and their matrix shows clefts in which bundles of wavy filaments may be present. With conventional osmium postfixation, the mucous droplets have a finely fibrillogranular matrix; the serous granules are homogeneously dense. Mucous cells additionally contain many small, dense granules that may be small peroxisomes, as well as aggregates of 10nm cytofilaments. Intercalated duct cells are relatively unspecialized. Striated ducts are characterized by highly folded basal membranes and vertically oriented mitochondria. Luminal surfaces of all of the secretory and duct cells have numerous microvilli, culminating in a brush borderlike affair in the striated ducts. The accessory gland has secretory endpieces consisting of mucous acini with small mucous demilunes. The acinar mucous droplets contain a large dense region; the lucent portion has punctate densities. Demilune mucous droplets lack a dense region and consist of a light matrix in which fine fibrillogranular material is suspended. A ring of junctional cells, identifiable by their complex secretory granules, separates the mucous acini from the intercalated ducts. The intercalated ducts lack specialized structure. Striated ducts resemble their counterparts in the principal gland. As in the principal gland, all luminal surfaces are covered by an array of microvilli. At least some of the features of the principal and accessory submandibular glands of the vampire bat may be structural adaptations to the exigencies posed by the exclusively sanguivorous diet of these animals and its attendant extremely high intake of sodium chloride. 0 1990 WILEY-LISS, INC.

INTRODUCTION

Because of their diverse feeding habits, Neotropical bats of the family Phyllostomidae have attracted considerable attention from biologists. Indeed, no other mammalian family matches the extent of ecomorphological radiation exhibited by the 130 phyllostomid species. The diverse diets of these bats are mirrored by the variability in the gross and microscopic anatomy of their digestive tract, as well as in their dentition and sensory organs (Phillips, 1971; Phillips et al., 1977, 1984; Phillips and Tandler, 1987; Findley and Wilson, 1982; Studholme et al., 1986, 1987). In particular, the salivary glands of phyllostomid bats vary to a n extraordinary degree a t the histological, histochemical, and ultrastructural levels (Phillips and Tandler, 1987; Tandler et al., 1990). It seems likely t h a t this structural heterogeneity is to some extent correlated with the great variation in phyllostomid diets. Frog-eating phyllostomid bats, for example, exhibit unique histological architecture in their accessory submandibular glands (Phillips et al., 1987). By virtue of its sanguivorous diet, the common vampire bat, Desmodus rotundus, is one of the most dramatic of the phyllostomids. These bats are indelibly stamped in popular imagination as sinister creatures of the night, the stuff of superstition and legend. Despite their “bad press,” vampire bats are of great interest to morphologists and physiologists alike because of their obligate sanguivory (Greenhall and Schmidt, 1988). The ingestion of a diet that consists wholly of blood poses numerous problems in homeostasis for these animals and can be expected to be related in a number of ways to salivary gland structure. For this reason, we have examined the principal and accessory submandibular glands of the common vampire bat by means of electron microscopy. MATERIALS AND METHODS

Adult common vampire bats (Desrnodus rotundus murinus) were obtained from a n established colony kept in the Department of Zoology, Cornell University, Ithaca, New York. The animals had been maintained

Received February 23, 1990. Accepted June 8, 1990. Dr. Toyoshima is now at the Department o f Oral Anatomy 11, Kyushu Dental College, 2-6-1, Manazuru, Kokurakita-ku, Kitakyushu 803, Japan. Address reprint requests t o Dr. Bernard Tandler, School of Dentistry, Case Western Reserve University, Cleveland, OH 44106.

1 3 ~TANDLEIZ E T AL.

Fig. 1. Photomicrograph of principal submandibular gland showing serous tubules with densely stained granules and lightly stained mucous endpieces. Toluidine blue. x 545. Fig. 2. Photomicrograph of a transversely sectioned striated duct in the principal submandibular gland. There is a brush borderlike structure surrounding the lumen. Toluidine blue. x 1,050.

Fig. 3. Electron micrograph of a secretory endpiece in the principal submandibular gland. Mucous cells occupy the left half of the micrograph. Mucous droplets are relatively large and moderately dense. Small dense bodies, perhaps microperoxisomes, are abundant between the mucous droplets. Cells in the serous tubule (right half of micrograph) contain fairly dense secretory granules. Microvilli are so numerous that the lumen appears to be occluded (this lumen is seen at higher magnification in Fig. 6). Reduced osmium postfixation. x 6,500.

