Cell and Tissue
Research
Cell Tissue Res. 213, 361-367 (1980)
9 by Springer-Verlag 1980
The Secondary Lamellae of the Gills
of Cold Water (High Latitude) Teleosts*
A Comparative Light and Electron Microscopic Study
R.B. Boyd 1, A.L. DeVries 2, J.T. E a s t m a n 3, and G.G. Pietra 1
1 Departments of Medicine and Pathology, University of Pennsylvania, Philadelphia, Pennsylvania,
USA;
2 Department of Physiology and Biophysics, University of Illinois, Urbana, Illinois, USA;
3 Department of Zoology and College of Medicine, Ohio University, Athens, Ohio, USA
Summary. The fine structure o f the secondary lamellae o f gills was examined in
two cold-water marine teleosts, the winter flounder, Pseudopleuronectes
americanus, and the antarctic cod, Trematomus borchgrevinki. In b o t h species
the overall lamellar fine structure is similar to that o f other marine teleosts. The
major variations in cellular organization involve the distribution o f b o t h the
"chloride cells" and the m u c o u s cells on the secondary lamellae o f P.
americanus. At winter water temperatures o f + 2 . 5 ~
significantly m o r e
chloride and m u c o u s cells are present than in s u m m e r with water temperatures
o f + 15.2 ~ C. B o t h cell types are routinely present t h r o u g h o u t the length o f a
secondary lamella as far as the lamellar tip. The chloride cells on the secondary
lamellae are always situated in the inner layer o f epithelium deep to the outer
pavement cells. T. borchgrevinki shows no apparent difference in the
distribution o f m u c o u s cells either at its n o r m a l water temperature o f - 1 . 9 ~ C or
at a temperature o f + 4 ~ C, the upper limit o f its thermal tolerance to which
some specimens were adapted in the aquarium. Chloride cells were never
observed on the secondary lamellae o f T. borchgrevinki. This suggests that low
environmental water temperatures m a y be related to the distribution o f m u c o u s
cells and chloride cells on the secondary lamella o f the teleost gill.
Key words: Gills - Chloride cells - M u c o u s cells - Winter flounder - Secondary
lamellae.
The fine structure o f the gills o f several species o f teleosts has been extensively
investigated (Hughes and G r i m s t o n e 1965; Newstead 1967; H u g h e s and W r i g h t
Send offprint requests to: Robert B. Boyd, Ph.D., Cardiovascular-Pulmonary Division, Department of
Medicine, 975 Maloney Building, Hospital of the University of Pennsylvania, 3600 Spruce Street,
Philadelphia, PA 19104, USA
* Supported in part be research grants from NSF, DPP 77-15612 and NIH HL-08805
Deepest appreciation is extended to Mr. Lewis W. Johns for his expert technical advice and to Mr.
Daniel C. Barrett and Ms. Susan M. Pharaoh for their assistance in the preparation of this manuscript
0302-766X/80/0213/0361/$01.40
362
R.B. Boyd et al.
1970). Studies dealing either with gill vascularization and respiration (Steen and
Berg 1966; Morgan and Tovell 1973), or "chloride cell" structure and function in
fresh or salt water environments (Keys and Willmer 1932; Philpott and Copeland
1963; Olson and Fromm 1973; Sardet et al. 1979) have also included descriptions of
the morphology of secondary lamellae. However, these studies refer only to
temperate marine and freshwater species.
Certain marine fishes normally inhabit waters with temperatures at or below the
freezing point of sea water during all or part of the year. The effects of low
environmental temperatures on the fine structure and distribution of gill cells have
not been investigated. To assess cellular adaptations and specializations at these
temperatures, we examined the secondary lamellae of two high latitude fishes, the
North Atlantic winter flounder, Pseudopleuronectes americanus, and the antarctic
cod, Trematomus borchgrevinki.
Materials and Methods
1. Animals
Winter flounder, Pseudopleuroneetes americanus, were captured by otter trawl in Sandy Hook Bay, New
Jersey. The winter specimens were taken from + 2.5~ waters, 22 ppt salinity, while the summer
specimens came from + 15.2 ~ C waters, 27 ppt salinity. Antarctic cod, Trematomus borehgrevinki were
caught by jig line, through the ice covering t h e - l . 9 ~ C waters of M c M u r d o Sound, Antarctica. A number
of T. borehgrevinki were adapted to + 4 ~ C water for five weeks in a plastic aquarium equipped with a
running sea water system. This temperature is near the upper limit ( + 6 ~ C) of normal thermal tolerance
of T. borchgrevinki (Somero and DeVries 1967).
