THE ANATOMICAL RECORD 255:105–115 (1999) Ultrastructure of the Parotid Gland in Two Species of Naked-Backed Bats BERNARD TANDLER,1 TOSHIKAZU NAGATO,2 AND CARLETON J. PHILLIPS3* 1Department of Biological Sciences and Institute of Environmental and Human Health, Texas Tech University, Lubbock, Texas 2Second Department of Oral Anatomy, Fukuoka Dental College, Fukuoka, Japan 3Department of Biological Sciences, Texas Tech University, Lubbock, Texas ABSTRACT Naked-backed bats of the genus Pteronotus (family Mormoopidae) occur in the Neotropics from Mexico through northern South America. These are relatively small-sized insectivorous species that frequently roost in caves. Eight specimens of naked-backed bats (Pteronotus parnellii) were livetrapped in Suriname and one in Cuba (P. quadridens). Their parotid glands were fixed in an aldehyde mixture designed for field work and postfixed in the laboratory with osmium tetroxide. Tissues were further prepared for electron microscopy by conventional means. The parotid glands of the two species of Pteronotusclosely resemble each other except for the substructure of their serous secretory granules. Serous granules in P. parnellii are bizonal, with a moderately dense inner matrix and an outer, denser corona or crescent. The matrix is occupied by laminae, flakes, and filaments in random array. In contrast, serous granules in P. quadridens consist of a uniform matrix that contains dense, usually stacked toroids or tubules either in random array or packed in bundles. A parotid gland from one specimen of P. parnellii contained an endpiece that consisted of cells that contained giant (up to 9 µm in diameter) serous granules. Serous cells in both species contain aggregates of small, uniformly dense, rod-like, membrane-delimited organelles as well as occasional bundles of cytofilaments. The endpieces are separated from intercalated ducts by a ring of granulated cells that contain secretory granules that often have a bull’s-eye configuration. Intercalated and striated ducts are typical in appearance, except that many of the cells in the latter contain small, dense secretory granules in their apical cytoplasm. The parotid glands in the two species of naked-baked bats differ slightly in terms of acinar secretory granule ultrastructure, but otherwise are fairly conservative. It is thought that the glands in these particular bats might represent the ‘‘basal’’ condition of the salivary glands of insectivorous bats and thus can serve as a reference point for making comparisons to the highly diversified (in terms of diet) phyllostomid bats. Anat Rec 255:105–115, 1999. r 1999 Wiley-Liss, Inc. Key words: salivary glands; secretory granules; bats; Pteronotus parnellii; Pteronotus quadridens It can be argued that salivary glands have played very important—perhaps ‘‘keystone’’—roles in the adaptive radiation of mammals. There is mounting evidence that the ultrastructure and function of these glands reflect a combination of evolutionary history (phylogeny), ecology, and adaptation (Phillips, 1996). The salivary glands of bats provide especially useful models for testing hypotheses r 1999 WILEY-LISS, INC. Grant sponsor: The National Institute of Dental Research (BT, CJP); Grant sponsor: The Carnegie Museum of Natural History (CJP); Grant sponsor: The United States Navy (CJP). *Correspondence to: Dr. Carleton J. Phillips, Department of Biological Sciences, Texas Tech University, Lubbock TX 79409– 3131. Received 30 April 1998; Accepted 19 July 1998 106 TANDLER ET AL. about the relationship between gland ultrastructure and diet (Phillips et al., 1993; Tandler and Phillips, 1998) because bats exhibit a remarkable diversity in diet. The naked-backed bats (also called ‘‘mustache’’ bats) of the genus Pteronotus are important in comparative studies for two reasons. These bats (and their relatives in the family Mormoopidae) are insectivorous, but as an evolutionary lineage they have been separate from other major groups of insectivorous bats for ⬃50 million years (Simmons and Geisler, 1998). Thus, although insectivorousness might be a shared ancestral diet, it has evolved along independent lines for a long period of time. Additionally, Pteronotus and its relatives share an ancestry with the Neotropical phyllostomid bats. Pteronotus thus offers a reference point (in systematic terms, a ‘‘sister’’ group) for comparison with the phyllostomid bats, which in turn exhibit the most diversified diets and salivary glands of any bat species (Phillips and Tandler, 1987; Phillips et al., 1993). In addition, one of the naked-backed bats, P. parnellii, has served as an important model for understanding auditory capabilities and echolocation in insectivorous bats (Pollak and Casseday, 1989). In terms of foraging behavior and diet, bats of the genus are characterized by enlarged fleshy lip pads and para-oral, bristle-like hairs that might serve to direct the flow of air into the mouth as the bat closes in on its prey (Nowak, 1991). Typically, bats in this genus feed on small, soft-bodied flying insects such as moths. MATERIALS AND METHODS Eight