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crovillus-lined intercellular canaliculi that extend close to the cell base. The serous cells have a central, spherical nucleus. The cytoplasm below and lateral to the nucleus is characterized by abundant granular endoplasmic reticulum (GER) and mitochondria. In some cells, the GER cisternae contain scattered intracisternal granules. The supranuclear cytoplasm contains a cluster of serous granules whose appearance, like that of the mucous droplets, is influenced by the manner in which the tissue has been postfixed. When conventional osmium mixtures are used, the granules are homogenously dense spheres (Figs. 3, 6); after exposure to ferrocyanide-reduced osmium, they often have a polygonal outline. Moreover, Iucent spaces or clefts appear in the granule matrix. These spaces are straight, often subtending the granule membrane, but occasionally intersecting to produce triangular patterns. The lucent spaces contain bundles of irregular, wavy filaments (Fig. 5); the finest of these filaments are 5 nm thick, whereas the thickest ones measure about 10 nm. The serous cells contain a number of lysosomelike bodies that usually include one or two lipid droplets. Another cytoplasmic structure, possibly a second variant of lysosomes, may be present in the serous cells. These are RESULTS oddly shaped and contain numerous dense laminae Principal Submandibular Gland (Fig. 7). The secretory endpieces consist of fairly long, An unusual feature of the serous tubules is that conbranched serous tubules that are capped a t their prox- stituent cells bear a great number of microvilli on their imal ends by relatively large mucous acini and that are luminal surface and on the cell surfaces that form the continuous at their distal ends with intercalated ducts intercellular canaliculi. So abundant are these struc(Fig. 1).In semithin epoxy sections stained with tolu- tures that the tubule lumina appear to be completely idine blue, the mucous cells are seen to be filled with occluded (Fig. 6). The individual microvilli are more large mucous droplets that have a light-blue colora- than 1 p,m in length and are approximately 80 nm in tion. Numerous tiny, densely stained granules are width. The cytology of the intercalated duct cells varies acstrewn among the mucous droplets. The simple cuboidal epithelial cells of the serous tubules are composed cording to their location. Those near the junction with of cells that contain darkly stained secretory granules, the serous tubules have many GER cisternae and some which are confined to the apical cytoplasm. The short serous-type granules, whereas the most distal duct intercalated ducts consist of simple low cuboidal epi- cells have only a few cisternae, but many free polythelium whose constituent cells contain an occasional somes, and are devoid of secretory granules (Fig. 8). small secretory granule. Striated ducts are composed of Unlike the granules in the serous tubules, the granules pseudostratified epithelium with sparse basal cells. in the intercalated ducts do not display a dichotomous The tall cells of these ducts bear a prominent brush response to osmium postfixation, remaining homogeborder on their luminal surface (Fig. 2). Excretory neously dense whatever osmium mixture is applied. Many microvilli are present on the free surfaces of the ducts closely resemble the striated ducts. The ultrastructural appearance of the mucous cells duct cells. depends on the nature of the fixation protocol: after The base of the striated ducts displays the usual arpostfixation in ferrocyanide-reduced osmium tetroxide, rangement of mitochondria and complexly folded the mucous droplets are moderately dense and homog- plasma membranes (Fig. 9). We report here for the first enous in structure (Fig. 3), whereas postfixation in con- time the presence of a gap junction between interlocked ventional osmium results in mucous droplets with a striated duct cells (Fig. 10). Scattered duct cells contain finely fibrillogranular matrix (Fig. 4). The droplets some small, moderately dense granules in their apical measure up to 1.25 km in diameter. Within the inter- cytoplasm (Fig. 12). Most cells lack granules, but constices of the closely packed mucous droplets are small, tain abundant vesicles (Fig. 11).As in more proximal dense granules measuring about 0.2 p,m in diameter. segments of the secretory unit, the tall duct cells bear In size and electron density, these granules resemble many microvilli on their luminal surfaces (Fig. 11); small peroxisomes of the sort found in the rat salivary because the striated duct lumina are of fairly large glands by Hand (1973). A peculiar feature of the mu- caliber, the microvilli appear as a brush border. The cous cells is that they contain aggregates of 10-nm fil- abundance of microvilli can be best appreciated in aments, usually near the cell base. These intermediate transverse sections through the ductular brush border filaments generally are spirally or sigmoidally ori- (Fig. 13). The microvilli seem t o lack the well-orgaented within an aggregation, and organelles and cyto- nized bundles of actin filaments that are so obvious in plasmic inclusions may be entirely excluded from such intestinal striated borders. Coated vesicles tend to be a collection (Fig. 4). The mucous cells border on mi- associated with the apical plasmalemma between mi-

