Morphology of the buccopharyngeal portion of the gill in the fathead minnow Pimephales promelas (Rafinesque).код для вставкиСкачать
THE ANATOMICAL RECORD 200:67-81 (1981) Morphology of the Buccopharyngeal Portion of the Gill in the Fathead Minnow Pimephales promelas (Rafinesque) ELIZABETH R. WALKER, STJ3PHEN F. FTDLER, AND DAVID E.HINTON Department of Anatomy, West Virginia Uniuenrity, Medical Center, Morgantown, West Virginia 26506 ABSTRACT Buccopharyngeal epithelium covering gill arches and gill rakers of the fathead minnow was studied by light microscopic, scanning, and transmission electron microscopic techniques. Mature mucous cells in goblet pattern and nonmucus containing cells were in the apical one-third of the tissue. The latter cells contributed to a surface microridge system which overlapped apices of goblet cells. The bottom of the epithelium was comprised of a continuous row of darkly stained basal epithelial cells. In this region, two to three epithelial cells of similar staining characteristics were piled up forming apical columns which partially encircled nests of lightly stained cells. A basal lamina and thick basement lamella of about 20 plies of orthogonally arranged collagen supported the epithelium. Numerous taste buds were seen in gill arches and rakers. Taste bud cellular components included marginal cells, light receptor cells, dark receptor cells, and basal cells. These were identical in all taste buds. Taste bud surface morphology differed between gill arch and raker. Pores of the former were depressed, while those of the latter were raised. Thick microvilli of taste pores were apical extensions of light cells, while smaller, more numerous microvilli were projections from dark cells. Teleost gill arches give rise to primary and secondary lamellae, the cells of which perform essential functions of respiration (Steen and Kruysse, 1964; Newstead, 1967; Hughes and Morgan, 1973) and osmoregulation (Maetz, 1971; Schmidt-Nielsen, 1974; Philpott and Copeland, 1963; Karnaky et al., 1976a,b; Kikuchi, 1977). Since these structures are in constant contact with the aquatic environment, they are susceptible to alterations in structure/ function following exposure to biologically active compounds such as metals (Baker, 1969; Rucker and Amend, 1969), pesticides (Eller, 19751, excretory waste products (Smith and Piper, 1975) and to acute and chronic changes in pH (Daye and Garside, 1976). Although the entire arch is normally removed a t necropsy, the nonrespiratory portion of the teleost gill has received little attention (Zander, 1903; Albright and Skobe, 1965). This portion includes buccopharyngeal surfaces of gill arches and gill rakers, forward extending projections into interarch spaces (internal gill slits), which form a primary filter in feeding and respiration (Smith, 1960). In addition, other important functions, including secretion 0003-276X/81/2001-0067$04.500 1981 ALAN R. LISS. INC. and sensory perception, require the differentiation of several epithelial cell types (Reutter, 1973; Reutter et al., 1974). This report relates our findings from a correlated light, scanning, and transmission electron microscopic study of the buccopharyngeal epithelium of the fathead minnow gill and is part of a detailed analysis of normal gill structure in this species. Selection of the fathead minnow was due to its extensive use as a n indicator species in aquatic toxicity studies (Brungs, 1969; Mount, 1968; Pickering and Gast, 1972). MATERIALS AND METHODS Fish Young adult, hatchery-reared fathead minnows (Pimphales promelas) of both sexes were maintained in flow-through stainless steel tanks provided with undergravel filtration using washed limestone. Water from the city utility was filtered over an activated charcoal bed and aerated via filtered house air. Water Received May 28,1980; Accepted October 16,1980. 