The uptake and distribution of radioselenium in the larvae of Fasciola hepatica and its snail host Lymnaea columella

Veterinary Parasitology, 21 (1986) 2 3 3 - - 2 4 5 Elsevier Science Publishers B.V., A m s t e r d a m -- Printed in The Netherlands

233

THE UPTAKE AND DISTRIBUTION OF RADIOSELENIUM IN THE LARVAE OF F A S C I O L A HEPATICA AND ITS SNAIL HOST L Y M N A E A COLUMELLA

HELEN HAN HSU'

Department of Preventive Medicine, NYS College of Veterinary Medicine, Cornell University, Ithaca, N Y 14853 (U.S.A.) ( A c c e p t e d for publication 2 O c t o b e r 1985)

ABSTRACT Han Hsu, H., 1986. The uptake and distribution o f radioselenium in the larvae of Fasciola hepatica and its snail host Lyrnnaea columella. Vet. Parasitol., 21: 233--245. Radiolabeled metacercariae of Fasciola hepatica were o b t a i n e d in vivo by incubating infected Lyrnnaea colurnella snails with 20 pCi r a d i o s e l e n o m e t h i o n e (TSSe-M) per snail in 5 ml of water for 5 h, or in vitro by incubating a batch of unlabeled F. hepatica metacercariae with 7sSe-M for 24 h. Radioassay showed that only 5% of the ~SSe-M was incorporated into maritas (juvenile flukes) f r o m the in vivo labeled metacercariae. The inner cyst wall o f in vivo labeled metacercariae contained 46% o f the t o t a l activity, o f which 21% was dissolved in the excysting m e d i u m . The outer, t a n ~ o l o r e d cyst wall c o n t a i n e d 49% o f the radioactivity. T h r o u g h d i f f u s i o n / a t t a c h m e n t , maritas f r o m in vitro labeled metacercariae could occasionally be labeled with 0.4% of the total radioactivity. However, the activity was lost after inoculation into the b o d y of mice. The o u t e r and inner cyst walls o f in vitro labeled metacercariae contained 92% and 7.6%, respectively, of the total activity. Microautoradiography d e m o n s t r a t e d that 7sSe-M was evenly distributed in the b o d y of marita and the cyst wall o f inner and outer layers f r o m an in vivo labeled metacercaria. A 9 × 4 p m rectangularly-shaped aggregate of Ag ° grains was present on the o u t e r periphery of the inner cyst wall. Microautoradiograpy of in vitro labeled metacercariae d e m o n s t r a t e d a significant c o n c e n t r a t i o n of Ag ° grains on the cyst walls. The ventral plug contained fewer Ag ° grains per unit area c o m p a r e d to the o t h e r p o r t i o n of the inner cyst wall. U p t a k e and distribution of ~sSe-M in the snail host were also studied. It appeared that rediae and cercariae tended to c o n c e n t r a t e the label in the foot, the mantle and the digestive gland. Little or no radioactivity was present in the areas where F. hepatica larvae were n o t found.

INTRODUCTION

The study of the uptake and distribution of radionuclides in Mollusca was limited until Gong et al. (1957) studied the uptake of fission products 1 Present address: Wyeth Laboratories, P.O. Box 861, Paoli, PA 19301, U.S.A.

0304-4017/86/$03.50

© 1986 Elsevier Science Publishers B.V.

234 and radionuclides in the marine clam. The ability of this clam to concentrate radionuclides suggested that it could serve as a potential biological indicator of low levels of radioactivity in sea water. In 1962, Johnson et al. studied the uptake, distribution and excretion of four radionuclides in Biomphalaria glabrata, the intermediate host of the trematode Schistosoma mansoni. It was shown that this snail takes up and concentrates 8SSr, SgFe, 86Rb, and 22Na, but S9Fe was the only isotope transferred to egg clutches in appreciable amounts. Cercariae of S. mansoni were labeled with radioselenium successfully by Knight et al. (1968), and Christensen et al. (1977): after exposure of infected snails to H2 7SSeO3 and 7SSe-M the label was rapidly incorporated into the snail tissue and the cercariae. The isotopic labeling of parasites is of significant value not only in tracing the migration routes within a host, but also in the investigation of immunologic interrelationships between the parasite and its intermediate or definitive hosts. To determine how fast Fasciola hepatica marita can migrate in the definitive hosts, radiolabeled larvae were obtained and the mechanisms of isotope uptake were studied in the present paper. MATERIALS AND METHODS