in good health by methods described previously (Wimsatt and Guerriere, 1961). The bats were anesthetized with chloroform and the various salivary glands removed. Specimens of each gland were fixed by immersion in either half-strength Karnovsky’s (1965) fixative or the triple aldehyde-DMSO fixative of Kalt and Tandler (1971);both mixtures were buffered with phosphate (pH 7.3-7.4). After 2 hr in aldehydes, the specimens were rinsed in buffered sucrose and postfixed for 2 hr in phosphate-buffered 2% osmium tetroxide (Millonig, 1961) or in ferrocyanide-reduced osmium (Hornick et al., 1984). Rinsing of the tissue in distilled water was followed by an overnight soak in aqueous 0.25% uranyl acetate. After rinsing in distilled water, the specimens were dehydrated in ascending concentrations of ethanol, passed through propylene oxide, and embedded in Epon-Maraglas (Tandler and Walter, 1977). Thin sections were stained with methanolic uranyl acetate (Stempak and Ward, 1964) and lead citrate (Venable and Coggeshall, 1965) and examined in a Siemens Elmiskop l a or 101 electron microscope. Semithin (1 km) sections were stained with toluidine blue (Bjorkman, 1962) and examined in a Zeiss Ultraphot 11.

Figs. 4-6.

SALIVARY GLANDS OF VAMPIRE EAT

crovilli. The basal cells are pyramidal in shape, contain randomly disposed bundles of cytofilaments, and have many hemidesmosomes on their basal surface. Except for their interlobular position, excretory ducts appear to be identical in morphology to striated ducts. Myoepithelial cells of conventional morphology are associated with mucous acini, serous tubules, and intercalated ducts but are absent from the remainder of the duct system. Although hypolemmal nerve terminals are extremely sparse in the mucous acini, they begin to increase in number in the serous tubules and become quite abundant in the intercalated and striated ducts (Fig. 14). These nerve terminals contain mostly small clear vesicles, but a few larger, dense-cored vesicles can also be present. Accessory Submandibular Gland

The accessory submandibular gland differs in histology from the principal gland. The secretory endpieces consist of mucous acini with small mucous demilunes (Fig. 15). The mucous acinar cells are filled with numerous mucous droplets that essentially are unstained by toluidine blue. In contrast, the demilune cells have a strongly stained cytoplasm and their mucous droplets, which are smaller than those in the neighboring acinar cells, are slightly metachromatic. A ring of metachromatic junctional cells intervenes between mucous acini and intercalated ducts. The latter are quite short but have a large lumen, so are much more prominent than in the principal submandibular gland. Striated ducts, like their counterparts in the principal gland, have a noticeable brush border. Ultrastructurally, the mucous acinar cells are seen to possess a more or less round, centrally placed nucleus (Fig. 16). GER is fairly abundant in the intranuclear cytoplasm, and the apical cytoplasm is occupied by many closely apposed mucous droplets. The latter consist of a punctate matrix with a dense, somewhat amorphous, eccentrically placed inclusion that may occupy fully half the granule volume (Fig. 17). The demilune cells (which, strictly speaking, are not demilunar, since they make extensive contact with the acinar lumen) have a basally situated, flattened nucleus. The mucous droplets have a homogeneously sabulous matrix with no dense inclusions (Fig. 18).Intercellular canaliculi with numerous microvilli course between demilunar and acinar cells. Junctional cells frequently are present at the junc-

Fig. 4.The basal region of a mucous cell in a specimen of principal submandibular gland that was subjected to conventional osmium postfixation. Mucous granules have a finely fibrillar content. There i s a large aggregate of circularly arranged 10-nm filaments; the dots in the center of the aggregate are transversely-sectioned filaments. x 27,400.

Fig. 5. A cluster of' serous granules in a specimen of' principal submandibular gland postfixed in reduced osmium. Several granules exhibit lucent clefts that contain fine filaments. x 23,700. Fig. 6 . Higher magnification of the lumen of the serous tubule seen in Figure 3. Microvilli are tightly packed together. These serous granules are homogeneously dense despite having been postfixed in reduced osmium. x 18,000.

