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Morphology of the buccopharyngeal portion of the gill in the fathead minnow Pimephales promelas (Rafinesque).

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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.
This study was supported in part by the
Water Research Institute, West Virginia University, with funds allocated under the Water
Resources Act of 1964 (PL 88-379)administered by the Office of Water Research and
Technology, U.S.Department of the Interior.
The work was done as part of Project A-037WVA, D. E.Hinton, Principal Investigator.
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