Parasitol Res (1988) 74:352-355
Parasitology
Fine structure and permeability of the metacercarial cyst wall
of Clinostomum marginatum (Digenea)*
Omer R. Larson 1, Gary L. Uglem 2, and Kook J. Lee 2
1 Department of Biology, University of North Dakota, Grand Forks, ND 58202, USA
2 Physiology Group, School of Biological Sciences, University of Kentucky, Lexington, KY 40506, USA
Abstract. Encysted metacercariae of Clinostomum
marginatum (Digenea) were obtained from tissues
of yellow perch, Perca flavescens. The outermost
wall (host response) as seen under electron micros-
copy consisted of a single, fibrous tissue layer,
10-25 gm thick. The tissue contained flattened fi-
brocytes, small fat deposits, and vacuoles embed-
ded between layers of collagen fibers. The cyst cav-
ity was filled with small vesicles, crystals, and de-
bris. No layer corresponding to the primary (para-
site-produced) cyst wall secreted by most species
of metacercariae was noted. To determine the per-
meability of the cyst wall, encysted worms were
incubated under initial rate conditions with [3H]
glucose, with and without the glucose transport
inhibitors phlorizin and phloretin. After incuba-
tion, the worms were mechanically excysted,
washed, and processed to determine glucose up-
take rates. Vmax and Kt were greater than those
obtained for worms excysted prior to incubation
with substrate. Moreover, the presence of phlorizin
or phloretin in the incubation medium had no ef-
fect on glucose uptake by encysted worms. Thus,
the selective permeability of the cyst wall permits
free diffusion of glucose to the cutaneous transport
systems of the worm, while restricting the move-
ments of phlorizin and phloretin.
Many digenean flukes develop as metacercariae in
the muscles'and connective tissues of second inter-
mediate hosts. These tissues are rich in glucose and
other dissolved organic nutrients. Although a few
* This study was supported by a grant from the National
Science Foundation (PCM-8203378) and by the Biology De-
partment, University of North Dakota.
Reprint requests' to: O.R. Larson
Research
9 Springer-Verlag 1988
species develop without encystment, most form a
cyst wall. Thus, the uptake of nutrients by encysted
forms could be influenced by the thickness, struc-
ture, and permeability of the cyst wall (Halton and
Johnston 1982).
The present report concerns the fine structure
and permeability of the metacercarial cyst wall of
Clinostomum marginatum. This larval stage is cos-
mopolitan in freshwater fishes; the adults occur
in the pharyngeal cavities of certain fish-eating
birds. Outside of North America, C. marginatum
is referred to as C. complanatum. The metacercaria
is well adapted for absorbing glucose through the
tegument. On the basis of the results obtained with
worms excysted before incubation in substrate,
glucose uptake was found to be accelerated by the
simultaneous operation of both facilitated diffu-
sion and active transport systems (Uglem and Lar-
son 1987). To determine how glucose uptake is
influenced by the cyst wall, we tested encysted
worms, with and without glucose transport inhibi-
tors, and compared the rates to those of excysted
worms. The fine structure of the cyst wall is also
described and compared with previous histologic
(Osborn 1911 ; Hunter and Dalton 1939) and histo-
chemical accounts (Kalantan et al. 1986).
Materials and methods
Yellow perch, Perca flavescens, infected with C. marginatum
were collected from Grace Lake, Minnesota (USA), during the
summer of 1986. The fish were kept on ice and dissected within
2 days of capture. Cysts were separated from host tissue and
washed three times in cold Tyrode's saline. Several cysts were
fixed in 2.5% glutaraldehyde and prepared for ultrastructural
examination using the methods of Uglem and Lee (1985).
Epoxy resin-embedded cysts were sectioned and examined in
a Hitachi 500 transmission electron microscope. For glucose
uptake studies, cysts in groups of two or three were preincu-
bated in Tyrode's saline at 25 ~ C. Any worm excysting sponta-
neously during preincubation was discarded and replaced with
O.R. Larson et at. : Metacercarial cyst wall of Clinostornum
Fig. 1. Transmission electron micrograph of the metacercarial
cyst wall and tegument (teg) of Clinostomum marginatum. The
cyst wall consists of fibrocytes with elongate nuclei (n), collagen
fibers (c), vacuoles (v), and electron-dense fat deposits (arrow).
The asterisk indicates a cyst cavity. Scale bar=4 gm
an encysted specimen from a reserve pool. After 15 min of pre-
incubation, the saline was removed by aspiration and 0.5 ml
prewarmed saline containing [3H]D-glucose (New England Nu-
clear), with and without 0. lmM phlorizin or phoretin, was add-
ed. After 5 min at 25~ C, the incubation was stopped by adding
5 ml ice-cold saline. Cysts were washed twice in cold saline
and mechanically excysted with fine needles under a dissecting
microscope. Excysted worms were washed three times, extract-
ed in 70% ethanol for 24 h, and dried for 24 h at 95~ C. Uptake
rates were calculated from the radioactivity in the ethanol ex-
tracts and the ethanol-extracted dry wt. of the worms. Differ-
ences were analyzed using a Student's t-test, with a value of
P < 0.05 considered significant.