68 ELIZABETH R. WALKER ET AL. quality was monitored weekly and maintained Coggeshall, 1965), and examined with either at lVC, 6.8 pH, 8-9 mgfliter dissolved 02,and an AEI model 802, RCA EMU 3G, or JEOL s 1 part per million ammonia nitrogen con- 100 CX electron microscope. centration. Fish were fed 1%of the biomass daily for 5 days each week using a dry comRESULTS mercial ration (Purina). In order to collect tissues for study, 15 healthy fish were “pithed” Gill arch and raker and their gills were rapidly immersed in fixPrimary light microscopic features of the ative for processing. buccopharyngeal surface of a gill arch are shown (Fig. 1). Lining epithelium over gill Light microscopy arches was thicker than that over gill rakers. Tissues were fixed for 24 hours in Bouin’s A lamina propria containing fibroblasts, confixative, rinsed overnight in 50% ethanol, and nective tissue fibers, blood, and lymphatic vestransferred to 70% ethanol for storage until sels separated epithelium from cartilage of the subsequent processing, which involved dehy- gill arch. The arrangement of blood and lymdration in graded ethanol solutions, clearing phatic vessels and musculature in the gill arch in xylene, and embedment in paraffin. The was similar to descriptions in other teleosts small size of the minnows (2.5-7.5 cm) made (Steen and Kruysse, 1964; Newstead, 1967; it possible to embed the entire fish in a single Hughes and Morgan, 1973; Morgan and Toblock for longitudinal or transverse serial sec- vell, 1973). tioning. Hematoxylin and eosin (H & E) stains The features of the buccopharyngeal surface of 5-7-pm-thick paraffin sections were used for on one of the four gill arches are shown (Fig. routine light microscopic examination. The 3). A double row of gill rakers projected from periodic acid-Schiff s reagent (PAS) stain was each arch. Each raker consisted of a cartilaused to selectively stain mucous granules and ginous core covered by epithelium (Fig. 1). At the basement membrane. low magnification, SEM revealed numerous bumps on gill arches and inner surfaces of rakTransmission electron microscopy (TEM) and ers (arrows, Fig. 3). By light microscopy (Fig. scanning electron microscopy (SEM) 2), these were parts of taste buds in the epiIn our laboratory, satisfactory fixation of te- thelium. leost tissues for SEM and TEM has been obBasal and intermediate regions of the tained (Hinton, 1975) when a buffer strength epithelium equal to two-thirds serum osmolality (Bone and Ryan, 1972) was used with either glutarHigh-resolution light microscopy of the bucaldehyde ( 2 4 % )or formaldehyde-glutaralde- copharyngeal epithelium (Fig. 2) revealed a hyde (McDowell and Trump, 1974). Following continuous row of darkly stained basal epithedetermination of serum osmolality (approxi- lial cells. At intervals dark cell cytoplasmic mately 280 milliosmoles) in the fathead min- processes partially encircled “nests” of lightly now, we routinely fixed gills in 4% phosphate- stained cells which we designated “clear cells” buffered (pH 7.4, 150-200 milliosmoles) glu- (Figs. 2 and 5). Clear cells were always sepataraldehyde. After 12 hours’ fixation, tissues rated from the basal lamina by the layer of were postfixed with phosphate-buffered 2% dark cells (Figs. 2 , 4 , and 5). osmic acid for 1 hour a t 4” C and dehydrated With TEM the region underlying basement in graded ethanol solutions. For SEM tissues membrane, designated basement lamella by were then critical-point dried, coated with 200 some authors (Nadol et al., 19691, contained A gold palladium, and observed with an ETEC approximately 20 layers of collagen fibrils orAutoscan or a Cambridge Stereoscan S4-10 thogonally arranged (i.e., adjacent layers oriscanning electron microscope. Dehydrated tis- ented approximately at right angles to each sues for TEM were processed through propyl- other) (Figs. 5 and 6). Dark basal epithelial ene oxide and embedded in Araldite-Epon cells showed a central nucleus, electron-dense (Luft, 1961). To correlate light and electron cytoplasm with rough endoplasmic reticulum, microscopic observations, 0.5-pm sections of scattered mitochondria, and numerous juncepoxy-embedded material were stained with tional complexes (Figs. 5 and 6). Clear cells toluidine blue (Trump et al., 1961) and viewed contained dense granules in a n electron-lucent with a li ht microscope. Thin sections cytoplasm, which lacked rough endoplasmic (900-1000 ) were stained with saturated ur- reticulum. Single and clustered ribosomes any1 acetate and lead citrate (Venable and were numerous. Nuclei were small, centrally 1 FATHEAD MINNOW GILL MORPHOLOGY located, and