naked-backed bats (Pteronotus parnellii) were live-trapped during field work in Suriname and one was obtained in Cuba (P. quadridens). Voucher specimens are in the research collections at the Carnegie Museum of Natural History and Texas Tech University, Lubbock. Of the South American bats, three were males and five were females; the specimen of P. quadridens was a female. The parotid glands were removed from the animals, which had been anesthetized with T-61 euthanasia solution (no longer marketed), and minced in fixative. Salivary glands from the Suriname specimens were fixed in a modified triple aldehyde-DMSO solution (Phillips, 1985) based on the fixative of Kalt and Tandler (1971). The specimen of P. quadridens was fixed in modified half-strength Karnovsky’s (1965) solution that contained DMSO and LiOH. After the initial fixation in aldehydes, all specimens were stored in 3% glutaraldehyde until refrigeration became available (Phillips, 1985). After thorough washing in buffered sucrose, the specimens were postfixed in phosphatebuffered 2% osmium tetroxide (Millonig, 1961a) and washed in distilled water. An overnight soak in acidified 0.25% uranyl acetate (Tandler, 1990) was followed by a second round of washing in distilled water. After dehydration in ascending concentrations of ethanol, the tissue blocks were embedded in Epon:Maraglas (Tandler and Walter, 1977). Thin sections were consecutively stained with acidified methanolic uranyl acetate (Tandler, 1990), then with either lead tartrate (Millonig, 1961b) or lead citrate (Venable and Coggeshall, 1965), and examined with either a JEOL 1200 EX (Tokyo, Japan) or a Siemens 101 or 1a Elmiskop (Berlin, Germany) electron microscope. Semithin sections were stained with methylene blue-azure II (Laczkó and Lévai, 1975), and examined with an Olympus Vanox. RESULTS In their general histology, the parotid glands of P. parnellii and P. quadridens are quite typical. Most of the serous secretory endpieces form acini, but some are tubuloalveolar. The intercalated ducts are short and difficult to detect, but the striated ducts are of usual length and prominence (Fig. 1).The apical surfaces of the serous cells face the endpiece lumen, and a typical system of microvillus-lined intercellular canaliculi courses down the lateral surfaces of these cells. The cell base is characterized by numerous slender basal folds; the intercellular space between these folds is occupied by a homogeneous dense material, which apparently is deposited in this location by exocytosis of coatamer vesicles. We have elsewhere described in detail this unusual basal apparatus (Nagato et al., 1998). In both species, the serous cells contain a full complement of secretory granules (Fig. 2). In a few cells in P. parnellii, these granules consist wholly of a structureless dense matrix. In most cells, however, the granules, the largest of which measure ⬃0.9 µm in diameter, have a well-defined, bizonal substructure (Figs. 3, 4). The matrix in such granules consists of a moderately dense core, and a marginally denser halo or crescent underlying the granule membrane. The inner core contains a variable number of apparently linear densities, but the true three-dimensional structure of these densities is difficult to decipher (Fig. 4). Certain of the densities probably have the form of flat, elliptical laminae or irregular flakes, whereas others are corrugated. In some granules, dot-like structures are visible. Some of these dots probably represent transversely sectioned rods, which would appear as laminae in longitudinal section. Other dots have a much smaller diameter than the thickness of the laminar densities or putative rods, and may represent cross-sectioned thin filaments. In a few cells, the granules may have only a partial outer halo, or may be lacking this component altogether. In this case, the core material fills the entire granule. The parotid serous granules of P. quadridens are quite different from those of its congeneric species. They tend to be larger (⬃1.3 µm in diameter) and their matrix lacks a bizonal appearance; it is homogeneous and of moderate density. At lower magnifications, the matrix is seen to contain many small densities, some of which are round, some of which are oblong (Fig. 5). At high magnification, it becomes evident that many of these densities actually consist of concentric, stacked, quite dense tori (Fig. 6). The outermost torus of each matrical inclusion is fitted with a series of short spinules (actually ribs) ⬃19–28 nm long. In other granules, the matrix includes an array of cylindrical structures measuring ⬃47 nm in diameter and with dense walls. These tend to be arranged in parallel in bundles, but the bundles are randomly disposed, so that these aggregates are sectioned in a variety of planes in the same granule (Figs. 7, 8). In transverse section, the cylinders are seen to be in a square packing arrangement (Fig. 8). In a very small portion of a single lobule from one of the eight specimens of P. parnellii in our sample, we encountered a few serous secretory cells that contained one or several giant secretory granules in addition to their complement of typical ones (Fig. 9). These outsized granules measure 7–9 µm in diameter; in addition to a giant one, a few cells had intermediate-sized granules. The matrix in the large granules is of the same general density as in PAROTID STRUCTURE IN NAKED-BACKED BATS Fig. 1. P. parnellii. Survey electron micrograph of the parotid gland showing the heavily granulated endpiece cells. Two segments of the same intercalated duct are indicated by arrows. 