In vivo radiolabeling of F. hepatica metacercariae and o f the snail host, L y m n a e a columella The F. hepatica used in this experiment was a laboratory strain maintained in laboratory-reared L. columella. Both parasites and snails originated from Baldwin Enterprises, M o n m o u t h , OR. Each 0.3--0.6 cm long, young L. columella was exposed to 5--8 miracidia of F. hepatica in 2 ml water for 24 h. The snails were maintained in aquaria at a temperature of 21--25°C and fed with Romain lettuce and chalk. After 45 days, the infected snails were exposed individually to 20 pCi 7SSe-L-methionine (Code SC 12, the Radiochemical Centre, Amersham) in 5 ml water for 5 h. The radiolabeled infected snails were washed with tap water several times to remove attached debris and absorbed radioactivity and then placed in a petr.i dish containing clean well water. After 0.5--12 h, the cercariae emerged from the labeled snail bodies and encysted on the b o t t o m or sides of the petri dish. Shedding was facilitated by placing the snails in a 4°C refrigerator for 5--10 minutes. The resulting metacercariae were immersed in tap water for two days to harden the tan-colored outer cyst walls and they were then radioassayed.

In vitro radiolabeling o f F. hepatica metacercariae Fifty unlabeled metacercariae were transferred to a 3-cm petri dish.

235 Approximately 2--3 ml tap water containing 20 ~Ci 7SSe-M was added to immerse all the metacercariae. After an incubation period of 24 h, these metacercariae were transferred into another dish and were washed 5--10 times with clean tap water from a squirt bottle. The metacercariae were determined to be radiolabeled by radioassay.

In vitro excystmen t o f metacercariae The outer tan-colored cyst wails of metacercariae were removed by applying light pressure with the point of a probe. The resulting transparent metacercariae were incubated in 0.5% pepsin in phosphate buffered saline (PBS) at pH 2, 37°C, for 20 min. The pepsin solution was decanted and the metacercariae were washed several times in indicating PBS, until the pH was slightly over 7.0. Excysting medium containing equal amounts of N/20 HC1, 0.8% NaC1 and 1% NaHCO3, plus 10% of total volume of bovine bile was added, and the metacercariae were agitated in a shaker bath (Precision, GCA Corporation) at 38°C, 100 strokes min -1 for 3--4 h. At the end of the incubation period the contents o f the beaker were poured into a petri dish and examined under the stereomicroscope. With proper illumination, actively moving maritas, e m p t y cyst walls and nonexcysted metacercariae could be easily distinguished.

7SSe-M uptake and retention by L. columella After the radiolabeled snails ceased shedding cercariae, the snails were pulled o u t intact from their shells with forceps. The digestive gland, gastrointestinal (G.I.) tract, heart, hermaphroditic organ and muscular tissue (including mantle, foot and head) as well as feces, were excised under the stereomicroscope and radioassayed. The separated snail tissues and feces were placed on a 200 × 250 mm cardboard for autoradiography. E m p t y shells were also radioassayed.

Retention of 7SSe-M in various compartments of radiolabeled F. hepatica metacercariae Several batches of washed, radiolabeled metacercariae were radioassayed. For each batch, the outer tan-colored cyst wails and the remaining transparent metacercariae were transferred to separate petri dishes by means of a capillary tube affixed on the needle of a 1-ml tuberculin syringe, and were radioassayed in a 7-counter. Transparent metacercariae were excysted in vitro. The resulting maritas, e m p t y cyst walls and the unexcysted metacercariae were separately transferred to filter papers. The location of these specimens was circled to facilitate subsequent evaluation of the autoradiograph, and each type of specimen was radioassayed.

236

Radioassay Samples of radioactive material were radioassayed between 127 mm diameter NaI(Th) crystals in a Nuclear Chigaco T o b o r Counter. All samples were c o u n t e d for a period of 10 min. Correction was made for background counts.

A u toradiography All cardboards or filter papers bearing tissues were covered with two layers o f p o l y e t h y l e n e film. The tissues were t hen flattened by the weight o f a lead brick and dried for 5--8 h at 60°C. The out er layer of p o l y e t h y l e n e film was changed to avoid possible c o n t a m i n a t i o n from the lead bricks and to prevent any remaining moisture from contacting the radiographic emulsion. In the dark r o o m , a 200 X 250 m m X-Omat AR film was placed on the to p of the cardboard or filter papers. Two layers o f aluminium foil were wrapped ar ound the entire package to exclude light, and a n o t h e r layer o f p o l y e t h y l e n e was added to exclude moisture. Films were exposed for 3 weeks at 5°C, then developed in an automatic processor (Kodak RP X-Omat Processor).

Microautoradiography o f radiolabeled F. hepatica metacercariae Sections of the o u t e r cyst walls and the remaining transparant radiolabeled metacercariae were prepared by stirring groups o f 50 metacercariae into two or three drops o f fresh rat blood. After coagulation, the blood was fixed in 10% neutral buffered formalin, e m b e d d e d in paraffin, and sectioned at 5 ~m. Dry slides bearing sections o f these metacercariae were dipped into liquid emulsion (Kodak, NTB-3) and placed on a flat surface. T w o minutes later, the dipped slides were put into small slide boxes containing anhydrous CaC12 in a small c o m p a r t m e n t . Two layers of aluminum foil were wrapped ar o u n d each box to ensure c om pl et e darkness, and the boxes were stored at 4°C. At the end of 2 weeks, the slides were taken out and developed in the d a r k r o o m .