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ture of acini and intercalated ducts. These cells have secretory granules that are smaller than those of the mucous cells. The granules consist of coarse, punctate densities that often appear to be connected by amorphous material, so that the densities seem to be layered in concentric rings (Fig. 19). Many of the granules additionally contain an aggregation of dense, finely particulate material of no particular shape or orientation. The Golgi complex in the junctional cells is quite large. Intercalated ducts consist of simple cuboidal epithelial cells of unremarkable cytology (Fig. 20). Some duct cells have a few serous-type secretory granules. Scattered microvilli are present on the luminal surface. Striated ducts in the accessory gland are nearly identical to those of the principal gland, differing only in the fact that secretory granules are absent from the duct cells and that the microvilli of their brush borders are shorter (Fig. 21). Myoepithelial cells are rather sparse in the accessory gland. Hypolemmal nerve terminals are present in the acini, but are far less common in the duct system of the accessory gland than they are in the principal gland. DISCUSSION

The exact origins and evolutionary relationships of vampire bats are uncertain. The common vampire bat, Desrnodus rotundus, and its relatives, Diphylla ecaudata and Diaemus youngi, are members of the Neotropica1 family Phyllostomidae. As a group, the phyllostomids are characterized by diversity in feeding habits, which range from insectivory to nectarivory (Hill and Smith, 1986). The salivary glands of vampire bats thus can be considered in terms of the unusual diet that these animals consume as well in comparison to glands in related species with different dietary habits. In the present investigation we sought to address several specific questions: 1) does the liquid nature of the vampire bat diet directly affect their salivary glands, 2 ) do the secretory cells have unique histological and ultrastructural features that might relate t o diet, 3) are there ultrastructural indications of the production of an enzyme that prevents or dissolves clots, and 4)what role(s) might the salivary glands play in physiological adjustments to ingestion of large quantities of sodium chloride? Vampire bats differ from all other mammals in that they subsist entirely on blood (Wimsatt and Guerriere, 1961; Greenhall, 1972; Wimsatt, 19781, which means that all of their food is in a liquid form. It has been shown that the consistency of the diet has a marked effect on the morphology and physiology of the rat parotid gland. Rats fed a liquid diet rather than pellets exhibit parotid gland atrophy (Hall and Schneyer, 1964; Sreebny and Johnson, 1968) characterized by a reduction in weight of the gland, a decreased content of RNA and amylase, and a significantly lowered rate of synthesis of secretory protein (Sreebny et al., 1971);not only is the protein concentration of the gland reduced, but several secretory proteins disappear altogether, and some of the remaining proteins show nonparallel decreases in concentration (Johnson, 1982). During early stages of the liquid diet, there is an increase in lysosomes in the acinar cells, accompanied by the presence of autophagic vacuoles and degenerating secretory granules, and even by necrotic cells (Wilborn and

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Fig. 7. An oddly shaped laminated body in a serous cell. Principal submandibular gland. x 49,700.

Fig. 9. Striated duct cells in the principal submandibular gland. Note the prominent microvilli. x 8,400.

Fig. 8. A portion of an intercalated duct some distance removed from its junction with a serous tubule. These duct cells lack secretory granules or basal membrane specializations, but microvilli are abundant. Principal submandibular gland. x 8,800.

Fig. 10. A gap junction immediately below a desmosome in the folded basal membranes in a striated duct of the principal gland. x 52,800.