were more heterochromatic than nuclei of dark cells (Figs. 4, 5,and 61. Light microscopic examination of the intermediate zone of epithelium showed a mixture of clear and dark cells (Fig. 2). In addition, PAS stains (not shown in figures)revealed occasional, small mucous granules. TEM of this region showed electron-dense epithelial cells, clear cells (Fig. 41, and immature mucous cells containing some mucous granules (arrows, Fig. 4). Rarely, small, rounded cells containing abundant rough endoplasmic reticulum but no mucous granules were seen (not shown in figures). The cytoplasm of these was suggestive of an immature mucous cell. Surface region of the epithelium Two types of cells were found a t the surface of the buccopharyngeal epithelium. A nonmucus-containing cell covered most of the surface. By TEM, this cell type (Fig. 9) had a central nucleus, cytoplasm containing numerous small, spherical, electron-dense granules (Fig. 8),and desmosomes. On their free surface these cells had regularly spaced, short cytoplasmic projections (Fig. 8). With SEM (Fig. 7) projections could be seen to form a pattern of small, tortuous ridges (microridge) over the cell surface. A more prominent ridge marked the boundary with adjacent cells (arrow, Fig. 7). Cross connections were seen between microridges. In both SEM (Fig. 7) and TEM (Fig. 8) microridges bore a fuzzy coat. A high concentration of cytoskeletal elements, apparently microfilaments (Bereiter-Hahn et al., 1979), made the microridge and immediately subjacent region of the cytoplasm appear more electron dense than the remainder of the cytoplasm (Fig. 8). In sections stained by PAS and toluidine blue (Fig. 2) or in TEM (Figs. 4 and 9) mature mucous cells filled with mucous granules were in the upper one-half of the epithelium. Rough endoplasmic reticulum and the nucleus occupied a small basal part of this cell. Mucous cells had no projecting structures to disrupt or contribute to the microridge pattern of the surface. Rather, smooth apical parts of these cells were extensively overlapped by processes of nonmucous cells, making the microridge system almost continuous (Fig. 9). Occasionally with SEM, blebs of mucous secretion were seen on the surface. Gill arch and raker The SEM features of one of the four gill arches are shown (Fig. 10). A double row of 69 gill rakers projected perpendicularly from each arch into the buccopharynx and formed a macrofiltering barrier over the internal gill slits. At low magnification (Fig. lo), SEM revealed numerous bumps on gill arches and rakers. Higher magnification of the epithelium overlying the gill arch showed rounded elevations (Fig. 11) composed of tall and short microvilli projecting above the microridge system (Fig. 12). Microridges of cells immediately surrounding the projecting microvilli were more tightly arranged than the microridge pattern of the other epithelial cells, as shown in Figure 7. However, the prominent ridge a t the cell boundary was maintained (arrow, Fig. 12). Light microscopy of sections through the gill arch (Fig. 13) showed the above-noted surface features to be the apical part of taste buds (Reutter, 1971,1973) in the epithelium. Taste buds extended from the basement membrane to taste pores located on the free surface. The central part of a taste bud was composed of elongated dark and lightly stained cells (Fig. 13). The microvilli, seen with the SEM (Fig. 121, were the apical extensions of both dark and light cells (Fig. 13). Gill rakers projected perpendicularly from the long axis of gill arches (Figs. 3 and 10) and consisted of a cartilaginous core covered by epithelium. The surface morphology of gill rakers differed from that over the gill arch. The distal tip of the raker formed a rounded cap which lacked a microridge system (Fig. 14). On the inner side of each gill raker (Figs. 3, 10, and 14) approximately 25 rounded elevations, papillae, were arranged in a double row (Fig. 14). Higher magnification of papillae (Figs. 15 and 16) revealed one rounded elevation, the apex of which was composed of a tuft of tall and short microvilli similar to those seen in the gill arch taste buds (Fig. 12). The surrounding cells exhibited a tightly arranged microridge system. When papillae were