2. Tissue Preparation
Individual gill arches were removed from each species. Several primary gill filaments were severed from
each arch, cut into 1 m m 3 pieces and placed in Karnovsky's fixative, pH 7.3, having an osmolarity of
approximately 800 mosmols. This osmolarity was necessarily high because the body fluids of these fish
are of greater ionic strength and also contain either peptide or glycopeptide antifreeze c o m p o u n d s
(DeVries 1971; D u m a n and DeVries 1974). All tissues were postfixed in 1% OsO 4 in 0.1 M cacodylate
buffer, en bloc stained with uranyl acetate in sodium maleate buffer, and embedded in Epon 812.
Embedding in flat molds ensured proper orientation and plane of sectioning of the secondary lamellae.
Thick sections (1-2 ~tm) were stained with 1% toluidine blue in 0.1% sodium borate. Sections with
silver interference colors were stained with uranyl acetate and lead citrate and examined in either a
Siemens 101 or a JEOL 100S electron microscope at 80 KV.
3. Cell Distribution Analysis
Representative thick sections from P. americanus were evaluated for orientation to ensure proper cross
sectional views of the secondary lamellae on each gill filament. Five animals from each temperature
( + 2.5 ~ C, + 15.2 ~ C) were used. The total number of epithelial cells in each section was determined by
counting individual nuclei. Mucous and chloride cells could be individually counted because of their
distinctive staining characteristics. The percentage of each cell type in the total population was
calculated. The corresponding cell populations from the two temperatures were analyzed by use of the
two sided t-test for the differences between means of two groups of data.
Secondary Lamellae of Gills of Cold Water Teleosts
w
363
Bt_
O
PC
VC
Fig. 1. Pseudopleuronectes. Double layer of respiratoryepithelium(EP) and supporting pillar cell (PC)
of a secondarylamella; BL basal lamina; VC vascular channel
Results
In both species the general morphology of the secondary lamellae is similar to that
of other marine teleosts. The lamellae are covered with a double layer of squamous
cells (respiratory epithelium) while the vascular channels within the lamellae are
lined with two different endothelia. The two layers of the respiratory epithelium
typically consist of outer pavement cells and inner undifferentiated cells resting on a
prominent basal lamina (Fig. 1). The pavement cells have a dense layer of filaments,
,,~0.5 lam thick, just below their surface and elaborately interdigitating lateral
borders with tight junctions at the cell/water interface. The pavement cell of both
species contains numerous mitochondria, an extensive Golgi apparatus, some
filamentous bundles, and considerable rough endoplasmic reticulum along with
some free ribosomes. The inner cells contain large nuclei and many mitochondria as
well as free ribosomes and a well developed rough endoplasmic reticulum.
The pillar cells o f the secondary lamellae separate the vascular channels and
support the respiratory epithelium (Fig. 1). The flangelike extensions of the pillar
cells function as the endothelial surface for most of the lamellar capillary bed.
Adjacent cells are joined by a junction resembling the punctate junction that
connects the cerebral capillary endothelium in the hagfish (Bundgaard et al. 1979).
No adhesion plates or nexus were observed. The basal lamina is continuous with
that of the marginal endothelial cells and primary filament epithelium. The
dominant features of the pillar cells are bundles of approximately 60A filaments,
364
R.B. Boyd et al.
Fig. 2. Pseudopleuronectes. Secondary lamella with typical mucous cell (MC), chloride cell (CC) and
pillar cell (PC). Note location of chtoride cell deep to extension (arrow) of outer pavement cell. • 4,800
Fig. 3. Pseudopleuronectes. Light micrograph showing chloride cell (CC) and numerous mucous cells
(arrows) on secondary lamellae, x 160
p r o b a b l y contractile (Bettex-Galland and Hughes 1973), seen t h r o u g h o u t the
cytoplasm.
The peripheral vascular channels o f the lamellae are lined with an endothelial
cell type characteristic o f a respiratory endothelium. This marginal endothelium is
somewhat thicker than the respiratory capillary endothelium o f mammals. It
contains fewer filaments than the pillar cell, numerous mitochondria and profiles o f
r o u g h endoplasmic reticulum. This cell also contains electron dense osmiophilic
Weibel-Palade bodies (Weibel and Palade 1964) and large vacuoles o f u n k n o w n
function. Cytoplasmic vesicles appear to be less a b u n d a n t than in m a m m a l i a n
Secondary Lamellae of Gills of Cold Water Teleosts
365
Secondary Iomella cell
distribution in winter flounder
100 --
,-.{_ P<O.O1
F
•
Winter temp.
- - ' ] S u m m e r temp.
In=5)
0
I--8
50
~////
z/z/.,
. . . .
]~ +S.E.