SALIVARY GLANLX OF VAMPIRK BAT

Schneyer, 1970). After three weeks on a liquid diet, acinar cell structure becomes stabilized; but the cells are considerably smaller than those in control rats, and they contain a relatively sparse population of secretory granules (Hand and Ho, 1981). All of these changes are thought to be neurally mediated and to be the result of reduced mastication (Schneyer, 1974); mastication appears to be the necessary stimulus for exocytosis of rat parotid-gland secretory granules to occur (Hand, 1972). Vampire bat salivary glands exhibit none of the degenerative changes associated with a liquid diet that have been observed in rat salivary glands, leading to the conclusion that there must be a signal other than mastication for salivary flow to commence. Although the act of biting may trigger a salivary response, this probably would be of insignficant duration. Wilkinson (1984)reported that upon return to their roost, successful predators share their bounty with unfed bats in their social group by regurgitation of their blood meal. Presumably, the beneficiaries of such largess ingest this second-hand blood without the necessity of biting. A more likely stimulus for salivary secretion may come from tongue movements during the act of feeding by the vampire. The vampire bat uses its razor-sharp teeth (Phillips and Steinberg, 1976) to scoop out a shallow hole in the skin, cutting into the dermal capillary bed, from which blood oozes into the excavation (Glass, 1970). Areas having numerous superficial capillaries, such as the inside of the nostrils, ears, and teats in domestic mammals, are often selected by vampire bats (Greenhall, 1988). Motion pictures show that during feeding the vampire tongue moves in and out with great rapidity (Villa-R, 1956). Although the precise innervation of the tongue and salivary glands has not been determined in the vampire bat, it is probable that branches of the same nerves (facial and glossopalatine) extend to each of these organs and that they are connected by a reflex arc. Thus the mechanical act of tongue extension and withdrawal may have the equivalent effect in vampires that mastication has on salivary-gland function in other animals. In her light microscopical description of the major salivary glands of Desmodus rotundus, DiSanto (1960) retained the histological terminology coined by Wimsatt (1956) in his report on salivary glands in the fruiteating bat, Artibeus jamaicensis. In most of the handful of mammals whose salivary glands had been described before 1960, mixed salivary glands had mucous acini or tubules that adjoined intercalated ducts and that were capped by serous demilunes. In Artibeus and Desmodus, however, the situation appeared to be reversed, that is, serous acini adjoined the intercalated ducts and were capped by mucous acini. In order to emphasize what was then considered to be a peculiar reversal of mucous and serous cells, Wimsatt (1956)referred to the cells adjacent to the intercalated ducts as “pseudomucous)) cells and the capping cells as “pseudoserous” cells. Our ultrastructural observations of the submandibular glands of the vampire bat, in agreement with the histochemical observations of Fava-de-Moraes (19651, show that this special terminology is unnecessary and that the so-called pseudomucous cells actually are serous cells and that the pseudoserous cells are mucous cells. Moreover, our observations on the submandibular glands of five species of Artibeus show a

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similar situation in these fruit bats, namely, that serous acini are capped by large seromucous demilunes (Tandler et al., 1986). It is now abundantly clear that secretory endpieces of mammalian salivary glands are composed of either one or two types of cells, these being serous, mucous, and seromucous; in mixed glands, any two of the three cell types can form the endpieces in any spatial relationship. Moreover, in some species the acini can be formed from one cell type and be capped by histochemically similar but ultrastructurally different cells of the same type. For example, in the cat submandibular gland the endpieces consist of mucous acini capped by mucous demilunes (Shackleford and Wilborn, 1970; Harrison, 1974). This species-determined flexibility in salivarygland architecture was noted before the turn of the century-see, for example, illustrations by Kiichenmeister (1895) and by Krause (1897)-but seems to have been overlooked by many modern students of salivary gland structure. The serous granules of the principal submandibular gland appear t o have a homogenously dense matrix when postfixed in conventional osmium mixtures. When, however, the granules are postfixed with ferrocyanide-reduced osmium tetroxide, they have clear spaces that usually contain a bundle of filaments. In recent studies of rabbit salivary glands, we have noted that serous granules in the submandibular gland, irrespective of the mode of fixation, contain tangles of filaments that are released into the serous tubule lumen during exocytosis (Toyoshima and Tandler, 1986a). In von Ebner’s glands of the rabbit, well-fixed specimens contain homogeneously dense seromucous granules, but when these granules are somewhat extracted as a consequence of less-than-optimal fixation, some may contain a bundle of filaments within a clear zone (Toyoshima and Tandler, 1986b)-we have not observed such granules in the process of exocytosis. It is possible that the secretory granules of both vampire bats and rabbits contain proteins with a propensity for linear polymerization; under the appropriate conditions of fixation, filaments become evident in the granule matrix. It has long been known that bite wounds inflicted by vampire bats continue to bleed for many hours after other types of wounds have formed clots and ceased bleeding (Ditmars and Greenhall, 1935; Mann, 1951), but the mechanisrn(s1whereby the free flow of blood is maintained has not been fully settled. Hawkey (1967, 1988) has shown that an inhibitor of blood platelet aggregation is present in the mixed saliva of D.rotundus. An earlier study by DiSanto (1960) found that extracts from the vampire bat principal submandibular gland, but not from the other major salivary glands, inhibit the clotting of blood. Based on these two observations, it seems likely that the principal submandibular gland is in fact the source of the platelet-inhibiting factor. DiSanto (1960) also found that a factor present in this gland was capable of partially dissolving already formed clots; she postulated that this factor is a protease that is able to digest fibrin. The morphology of the serous tubules in the principal submandibular gland suggests that they are the site of synthesis of enzymes for export. Studies of “submandibular gland” (presumably pooled principal and accessory glands) ex-

Figs. 11-13.