viewed in section (Fig. 171, each contained a taste bud with elongated dark and light cells. Microvilli from these projected through the taste pore and were seen on the buccopharyngeal surface (Fig. 16). Taste buds Light microscopic examination of Eponembedded gill arches showed the relationship of taste bud to adjacent structures (Figs. 2,13, and 17). Each taste bud extended from a cupshaped concavity of the basement membrane to the surface of the epithelium. Light and dark cells occupied the central portion of taste 70 ELIZABETH R. WALKER ET AL. bud with microvilli extending above the epithelial surface (Figs. 2, 13, and 17). Nerve fibers (Fig. 13) passed from lamina propria through the basement membrane to the base of taste bud cells. Light microscopy showed little morphologic difference between gill arch and gill raker taste buds-only that the former had a depressed and the latter a raised taste pore. The arch and raker taste buds were similar in their fine structure. Their constituents were marginal cells, basal cells, nerve plexus, dark cells, and light cells. Marginal cells (Fig. 18), at the boundary of individual taste buds, were flattened epithelial cells, with an electrondense cytoplasm and a nucleus with peripherally clumped heterochromatin. The region underlying the basement membrane lacked the layers of orthogonally arranged collagen (Fig. 6) and was interrupted at various sites by fibers of the nerve plexus. Basal cells (Fig; 2) lay between the basement membrane and light and dark cells. Basal cells were surrounded by unmyelinated nerve fibers, which passed around the cells to end a t the bases of light and dark cells. Cytoplasmic processes from dark cells (Fig. 18) extended around tbe basal part of light cells to reach the nerve plexus. Details of the synaptic connections between the nerve plexus and the various cell types of the taste bud will be reported later. Light cells, so named because of their electron-lucent cytoplasm, showed elongated mitochondria and numerous membrane-limited spaces. The latter were seen as groups of small vesicles distally (Figs. 19 and 21) but formed large channels in middle and basal portions of light cells (Figs. 18 and 19). The nucleus contained large electron-lucent regions (Fig. 18) which contrasted with scattered clumps of heterochromatin. A few microtubules were seen in light cells (Fig. 19). From each light cell, a single large microvillus extended above the epithelial surface (Fig. 21). This corresponded to the large clublike microvilli observed with SEM (Fig. 22) in both gill arch (Figs. 12 and 22) and gill raker taste buds (Fig. 16). About 20 of these thick microvilli emerged from each taste pore (Fig. 22). In addition, microvilli of light cells lacked surface coat seen on dark cells (see below) (Fig. 21). The dark cells of taste buds extended from the basement membrane to the taste pore. The nuclei of these cells were restricted to basal parts of the cell, and were more electron-dense than nuclei of light cells (Fig. 18). Cytoplasm contained elongated mitochondria, numerous bundles of longitudinally oriented microtubules, and granules (secretory?) (Figs. 19 and 20). Some granules had both electron dense and lucent contents (Fig. 19) while contents of others were uniform in density (Fig. 19). Small microvilli, covered with a cell coat material, extended from the apical end of the cell (Fig. 19 and 21). When observed with SEM (Fig. 22) these small microvilli were two to three times more numerous than large microvilli from light cells. Both large and small microvilli were fbund in gill arch (Figs. 12 and 22) and gill raker taste buds (Fig. 16). DISCUSSION The few ultrastructural observations of teleost epithelia emphasize palatal mucosa as studied in the stickleback, Gasterosteus acuhatus (Whitear, 19711, the tropical catfish Corydoras juZZi, and Helostoma temmincki, the kissing fish (Albright and Skobe, 1965). Only Reutter et al. (1974) have described the gill buccopharyngeal epithelium a t the ultrastruc- Abbreviations A, gill arch LC,light taste cell BC, basal taste cell BP, buccopharynx BE, basal epithelial cell BL, basement lamella BM, basement membrane CB, cross bridges CH, channel CL, clear cell DC,dark taste cell G, granule GR, gill