A
~ / I I A
~///A
"///4
N
P<O.03
NEP
MC
CC
Cell type
Fig. 4. Seasonal changes in specific cell distribution in secondary lamellae of Pseudopleuronectes
expressed as percent of total ceils present. EP epithelial cells; M C mucous cells; CC chloride cells
endothelium. Adjacent cells are occasionally connected by a type of junction more
chracteristic of nonendothelial epithelial cells.
The secondary lamellae o f both species have two unusual features: (1) Scattered
throughout the respiratory epithelium o f P . americanus and T. borchgrevinki from
all temperature regimens are numerous large, ovoid mucous cells (Fig. 2). (2) P.
americanus also has numerous mitochondria-rich "chloride cells" distributed along
the entire length of a secondary lamella as far distally as the lamellar tip (Fig. 3). In
all P. americanus examined, the chloride cells on the secondary lamellae are always
situated in the inner layer of epithelium (Fig. 2). Chloride cells are absent on the
secondary lametlae of T. borchgrevinki, but occur in the interlameltar pits and on
the gill filaments.
Comparison of the lamellae from P. americanus indicates that fewer mucous
and chloride cells are present in fish from warmer water. To determine the effect of
thermal history on cell population, we counted the total number and types of cells
on random lamellar cross sections. Cell distributions of five P. americanus from each
temperature are presented in Figure 4. The means for the total number of secondary
366
R.B. Boyd et al.
lamellar cells are 944 from each cold temperature fish and 972 from each warm
temperature fish. There is no significant difference between the means of the total
numbers of cells. However, we observed a significant decrease in the percentage of
both mucous and chloride cells in P. americanus from warmer waters.
We did not quantify cell types on the secondary lamellae of T. borchgrevinki.
However, mucous cell populations appeared to remain constant upon + 4~
acclimation. Furthermore, mucous cells seem to be as common in T. borchgrevinki
as they are in P. americanus from + 2.5~ water.
Discussion
The differences in the organization of secondary lamellae of gills of Pseudopleuronectes and Trematomus from that of marine teleosts from temperate water possibly
represent adaptations to the low environmental temperatures normally experienced
by these species. Although the presence of chloride cells on secondary lamellae has
been previously reported (Hughes and Wright 1970), they are more abundant in P.
americanus. Moreover, more chloride cells are present during the winter when the
fish is feeding less (Umminger and Mahoney 1972) and its metabolism is depressed
(Rowell et al. 1975) than during the summer period when the fish has an increased
ionic intake because of ingestion of invertebrates and sea water through feeding.
Electron microscopic studies by Morgan (1974) indicate that chloride cells
normally differentiate from the epithelial covering of developing secondary
lamellae and that these cells are more numerous in developing gills than in the adult.
Laurent and Dunel (1980) showed that the inner epithelial cells of secondary
lamellae retain the potential to differentiate into chloride cells. Mattheij and
Stroband (1971 ) found that prolactin stimulates the differentiation of chloride cells
from secondary lamellar epithelium in adult Cichlasoma biocellatum maintained in
25 % sea water. I f such chloride cells o f P . americanus in winter temperature are in
some stage of differentiation, they do not function as mature chloride cells.
In P. americanus an unknown mechanism, possibly endocrine, may trigger
proliferation of immature chloride cells during the colder months when the fish is
subject to less osmotic stress and the sea water salinity is fairly constant. As the
water warms to summer levels, and the fish becomes more active metabolically, the
chloride cells on the secondary lamellae mature and migrate to the primary filament
epithelium. Therefore, fewer chloride cells are present on the secondary lamellae,
but the total chloride cell population of the entire gill epithelium has increased. This
would be compatible with the data of Conte and Lin (1967) indicating that, with
increasing salinity, the total chloride cell count of the gill epithelium increases.
The mucous cells on the secondary lamellae of teleost gills produce a film that
probably protects the gills against physical abrasion and also functions as an
antibacterial agent (Hughes and Wright 1970). Umminger and Kenkel (1975)
showed increased mucous cell activity in Fundulus heteroclitus acclimated to - 1~ C
in salt water. Our data indicate that gills ofP. americanus, taken from near 0 ~ C sea
water, contain a significantly higher number of mucous cells than those in
temperate sea water. Secondary lamellae of T. borchgrevinki from both - 1.9 ~ C
and + 4 ~
water, also have abundant mucous cells. A similar mucous-cell
Secondary Lamellae of Gills of Cold Water Teleosts
367
distribution was noticed in other antarctic fishes inhabiting - 1 . 9 ~
sea water.
Among these species are T. loennbergii, T. nicolai, Gymnodraco acuticeps and
Dissostichus mawsoni. Whether or not these extra mucous cells have a specialized
function at near 0~ temperatures has yet to be investigated.
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Accepted August 19, 1980