SALIVAItY G1,ANDS OF VAMPIRE EAT

Fig. 14. The base of a striated duct in the principal submandibular gland showing a nerve terminal (arrow) in the midst of rod-shape mitochondria and folded plasma membranes. The terminal contains both clear and dense-cored vesicles. X 25,000.

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Fig. 15. Photomicrograph of a secretory endpiece in a n accessory submandibular gland. A small demilune is indicated by the arrow. An intercalated duct (ID) lies adjacent to the acinar cells. Toluidine blue. x 1,000.

tracts have shown the presence of substantial amounts cluded that they may be histogenic precursors of acinar of protease, which was not identified as to type (Jun- cells. The observation that similar cells occur not only queira et al., 1973). Since these extracts were devoid of between serous tubules and intercalated ducts, but also amylase or lysozyme, an antifibrin protease conceiv- between seromucous acini and serous tubules of the ably could be a major secretory product of the serous rabbit submandibular gland casts some doubt on the cells in the principal submandibular gland. Other in- hypothesis that these cells are the progenitors of acinar vestigations have revealed that a plasminogen activa- cells; rather, they may be fully differentiated secretory tor, termed desmokinase, also is present in vampire bat cells that contribute their unique secretory products to saliva (Hawkey, 1967,1988). The vampire bat salivary the forming saliva (Toyoshima and Tandler, 1986a). plasminogen activator (PA) is probably synthesized Junctional cells intervening between acinar cells and and exported by the parotid and principal and acces- intercalated duct cells have previously been noted in sory submandibular glands (Hawkey, 1988; Gardell et two other species of bats, namely, the Japanese horseal., 1989). Vampire bat PA recently has been isolated shoe bat, Rhinolophus ferrumequinurn (Mineda, 1977, and its cDNA sequenced (Gardell et al., 1989). In the 1978) and Kuhl’s pipistrelle, Pipistrellus Kuhli (Azzali presence of fibrin I, vampire bat PA has a much higher et al., 1986). Similar cells also are found in human activity toward plasminogen than does the typical salivary glands (Lantini et al., 1988). The fact that mammalian tissue-type plasminogen activator (Gar- junctional cells now have been encountered in five dell et al., 1989). In our view, an understanding of the mammalian orders (Chiroptera, Primates, Rodentia, evolution and expression of the vampire bat salivary Lagomorpha, Artiodactyla) should alert microscopists PA gene(s) will shed light on the phylogenetic relation- to the possibility that such cells exist in other groups of mammals as well. ships of these animals to other bats. An apparently unique feature of vampire bat subIntercalated ducts of the vampire bat accessory submandibular gland are relatively simple in structure, mandibular glands, both principal and accessory, is the but they are preceded by a cell type-the so-calledjunc- presence on all luminal surfaces of a vast array of mitional cells-that differs from both duct cells and aci- crovilli, culminating in a brush border in the striated nar cells. These cells contain unusual secretory gran- and excretory ducts that is virtually indistinguishable ules. They were originally noted in several species of ruminants (Takano et al., 1976) and studied in detail in the rat by Qwarnstrom and Hand (19831, who conFigs. 16-18 overleaf.

Fig. 11. The luminal surface of a striated duct cell in the principal gland showing the brush borderlike array of microvilli. Small vesicles are present in the apical cytoplasm. X 20,700.

Fig. 16. Secretory endpieces of the accessory gland. A demilune cell with homogeneous mucous droplets is a t the left. Remaining cells are mucous acinar cells; these contain structured secretory droplets. Note that although the lumen has many microvilli, these structures are not so numerous as in the principal gland. x 5,700.

Fig. 12. Apical granules from a striated duct cell. Principal submandibular gland. x 60,000.

Fig. 17. Mucous droplets in a n acinar cell of the accessory gland. The droplets each have two distinct components: a coarsely fibrillar matrix and a homogeneously dense inclusion. x 20,400.

Fig. 13. Transverse section through the microvillous border of a striated duct cell in the principal gland, underscoring the resemblance of this array to a renal brush border. x 70,600.

Fig. 18. Mucous droplets in a demilunar cell of the accessory gland. In contrast to those in acinar cells, these droplets lack dense inclusions and consist wholly of a finely fibrillnr matrix. x 40,000.

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Figs. 16-18.