Results
M a t u r e m e t a c e r c a r i a l cysts o f C. m a r g i n a t u m w e r e
spherical o r slightly oval, m e a s u r i n g a b o u t 2 - 3 m m
in d i a m e t e r . T h e c y s t wall c o n s i s t e d o f a single
layer o f f i b r o u s tissue, 1 0 - 2 5 g m t h i c k (Fig. 1).
C o m p r e s s e d f i b r o c y t e s w i t h e l o n g a t e nuclei w e r e
353
t
A /
/
, 0
0 2 4 6 8 10
8
B
"
4
o o
0 2 4 6 1
~Glucose'] , mM
Fig. 2A, B. [3H]Glucose uptake (V, gmol/g ethanol-extracted
dry wt. per h) by encysted metacercariae (e-e) of C. margina-
turn. In A, the slope of the line between 5 and 10 mM glucose
(lowest dashed line), fitted by regression analysis (r = 0.98), rep-
resents the rate of glucose diffusion into the worm. The upper-
most dashed line, fitted by regression analysis (r=0.99) of the
observed uptake by encysted worms, between 0.1 and 1.0 mM,
was considered an estimate of glucose diffusion through the
cyst wall (see text). Included in A, for comparison are the ob-
served [3Hlglucose uptake rates (o-o) and diffusion (middle
dashed line) for worms excysted prior to incubation with sub-
strate (from Fig. 1 in Uglem and Larson 1987). The two curves
in B represent mediated uptake by encysted (e-e) and excysted
worms (o o), obtained by subtracting diffusion from the ob-
served values. Each point is the mean _+SE of three replicates
e m b e d d e d b e t w e e n thin, i r r e g u l a r layers o f colla-
gen fibers. This tissue w a s c h a r a c t e r i z e d b y i r r e g u -
larly s h a p e d spaces o r v a c u o l e s a n d o c c a s i o n a l de-
p o s i t s o f fat. T h e cyst c a v i t y c o n t a i n e d f l o c c u l e n t
m a t e r i a l , vesicles, crystals, a n d debris ( n o t s h o w n
in Fig. 1).
[ a l l ] G l u c o s e u p t a k e b y e n c y s t e d w o r m s dis-
p l a y e d s a t u r a t i o n kinetics b e t w e e n 1 a n d 5 m M
s u b s t r a t e c o n c e n t r a t i o n s (Fig. 2 A ) . T h e slope o f
the line b e t w e e n 5 a n d 10 m M (lowest d a s h e d line)
w a s c o n s i d e r e d the d i f f u s i o n c o m p o n e n t . D i f f u -
sion-corrected rates (i.e., mediated uptake;
Fig. 2 B ) were o b t a i n e d b y s u b t r a c t i n g d i f f u s i o n
f r o m the o b s e r v e d values. T h e m e d i a t e d u p t a k e
rates (in g m o l / g e t h a n o l - e x t r a c t e d d r y wt. p e r
354
hour) below 5 m M were 0.35 (0.1 mM), 0.56
(0.25 mM), 1.14 (0.5 mM), 2.06 (0.75 raM), 2.90
(1.0 mM), and 4.38 (2.5 mM). These values were
paired (0.35 and 2.06; 0.56 and 2.90; 1.14 and 4.38)
and analyzed using the algebraic method of Neame
and Richards (1972); Vm,~ was 11.5_+ 1.95 ~tmol/g
ethanol-extracted dry wt. per h, and Kt was
3.7_+ 1.19 mM.
The inhibitory potencies of 10 m M unlabeled
glucose, 0.1 m M phlorizin, and 0.1 m M phloretin
on the uptake of 0.1 m M [3H]glucose were deter-
mined. The uptake rate (in ~tmol/g ethanol-extract-
ed dry wt. per hour) in the absence of inhibitors
(0.29) was inhibited 100% (i.e., 0.04) by unlabeled
glucose, but was not significantly affected when
either phlorizin (0.31) or phloretin (0.33) was pres-
ent in the medium bathing the cysts.