raker GS,gill slits H%E,hematoxylin and eosin IR,interraker region of gill arch Lp,lamina propria MC, marginal cell MI, mitochondria MR, microridge MT, microtubule MU, mucow cell MV, microvilli NP, nerve plexus PA. oaoilla SE,' kf'ace epithelial cell TB, taste bud To1 B1,toluidine bluestained Epon-embedded material TP, taste pore VE,vesicle Fig. 1. Section through gill arch, primary lamellae, and eeeondary lamellae. In buecopharynx, epithelium covers gill rakers and interraker region of gill arches. h&e, x 50. Fig. 2. Gill arch epithelium. Basal cells encircle ‘nests” of clear cells. Mature mucous cells are loeated in surface region of epithelium. A taste bud extends from basement membrane to the surface. To1 bl, X 600. Fig. 3. Buccopharyngeal surface of one gill arch. A double row of gill rakers projects from each gill arch. Numerous taste buds ( t ) are Seen on this surface. x 175. 72 ELIZABETH R. WALKER ET AL. Fig. 4. Buccopharyngeal epithelium. Mueous cells ( t ) are seen in upper one-half of epithelium. x 1,300. Fig. 5. Basal region of epithelium. Basal epithelial cells rest on basement membrane. Cytoplasmic processes of these cells partially surround “nests” of clear cells. x 2,750. Fig, 6. Basal region of epithelium. Basement lamella underlying basement membrane consists of many layers of collagen in orthogonal pattern. x 11,250. FATHEAD MINNOW GILL MORPHOLOGY 73 Fig. 7. Epithelial cell surface. Free surface of epithelial cells are shaped in folds forming a micmridge system. Note cross bridges between microridges. x 17,500. Fig. 8. Surface epithelial cell. Surface epithelial cell has cytoplasmic granules. Surface coat material can be Seen on sectioned microridges. x 18,750. Fig. 9. Surface region of epithelium. Cytoplasmic projections from surface epithelial cells overlap apical portion of mature mucous cell. x 4,400. 74 ELIZABETH R. WALKER ET AL. Fig. 10. Buccopharyngealsurface of gills. Gill rakers project from each arch interdigitating with rakers from adjacent arch. x 18. Fig. 11. Surface of gill arch. Epithelium covering gill arch contains rounded elevations. x 300. Fig. 12. Surface view of single rounded elevation. Cell boundaries of surface epithelial cells are delineated by more prominent mimridges. Emerging from the elevation are short and tall microvilli comprising the taste pore of a taste bud. x 7,500. Fig. 13. Longitudinal section through interraker taste bud of gill arch. Basement membrane forms cup around base of taste bud. Nerve plexus extends from lamina pmpria through basement membrane to base of light and dark receptor cells. Microvilli project from apical portion of the receptor cells through taste pore. To1 bl, x 960. FATHEAD MINNOW GILL MORPHOLOGY 75 Fig. 14. Surface view of gill rakers. The tip of each raker has a cap lacking the characteristic microridge system. A double row of raised projections, termed papillae, line each raker. x 15. Fig. 15. Surface of paillae on gill raker. Each papilla contains a taste bud with surface projections. x 1,500. Fig. 16. Gill raker papilla. A tuft of tall and short microvilli project from the taste pore in each papilla. "he free surface of the epithelial cells shows the characteristic microridge system. X 3,000. Fig. 17. Longitudinal section through gill raker taste bud. Taste bud extends from basement membrane to free surface of epithelium. Microvilli from dark and light cells project out of taste pore to point above epithelial surface. To1 bl, x 960. 76 ELIZABETH R. WALKER ET AL. FATHEAD MINNOW GILL MORPHOLOGY tural level. The similarities of the fathead minnow buccopharyngeal epithelium, to teleost skin, and gill respiratory epithelium led us to compare these three. Further justification for this comparison lies in the common interface of these surfaces with the aqueous environment and their exposure to the same ionic, chemical, and mechanical stress. The cell types of the epithelium to be considered are filament-containing, goblet mucous, immature mucous, undifferentiated, and the cells of the taste buds. Another feature to be considered is the prominent basement lamella underlying this epithelium. Filament-containing cells have been regarded as the most numerous cell type in teleost skin and oral epithelium (Henrikson