800⫻. Fig. 2. P. parnellii. An endpiece cell at higher magnification. It is 107 underlaid by a process of a myoepithelial cell (MC). A transversely sectioned intercellular canaliculus (ICC) is present at the lateral border of the cell. 10,100⫻. 108 TANDLER ET AL. Fig. 3. P. parnellii. A cluster of serous granules in an endpiece cell. Although these granules exhibit minor variation in substructure, they all have the same basic morphology. 34,500⫻. Fig. 4. P. parnellii. A serous granule at high magnification. It contains two zones of marginally different density. The lighter zone contains what appear to be profiles of short laminae. 80,500⫻. PAROTID STRUCTURE IN NAKED-BACKED BATS Fig. 5. P. quadridens. A cluster of serous granules in an endpiece cell. The morphology of these granules clearly differs from that of P. parnellii. 39,500⫻. 109 Fig. 6. P. quadridens. A serous granule at high magnification. The dense inclusions consist mainly of stacked toroids that exhibit an axial periodicity. 70,500⫻. 110 TANDLER ET AL. Fig. 7. P. quadridens. In this serous granule, the dense inclusions consist of twisted, tubular forms. 66,000⫻. Fig. 8. P. quadridens. In this serous granule, the dense tubules are in a closely packed, parallel array. They are seen in both transverse and longitudinal section. The lucent space at the bottom of the granule is due to the artifactual separation of the granule from the embedding medium. 66,200⫻. Fig. 9. P. parnellii. A serous endpiece in which several of the constituent cells harbor giant secretory granules. Small and intermediate size granules are also present in the same cells. 5,200⫻. Fig. 10. P. parnellii. The edge of a giant granule showing some of the faint inclusions in the granule matrix. 31,800⫻. PAROTID STRUCTURE IN NAKED-BACKED BATS 111 Fig. 11. P. parnellii. The basal cytoplasm of a serous cell with a collection of dense, membrane-delimited rods. 33,000⫻. Fig. 12. P. parnellii. Two sheaves of cytofilaments in a serous cell. 64,000⫻. Fig. 13. P. quadridens. A granular cell that separates the endpiece from the intercalated duct proper. The substructure of the granules is completely different from that of granules in the serous cells or in the intercalated duct cells that follow. 15,600⫻. normal-sized ones and tends to be homogeneous, but small vesicles with lucent borders are randomly arrayed at the periphery of the matrix of the enlarged granules (Fig. 10). Because most serous cells in our specimens are replete with secretory granules, both the rough endoplasmic reticulum (RER) and Golgi apparatus are relatively inconspicuous. The RER is manifested as some scattered cisternae; free ribosomes are abundant. The Golgi apparatus consists of three or four parallel saccules, several of which may be moderately dilated. A few coated vesicles sometimes are present near the Golgi saccules, but in the main condensing vacuoles are absent. In both species, the serous cell cytoplasm contains collections of loosely aggregated dense rods (Fig. 11). These structures are delimited by a single membrane and contain a homogeneous dense material. They are up to 0.7 µm long and 60 nm in diameter. These structures have no obvious relationship to any cytoplasmic organelle. Some serous cells in P. parnellii contain cytoplasmic bundles of parallel cytofilaments; each filament measures ⬃10 nm in thickness (Fig. 12). Intervening between secretory endpieces and intercalated ducts are rings of intermediate granular cells. These contain granules that frequently have a bull’s-eye configuration (Fig. 13). In some granules, the substructure is less regular. Intercalated ducts are simple affairs. They consist of a single layer of cuboidal epithelium that lacks distinguish- ing features and is underlaid by myoepithelial cells (Fig. 14). The duct cells have a relatively large nucleus and a few scattered mitochondria. Except for several short microvilli, they lack surface modifications. A few secretory granules are present in the duct cells of P. quadridens, but are absent from the same site in P. parnellii. The granules in the former bat are up to 0.9 µm in diameter and of low matrix density; some contain a spherule (Fig. 15). Striated ducts in the parotid glands of both kinds of naked-backed bats in general are similar in structure to those in a variety of bats (Tandler et al., 1989; Tandler, 1993) (Fig. 16). In addition to the usual basal array of folded plasma membranes and vertically oriented mitochondria (Fig. 17), some of the duct cells feature a juxtaluminal band of small, dense, structureless granules (Fig. 17). This band of apical granules is