Discussion
The metacercarial cyst wall of C. marginatum is
similar, with minor variations, to those of other
species of Clinostomum. There is no inner, acellular
or hyaline layer corresponding to the primary or
parasite-induced cyst wall often seen in other spe-
cies of metacercariae (for reviews see Erasmus
1972; Cheng 1973; Smyth and Halton 1983). In
Clinostomum, collagen is the major component of
these walls, which range in thickness from
10-25 txm in C. marginatum to 2 9 4 0 ~tm in C. tila-
piae (Asanji and Williams 1973). Cyst wall thick-
ness, however, can vary greatly with the age of
the larva, site of infection, and species of fish host
(Hunter and Dalton 1939). Cyst walls formed near
external surfaces of the host are usually thicker
than those lodged in deeper tissues, due to the par-
tial incorporation of host dermis. C. complanatum
does not induce cyst wall formation in some species
of fish (Siddiqui and Nizami 1982), but when the
wall is present, it can be shown by histochemical
tests to consist of an inner layer of collagen and
an outer layer of basic proteins with fibrocytes
scattered throughout (Kalantan et al. 1986). Al-
though our electron microscopic observations of
the cyst wall sections confirmed the distribution
of flattened fibrocytes, they did not reveal inner
and outer layers. Irregular lamellae of collagen
fibers were distributed throughout the entire cyst
wall. Assuming that C. eomplanatum is synony-
mous with C. marginatum, structural variations
probably reflect host differences (Cyprinodontidae
versus Percidae).
Results of the transport experiments indicated
that when the concentration of glucose in the incu-
bation medium is low (i.e., 0.1-0.5 raM), glucose
O.R. Larson et al. : Metacercarialcyst wall of Clinostomum
uptake by the worm is limited by the rate of its
diffusion through the cyst wall. This lower uptake
could also be due to the smaller surface area avail-
able for absorption, because an encysted worm is
folded, somewhat compressed, and nearly motion-
less. Indeed, apparently reflecting the difference in
surface area available for absorption, the rate of
glucose diffusion into excysted worms was nearly
three times that into encysted ones. However, en-
cysted worms took up more glucose at the higher
concentrations (i.e., 5-10 mM) that saturated the
transport systems. Thus, the depressed uptake at
lower concentrations appears to be associated
more closely with the permeability limits of the
cyst wall than with effective surface area and/or
movements of the worm. Moreover, since diffusion
is unsaturable and thus linear with concentration,
the cyst wall would limit glucose uptake by the
worm only when external glucose is low. This inter-
pretation is consistent with the linear uptake of
glucose by encysted worms, between 0.1 and
1.0 m M (uppermost dashed line in Fig. 2A), which
represents an estimate of the glucose diffusion rate
across the cyst wall.
The higher K, calculated for the encysted
worms is due in part to the depressed uptake rates
at low glucose concentrations, as mentioned above.
It is not clear, however, why encysted worms have
this greater transporting capacity (i.e., Vmax). Be-
cause the incubations were conducted under initial
rate conditions, transport is either stimulated in
encysted worms or inhibited when the worm sur-
face is exposed directly to saline. A similar differ-
ence occurs in metacercariae of C. campanulatum,
which incorporate more label from [14C]glucose
in 1 h in vivo (i.e., encysted) than they do in vitro
(i.e., excysted) (Thomas and Gallicchio 1967). It
was suggested that [14C]glucose uptake in vitro
was inhibited because the worms were exposed to
a "very unnatural environment" (i.e., saline).
Thus, metacercariae of both C. marginatum and
C. campanulatum are better able to take up glucose
from a bathing medium when separated from that
medium by a cyst wall.
Vascularization of metacercarial cyst walls of
C. marginatum was first described by Osborn
(1911), and the glucose-transporting capacity of
the encysted worms correlates well with the blood
glucose concentrations in various fish hosts. Dean
and Goodnight (1964) have compared black bull-
heads (Ictalurus rnelas), bluegills (Lepomis macro-
chirus), largemouth bass (Micropterus salmoides),
and white crappie (Pomoxis annularis), all accept-
able hosts for C. marginatum. They found that
blood glucose ranged from about 2.5 m M in non-
O.R. Larson et al. : Metacercarial cyst wall of Clinostornum
exercised largemouth bass at 20 ~ C to over 10 m M
in white crappie exercised at 5~ C; most concentra-
tions were near 5raM. In this range, most
(85%-95%) of the glucose absorbed by the worm
would enter via the transport systems. Moreover,
since the rate of diffusion through the cyst wall
exceeds the rate of transport into the worm, the
cyst wall would not restrict the worm's ability to
acquire glucose.
Halton and Johnston (1982) have suggested
that the ability of an encysted larva to communi-
cate with its external environment is dependent on
the thickness of the cyst wall. They observed that
encysted metacercariae of Bueephaloides graciles-
cens with thin cyst walls incorporated more
[3H]thymidine from a bathing medium than did
those with thick walls. The present results indicate
that permeability also depends on the size of the
probing molecule and the sieving properties of the
cyst wall. For example, the active transport and
facilitated diffusion systems for glucose (180 dal-
tons) in metacercariae of C. marginatum are known
to be inhibited by the plant glycoside phlorizin (476
daltons) and its aglycone phloretin (274 daltons),
respectively (Uglem and Larson 1987). However,
when these molecules were added along with
[3H]glucose to the medium bathing encysted
worms, glucose uptake was unaffected. Apparent-
ly, the cyst wall is selectively permeable, permitting
the rapid diffusion of glucose to the worm surface,
while restricting the movements of phlorizin and
phloretin.
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Accepted September 12, 1987