and Matoltsy, 1968a; Whitear, 1971; Merrileev, 1974).As the name implies, this cell type contains numerous filaments. The central nucleus is immediately surrounded by a cuff of clustered organelles. Numerous desmosomes also characterize this cell type, which is found in basal and midregions of the fathead buccopharyngeal epithelium. The surface epithelial cells in teleost skin and oral epithelium usually are classified with the filament-containing cells, but have the specialization of a microridge pattern. This microridge system in the gill buccopharyngeal region is similar to that in teleost skin (Hawkes, 1974; Merrilees, 1974) and in the head gut of Xiphorus helleri, the swordtail fish (Reutter et al., 1974).The latter study reported similar cross bridges between microridges to those which we observed. In the respiratory portion of the gill, the fathead minnow exhibits a shallow microridge pattern on the primary lamellae of the gill, as do many other species of teleosts (Hughes and Wright, 1970; Hughes, 1979; Olson and Fromm, 1973). Unpublished work in this laboratory has shown that minnow primary lamellar ridge pattern is much less pronounced than that of skin or buccopharyngeal epithelium, and that secondary lamellae do not exhibit any regular microridge pattern. 77 It is thought that the microridge system functions to prevent washing away of the mucous coat from the epidermal, buccopharyngeal, and respiratory surfaces (Hughes and Wright, 1970; Hawkes, 1974). In addition to retaining secretions, the increase in surface area may better enable the surficial tissue to function in exchange reactions. Although some investigators question the role of microridges in gas exchange (Hughes, 1979), Olson and Fromm (1973) reported that microridges increased absorptive surface of cells in gill of rainbow trout Salmo gairdneri by 2.5 times. Bereiter-Hahn et al. (1979) observed structural and functional similarities between microridges and microvilli in cell culture. Goblet mucous cells are the most common mucus-producing cell in teleost skin, oral epithelium, and gill interlamellar region. These cells vary in number depending upon location and species (Henrikson and Matoltsy, 1968b). The electron microscopic analysis of buccopharyngeal epithelium in the present study showed mature goblet cells in the superficial one-third. This finding correlated with the histochemical analysis in which stainable mucous granules were numerous in the superficial layer, rare in the intermediate zone, and lacking in the basal zone. As in other teleost species, the fathead minnow goblet mucous cells were seen on the surface of the primary lamellae and in the interlamellar region between secondary lamellae. Immature mucous cells similar to those of this study have been described in the basal and intermediate zones of epidermis from various teleost species (Wellings et al., 1967; Henrikson and Matoltsy, 196813; Whitear, 1971; Merrilees, 1974; Hawkes, 1974). Rounded cellular profiles, a cytoplasm of medium to high electron density, abundant parallel membranes of rough endoplasmic reticulum, and absence of cytofilaments identify them. A progression from cells with abundant rough endoplasmic reticulum, to cells with rough endoplasmic reticulum and a few Golgiassociated mucous vesicles, to cells with pack- Fig. 18. Taste bud. Nuclei of light and dark cells are basal in location. Cytoplasm of light cells contains membranelined cisternae. Dark cell cytoplasmic processes go around light cells to reach nerve plexus which extends through basement membrane. Marginal cells are located around periphery of taste bud. x 1,600. Fig. 19. Light and dark cells. Dark cell cytoplasm contains granules. Light cell is characterized by electron-lucent cytoplasm, elongated mitochondria, and membrane-boundchannels near the outer cell membrane. x 13,500. Fig. 20. Light and dark cells. Dark cell is characterized by cytoplasm filled with longitudinally oriented microtubles. Light cell has an electron-lucent cytosol, elongated mitochondria, a few mimtubules, and many membrane-bound cietemae. x 40,OOO. ELIZABETH R. WALKER ET AL. Fig. 21. Taste bud apical region. The apical region of the light cell contains small vesicles and terminates in a single