SA1,TVARY GLANDS OF VAMPIRE RAT

Fig. 19. A junctional cell at the meeting point of the mucous acinus with an intercalated duct, This cell can he distinguished from acinar cells because of its granules, which contain a dot-like matrix. x 14,900. Fig. 20. The junction between an intercalated duct and a striated duct in the accessory gland. The former consists of cuboidal cells that

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surround a conspicuous lumen. The lower half of the illustrated structure consists of the basal portion of a striated duct cell. x 3,000. Fig. 21. Transversely sectioned striated duct in the accessory gland. Note abundance o f luminal microvilli. x 2,000.

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in morphology from that in proximal convoluted tu- ous nerve terminals, their constituent cells are linked bules of the kidney. We have examined the major sal- by gap junctions. Although gap junctions are being reivary glands of nearly 180 species of bats, a s well a s ported in salivary glands a t a n increasing rate (see those of many other kinds of mammals, and have not review by Nagato and Tandler, 1986), they have been encountered the profusion of microvilli that character- described only in association with acinar cells. Our obizes the vampire bat glands. The necessity for this pan- servations of gap junctions in striated ducts are the oply of microvilli probably is related to the unique diet first in t h a t location. It may be that gap junctions in of D. rotundus. These bats are voracious feeders and striated ducts are to some extent masked by the welter daily may consume a n amount of blood that equals of plasma membranes that make up the basal regions twice their body weight (Wimsatt and Guerriere, of such ducts, but we have never encountered these 1962). On a unit body weight basis, this means that junctions in any of the other species that we have exthese animals are taking on a huge quantity of sodium amined with the same care that we have expended on chloride and protein. The concentration of NaCl is fur- the vampire bat glands. The presence of gap junctions ther raised by the fact that upon initiation of feeding, in vampire bat striated ducts reinforces the notion that the animals produce copious amounts of dilute urine; the duct cells are highly responsive to nervous control during the later hours of diminishing urine flow, the in a n integrated fashion. osmolality of the urine rises to very high mean levels ACKNOWLEDGMENTS (4,656 mOsm/kg H20), reaching peak levels greater than 6,100 mOsm (McFarland and Wimsatt, 1969). Financial support fox this project came from NIDR Horst (1968) has shown t h a t the urine concentrating grant R01 DE 07648 (Tandler and Phillips). We appreability of Desmodus is not due to any innovations in ciate the assistance of our colleague, the late William kidney structure but rather to physiological adapta- A. Wimsatt, who supplied live vampire bats from his tions. colony at Cornell University; this work is dedicated to It is generally accepted that the formation of saliva his memory. Technical assistance was provided by in mammals is a two-step process: the initial saliva Carol Ayala; secretarial assistance was provided by elaborated by the secretory endpieces has plasmalike Hofstra University. concentrations of electrolytes, which are progressively LITERATURE CITED modified, usually by resorption, as the saliva moves through the duct system, yielding a final saliva that is Azzali, G., G. Romita, and R. Gatti 1986 Fine struttura ed aspetti stagionali della ghiandola parotide. Arch. Ital. Anat. Embriol., hypotonic (Schneyer et al., 1972). Clearly, it would be 91:257-300. of advantage to vampire bats to produce a hypertonic Bjorkman, N. 1962 Low magnification electron microscopy in histosaliva rather than a hypotonic one in order to reduce logical work. Acta Morphol. Neerl. Scand., 4.344-348. their enormous load of salt after feeding. The salivary Cope, G.H. 1977 Ultrastructure of the innervation of the ducts within the rabbit parotid gland. Arch. Oral Biol., 22;715-719. glands, however, are fitted with numerous microvilli, P. 1960 Anatomy and histochemistry of the salivary glands which structures are more geared to absorption than to DiSanto, of the vampire bat, Desmodus rotundus murinus. d. Morphol., excretion. It is possible that the ducts a r e involved in 106:301-336. selective resorption of a n electrolyte that is being Ditmars, R.L., and A.M. Greenhall 1935 The vampire bat. A presentation of undescribed habits and review of its history. Zoologica, dragged along by the sodium chloride in the initial 19:53-75. saliva. Determination of the function of the microvilli Fava-de-Moraes, F. 1965 Anglus dados morfologicos associados ao esin D. rotundus salivary glands must await the availtudo histoquimico dos polissacarideos em glandulas salivares de animais pertencentes as seguints ordens: Marsupialia, Chiability of data on the electrolyte composition of vamroptera, Primates, Edentata, Lagomorpha, Rodentia, Carnivora e pire bat saliva during and after feeding. Artiodactyla (Mammalia). Rev. Fac. Odont. S. Paulo, 3t231-290. The importance of the striated ducts, at least those in Findley, J.S., and D.E. Wilson 1982 Ecological significance of chithe principal gland, in the physiology of the vampire ropteran morphology. In: Ecology of Bats. T.H. Kunz, ed., Plenum bat is underscored by their lavish innervation. The Press, New York, pp. 243-260. usual pattern of occurrence of hypolemmal nerve ter- Gardell, S.J., L.T. Duong, R.E. Diehl, J.D. York, T.R. Hare, R.B. Register, J.W. Jacobs, R.A.F. Dixon, and P.A. Friedman 1989 Isolaminals in salivary glands is that they are present in tion, characterization, and cDNA cloning o f a vampire bat salimodest numbers in acini, somewhat more abundant in vary plasminogen activator. J. Biol. Chem., 264:17947-17952. intercalated ducts, and extremely rare in striated ducts Glass, B.P. 1970 Feeding mechanism of bats. In: About Bats. B.H. Slaughter and D.W. Walton, eds. Southern Methodist University (Cope, 1977; Kidd and Garrett, 1979). In the vampire Press, Dallas, pp. 84-92. bat, there is a steady increase in number of nerve ter- Greenhall, A.M. 1972 The biting and feeding habits of the vampire minals from acinus to ducts, with the greatest number bat, Desmodus rotundus. J. Zool. (Lond.), 168:451-461. of these terminals occurring in the striated ducts; a Greenhall, A.M. 1988 Feeding behavior. In: Natural History of Vampire Bats. A.M. Greenhall and U. Schmidt, eds. CRC Press, Boca similar pattern has been noted in the accessory subRaton, pp. 111-131. mandibular gland of the little brown bat, Myotis lucifu- Greenhall, A.M., and U. Schmidt 1988 Natural History of Vampire gus (Tandler et al., 1989). The change in electrolyte Bats. CRC Press, Boca Raton. and water content of vampire bats during and after Hall, H.D., and C.A. Schneyer 1964 Salivary gland atrophy in rat induced by liquid diet. Proc. SOC.Exp. Biol. Med., 117:789-793. feeding are so dramatic that these animals must have A.R. 1972 'The effects of acute starvation on parotid acinar finely tuned homeostatic mechanisms in all of their Hand, cells. Ultrastructural and cytochemical observations on ad libihydro-ionic organs-the presence of nerve terminals in tum-fed and starved rats. Am. J. Anat., 135:71-92. the striated ducts, which presumably are heavily in- Hand, A.R. 1973 Morphologic and cytochemical identification of peroxisomes in the rat parotid and other exocrine glands. J. Hisvolved in transport processes, undoubtedly permits tochem. Cytochem., 21:131-141. rapid adjustments to the exigencies of food intake and Hand, A.R., and B. Ho 1981 Liquid-diet-induced alterations of rat diuresis. parotid acinar cells studied by electron microscopy and enzyme Not only are the striated ducts provided with numercytochemistry. Arch. Oral Biol., 26:369-380.