more prominent in P. parnellii than in P. quadridens, but the granules are more distinct in the latter. The granular duct cells may alternate with cells of similar overall morphology, but that lack apical granules altogether (Fig. 18). DISCUSSION Compared to serous granules in the serous endpieces of the parotid gland of many other species of bats (Phillips et al., 1987; Tandler and Phillips, 1993), those in the parotid of Pteronotus have a very unusual substructure. We previously attempted to explain granule substructure by postu- 112 TANDLER ET AL. Fig. 14. P. quadridens. An intercalated duct sectioned in a slightly oblique transverse plane. The cuboidal epithelial cells of the duct lack secretory granules or cytoplasmic specializations. It may be inferred that this section passes through an intermediate segment of the duct. 5,400⫻. Fig. 15. P. quadridens. Secretory granules in a proximal cell of an intercalated duct. They consist of a dense, ragged, eccentrically placed spherule suspended in a relatively light matrix. 27,000⫻. lating a self-assortment mechanism for the different molecules in the granule matrix, leading to the production of patterning based on distribution of electron-dense components. This supposition is now backed by some experimental evidence (Takano et al., 1991, 1993). If such a mechanism is operative in the serous granules of the nakedbacked bat, it must be an extraordinarily complex one, given the diversity of structural components in the mature granules of P. parnellii, i.e., dense and less-dense zones, flat and corrugated dense laminae, dense rods, and thin filaments. The serous granules of P. quadridens also have an extremely unusual substructure. Spiny inclusions of somewhat similar type also have been noted in the submandibular serous cells of the little brown bat, Myotis lucifugus (Tandler and Cohan, 1984), but as in P. quadridens, the putative spines turned out to be ribs in three dimensions; however, the inclusions in the granules of the former are quite different in appearance from those in the granules of P. quadridens. Unfortunately, we were unable to trace the morphological concomitants of secretion of the complex granules in either species of Pteronotus, because almost all endpiece cells were crammed with stored mature granules; that is, these cells were not actively secreting new granules. Although the composition of the parotid secretory granules in Pteronotus is unknown, their electron-density points to the possibility of an enzyme-rich content (Junqueira et al., 1973). In a previous study (Phillips et al., 1998), we screened P. parnellii with a polyclonal antiserum to lysozyme; the parotid gland acinar and intercalated duct cells were negative. This negative finding is interesting because the parotid glands of some species of insectivorous bats exhibit considerable lysozyme-like immunoreactivity. The most likely difference is that species of Pteronotus feed on insects that are soft-bodied (low in chitin). The presence of giant secretory granules in a subset of serous cells of a parotid gland in a single individual of P. pteronotus is unprecedented in the annals of salivary gland research. Although giant granules have been reported to occur in the striated and excretory ducts of the parotid and submandibular glands of the slow loris (Tandler et al., 1996), practically every ductal cell in these specific glands is involved in the elaboration of grossly enlarged granules; this clearly is a manifestation of a mechanism that regulates granule size in each and every duct cell. Giant secretory granules up to 8 µm in diameter sporadically occur in serous glands underlying the mucosa of human nasal turbinates (R.A. Erlandson, personal communication). Their occurrence in only a few cells in just one specimen of P. parnellii and in some human nasal glands is difficult to explain. The propinquity of the affected serous cells suggests that they represent a clone of endpiece cells. A recent study by Redman (1995) has shown PAROTID STRUCTURE IN NAKED-BACKED BATS Fig. 16. P. parnellii. A portion of a striated duct. The cells have both basal mitochondria and apical, dense secretory granules. 8,400⫻. Fig. 17. P. parnellii. The base of striated duct cells showing the vertically oriented, rod-shaped mitochondria alternating with folded plasma membranes. 15,600⫻. 113 Fig. 18. P. parnellii. The apices of two adjacent striated duct cells. The cell on the right contains a panoply of secretory granules, whereas its immediate neighbor on the left lacks these structures altogether. 20,000⫻. 