tall, thick microvillus. Dark cells have smaller, more numerous mimvilli, covered by cell coat material. x 14,400. Fig. 22. Taste pore surface view. Taste pore shows a few tall, thick microvilli and numerous small microvilli. x 21,000. FATHEAD MINNOW GILL MORPHOLOGY ets of mucous vesicles in a mature goblet pattern has been proposed (Henrikson and Matoltsy, 196813; Merrilees, 1974). In our specimens of buccopharyngeal epithelium, intermediate forms between the above-described immature and mature (goblet) mucous cells were rare. Thus, our findings to date cannot refute or confirm a similar progression of stages in buccopharyngeal goblet cell development. ”Undifferentiated or “stem” cells occurring in nests between basal cells and filament-containing cells of the minnow buccopharyngeal epithelium are apparently unique to this site. Trimary” cells, lying between claviform processes of basal cells, were described in the skin of pike (Merrilees, 1974). However, these “primary” cells showed characteristics of immature mucous cells, including rough endoplasmic reticulum, not seen in the undifferentiated cells of our study. In this study the electron-dense granules of undifferentiated cells were similar in size and appearance to those of the surface epithelial cells. However, no filaments were observed in these stem cells, which would suggest their eventual development as surface epithelial cells. Thus, the relationship of these cells to definitive, differentiated surface cell types has not been determined. Another “undifferentiated cell type, the basal cell, was morphologically similar to the basal cell in teleost epidermis and oral epithelium (Henrikson and Matoltsy, 1978a; Whitear, 1971; Merrilees, 1974). From their numerous cytoplasmic filaments, these cells are generally classified with the other filament-containing cells. Initial studies underway in our laboratory to determine the effect of acid mine water upon skin, buccopharyngeal, and respiratory epithelium of the fathead minnow, show increased mucus production, induced by intermittent exposure to acid. Such an “induced” epithelium may help determine the role of undifferentiated cells in development of specific adult cell types. Two cell types commonly encountered in teleost epidermis were not seen in the buccopharyngeal epithelium. The first of these is the club cell, a large unicellular gland present in the middle layer of teleost epidermis (Henrikson and Matoltsy, 1968~).The second cell not seen in gill buccopharyngeal epithelium is the chloride cell. We have seen chloride cells on gill primary and secondary lamellae of fathead minnows where they are easily recognized by their abundant mitochondria and agranular 79 endoplasmic reticulum (Kessel and Beams, 1962). The description by Albright and Skobe (1965) of “differentiated cells” in palatal epithelium of Corydoras is identical to that of chloride cells. Furthermore, chloride cells have been found in oral epithelia but not the buccopharyngeal region of other teleosts (Whitear, 1971). Cuticle was not seen in the minnow buccopharynx in the present study. We observed dense granules within surface cells similar to those described in oral epithelium (Albright and Skobe, 1965) and skin (Whitear, 1971), where they have been postulated to contribute to the formation of an overlying cuticle. Our scanning and transmission electron microscopy however, revealed no cuticle. Basement lamella, an orthogonal array of collagen fibrils underlying the basement membrane, has been described in the skin of some aquatic vertebrates (Nadol et al., 1969; Henrikson and Matoltsy, 1968a). Since the basement lamella varies in thickness in different regions and may not exist in some species, a n alternate view of this layer is as an extension of the collagen layer of the basement membrane with no special designation as the basement lamella. Albright and Skobe (1965) reported a thick, well-organized basement lamella in the lamina propria of palatal epithelium of the tropical catfish, but not in the kissing fish. Morgan and Tovell (1973), in a study of the gill of trout (Salmo gairdneri), described the collagen layer underlying the buccopharyngeal epithelium as being continuous with the connective tissue core of the gills, but