SALIVARY GLANDS OF VAMPIRE BAT Harrison, J.D. 1974 Salivary glands of the cat: a histochemical study. Histochem. J., 6:649-664. Hawkey, C. 1967 Inhibitur of platelet aggregation present in saliva of the vampire bat I1esmodu.s rutundus. Brit. J. Haematol., 13: 1014-1020. Hawkey, C.M. 1988 Salivary antiheinostat,icfactors. In: Natural History of Vampire Bats. A.M. Greenhall and 11. Schmidt, eds. CRC Press, Boca Raton, pp. 133-141. Hill, J.E., and J.D. Smith 1986 Bats: A Natural History. university of Texas Press, Austin. Hornick C.A., A.L. Jones, G. Renaud, G. Hradek, and R.J. Have1 1984 Effect of chloroquine on low-density lipoprotein catabolic pathway in rat hepatocytes. Am. J. Physiol., 246rGI87-Gl94. Horst, R. 1968 Observations on the structure and function of the kidney of the vampire bat, Desmodus rotundus murinus. Ph.D. dissertation, Cornell University, pp. 1-123. Johnson, D.A. 1982 Effect of‘a liquid diet on the protein composition of rat parotid saliva. J. Nutr., 112:175-181. Junqueira, L.C.U., A.M.S. Toledo, and A.I. Doine 1973 Digestive enzymes in the parotid and submandibular glands of mammals. An. Acad. Brasil Cienc., 45,629-643. Kalt, M.R., and B. Tandler 1971 A study of fixation of early amphibian embryos for electron microscopy.