114 TANDLER ET AL. that salivary acinar cells are fully capable of selfreplication. We posit the following scenario: A somatic mutation took place during mitosis of a serous cell and was propagated through several rounds of cell division, forming a clone. This mutation, while not having immediate overt effects, rendered the granule-elaborating cytological machinery capable of producing huge granules. Mroz and Léchene (1986) have shown that pancreatic zymogen granules can differ markedly in protein composition and Schick et al. (1984) have demonstrated that diet can affect the composition of pancreatic exocrine granules. If the naked-backed bat in question consumed just the right combination of nutrients, the production of megagranules might have been triggered in the susceptible, mutated cells. If a mutation of this type had occurred in the gametes before fertilization or during embryonic development of the bat, then the presence of giant granules would be a widespread phenomenon, rather than being confined to a small group of neighboring serous cells. The precise mechanism whereby giant secretory granules are engendered is unknown. One obvious way might be by fusion of smaller granules, a process that has been documented in cells of the anterior pituitary gland (Senda et al., 1998). Some simple computations are enough to dispel this notion. A giant granule has a volume roughly equal to a thousand normal-sized granules, but because of surface area to volume ratios, a thousand normal granules collectively have a surface area about 2.5 times greater than a single megagranule. This means that if fusion occurs, a huge amount of limiting membrane becomes superfluous and must be eliminated. The relatively few small vesicles within the matrix of the giant granules may be the relics of such an elimination process, but these by themselves cannot account for all of the unneeded membrane. Similar internal vesicles are present in the giant ductular granules of the parotid and submandibular glands of the slow loris (Tandler et al., 1996). However, inspection of the serous cell cytoplasm fails to reveal either autophagic vacuoles or of pools of membrane, indicating that granule fusion is not responsible for the production of giant granules. In normal exocrine cells, the terminal expansions of the inner Golgi saccules become filled with secretory product and, when a certain size range is reached, separate from the Golgi to give rise to mature secretory granules. In the affected cells in the parotid gland of P. parnellii, the scission process might be compromised, so that the terminal dilatations continue to grow by acquisition of additional secretory product, until they reach huge proportions. Then a nascent giant granule might separate from the Golgi apparatus by virtue of its mass, or the entire Golgi saccule, having been filled to bursting with secretory product, is cast out in toto into the cytoplasm. In general, acinar secretory cells are similar within a given salivary gland, giving the impression that all of these cells represent but a single population. An earlier ultrastructural study of the human submandibular gland (Tandler and Erlandson, 1972) documented the presence in the same gland of two serous cell phenotypes with secretory granules that exhibited two widely differing substructural patterns. Using histochemistry, we found that only scattered individual acinar cells in the parotid gland of the sac-winged bat, Saccopteryx bilineata, were immunoreactive to antilysozyme antiserum (Phillips et al., 1998). Although such data do not prove that some cells are regulated independently from others, the results raise the possibility that there can be more than one ‘‘class’’ of acinar cells in a serous gland. In addition to being an interesting issue in evolutionary terms, e.g., the possibility of intragland diversification in secretory product, these observations also call attention to the dynamics of gland development. The possibility of secretory diversification and subdivision of the parotid gland acinar cell population has been discussed by Denny et al. (1993). Granular cells positioned between secretory endpieces and intercalated ducts have been observed in salivary glands from five different mammalian orders. The only chiropteran other than naked-backed bats whose salivary glands are reported to contain such cells is the common vampire bat, Desmodus rotundus, where they are present in the principal submandibular glands (Tandler et al., 1990). The precise status of these cells remains to be established. The striated ducts in the two Pteronotus species are quite typical of bat salivary glands. Not only do they have the usual basal array of mitochondria and folded plasmalemma, but they sporadically have an abundance of small, apical secretory granules, in this respect paralleling the situation in the excretory ducts of the parotid gland of the little brown bat, in which organ granular cells alternate with nongranular ones (Tandler and Cohan, 1984). These ducts appear to be able to modify the initial saliva by liberating significant amounts of organic secretion into it, as well as by active transport of electrolytes. ACKNOWLEDGMENTS We appreciate the field participation of H.H. Genoways (University of Nebraska) and R.J. Baker (Texas Tech University). LITERATURE CITED Denny PC, Chai Y, Klauser DK, Denny PA. 1993. Parenchymal cell proliferation and mechanisms for maintenance of granular duct and acinar cell populations in adult male mouse submandibular glands. Anat Rec 235:475–485. Junqueira LCU, Toledo AMS, Doine AI. 1973. 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