not a basement lamella. In this study, because the collagen was highly organized and consisted of approximately 20 plies, the region underlying the fathead minnow buccopharyngeal epithelium was designated a basement lamella, most similar to the basement lamellae described underlying some species of teleost skin (Henrickson and Matoltsy, 1968a) and palatal epithelium (Albright and Skobe, 1965). Taste bud Our SEM data suggest two structurally different taste bud types in the buccopharyngeal cavity of the fathead minnow. Using the taste bud classification of Reutter (19731, type I1 (raised) and I11 (depressed) taste buds were found on the buccopharyngeal surface of the gill-type I1 in the gill rakers, and type I11 buds in the gill arches. Taste buds of both types showed tall and short receptor microvilli. The 80 ELIZABETH R. WALKER ET AL. surface morphology of receptors was similar to that described in sword-tail type I1 and type I11 taste buds (Reutter, 1973). Taste bud is the term used to describe chemoreceptors and mechanoreceptors in fish and is based on ultrastructural similarity to descriptions of taste organs in other vertebrates (Graziadei, 1968; Farbman, 1965). Widely distributed, taste buds of fishes have been described in headgut, palate, oral papillae, oropharynx, and, in some species, within epidermis over the entire body including the tail region (Reutter et al., 1974;Ono, 1980;Bardach and Villars, 1974).Taste buds in barbels of various catfishes have received the most attention. Descriptions exist for Amiurus nebulosus (Reutter, 1971, 1978); Clarias batrachus and Kryptopterus bicirrhus (Welsch and Storch, 1969);Zctalurus pumtutus (GroverJohnson and Farbman, 1976; Joyce and Chapman, 1978);and Corydoras paleatus (Fujimoto and Yamamoto, 1980).In the above, four cell types have been commonly encountered. These include (1) marginal cells, peripherally situated epithelial cells, thought to represent transitory forms between epidermal and sensory cells; (2) basally positioned light ovoid cells, a sensory or ganglionic cell ( h u t ter, 1971, 1978) located a t the bases of light and dark cells and resting upon the basement membrane; (3) elongated light cells usually referred to as receptor cells (Fujimoto and Yamamoto, 1980;Welsch and Storch, 1969)with one club-shaped microvillus and little to no cell coat material; and (4)elongated dark cells, supportive cells (Fujimoto and Yamamoto, 1980)with small microvilli and a network of fibrillar and punctate cell coat. Taste buds in buccopharyngeal epithelium of the fathead minnow closely paralleled the above descriptions for similar structures in other tissue sites of various fishes. All the above cell types were seen. The electron-lucent nature of cytosol coupled with the large smooth membrane-delimited channels in light cells of the present study, suggested cellular injury in which high-amplitude swelling of endoplasmic reticulum is a common feature (Trump and Arstila, 1975).Welsch and Storch (1969)and Grover-Johnson and Farbman (1976) made similar observations in catfish taste buds. Mammalian taste bud cells are continually changing, dying and being replaced (see review by Farbman, 1965) and life spans from 3 to 5,up to 12 days have been reported (Biedler and Smallman, 1965; Crisp, 1975). Cell turnover studies in taste buds of channel catfish (Ictulurus punctatus) barbels indicated a temperature dependance with cell life spans of 15 and 12 days a t 22 and 30"C respectively (Raderman-Little, 1979).The close similarity of the smooth membrane-delimited vesicles and channels to cisternae of smooth endoplasmic reticulum has led some workers (GroverJohnson and Farbman, 1976) to the conclusion that these were cellular organelles. However, most recent work (Fujimoto and Yamamoto, 1980),employing lanthanum nitrate, showed vesicles, saccules, and channels to be invaginations of plasma membrane. We saw material in laterally placed channels of light cells which resembled cell coat. However, further correlated histochemical and cytochemical studies are underway to determine the nature of these structures. ACKNOWLEDGMENTS We thank Mr. Phillip Allender and Ms. Barbara Foster for technical assistance. 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