Evaluation o f in vivo and in vitro labeling techniques Seven 1-year-old CD-1 male mice were divided into two experimental groups and one c o n t r o l group. Three mice were dosed by gastric intubation with 10 in vivo radiolabeled metacercariae containing radioactivity o f 13 + 4 cpm/metacercaria and three mice were similarly dosed with 10 in vitro radiolabeled metacercariae averaging 374 + 4 cpm/metacercariae. One mouse f r om the c ont r ol group was dosed with 10 non-radiolabeled metacercariae. All of the o u t e r layers of metacercarial cysts were removed

237 b e f o r e r a d i o a s s a y . Mice w e r e a n e s t h e t i z e d w i t h p e n t o b a r b i t o n e s o d i u m ( 0 . 2 5 m g ml - I ) b e f o r e i n o c u l a t i n g t h e m e t a c e r c a r i a e to p r e v e n t struggling. E i g h t d a y s a f t e r i n o c u l a t i o n , all o f t h e m i c e w e r e sacrificed b y i n h a l a t i o n o f CO2 and n e c r o p s y was p e r f o r m e d . A t this t i m e , t h e i n f e c t e d livers s h o w e d o b v i o u s m i g r a t o r y t r a c t s o f m a r i t a s o n t h e surface a n d in t h e p a r e n c h y m a . All t h e livers w e r e r e m o v e d f r o m t h e carcasses a n d p l a c e d o n 2 0 0 × 2 5 0 m m c a r d b o a r d s . E a c h c a r d b o a r d held t w o livers a n d was d o u b l e - w r a p p e d w i t h a p o l y e t h y l e n e film. T h e livers w e r e f l a t t e n e d w i t h a lead brick to m a k e squash p r e p a r a t i o n s . T h e squash p r e p a r a t i o n s w e r e pressed d o w n w i t h t w o or t h r e e lead bricks a n d w e r e left in t h e f r e e z e r f o r /> 24 h to let the tissues s p r e a d o u t and h a r d e n . A f t e r r e m o v i n g t h e liver squashes f r o m t h e freezer, t h e o u t e r l a y e r o f t h e p o l y e t h y l e n e film was t a k e n o f f t o avoid possible c o n t a m i n a t i o n f r o m t h e lead bricks. T h e squashes w e r e p l a c e d in t h e o v e n (60°C) f o r a n o t h e r 5 h to dry. In vitro e x c y s t a t i o n was p e r f o r m e d o n t h e s a m e b a t c h e s o f in vivo a n d in vitro r a d i o l a b e l e d m e t a c e r c a r i a e t h a t h a d b e e n given t o the mice. A u t o r a d i o g r a p h s o f t h e m a r i t a s a n d t h e liver squashes w e r e set up a n d d e v e l o p e d the same day. RESULTS In vivo labeled metacercariae T h e in vivo labeled m e t a c e r c a r i a w i t h o u t t h e o u t e r c y s t wall c o n t a i n e d an average r a d i o a c t i v i t y o f 77 + 36 c p m / m e t a c e r c a r i a . T a b l e s I and II ilTABLE I 7SSe-M in metacercariae with and without outer cyst walls Batch no.

Metacercariae with outer cyst walls

Metacercariae

% of 7SSe-M in the

without outer cyst walls metacercariae without

outer cyst walls No.

vSSe-M No. (cpm/individual)

7sSe-M (cpm/individual)

(%)

1 In vivo 2 labeled 3 4

6 15 46 10 14

200 116 119 108 79

4 10 10 8 8

100 56 63 48 46

50 48 53 44 58 (51 _+5)*

In vitro 1 labeled 2

33 20

1964 5684

24 20

182 350

9 6 (8 -+ 2)*

* M e a n -+ s.d.

238 T A B L E II R e t e n t i o n of 75Se-M in various c o m p a r t m e n t s of in vivo a n d in vitro labeled m e t a c e r cariae w i t h o u t o u t e r cyst walls No. assayed per B a t c h

Metacercariae (cpm)

E m p t y cysts (cpm)

Maritas (cpm)

I n vivo labeled

5 2

139 642

72 322

15 62

In vitro labeled

10

190

88

10

7SSe-M in e m p t y cyst

7sSe-M in m a r i t a

(%)

(%)

52 50 (51_+1)*

11 10 (10_+7)*

46

5

* M e a n _+ s.d. In vitro labelled 92%

~ .

In vivo labelled /

~

~

cuter cyst w a l l - -

49,~,

ventral plug Fig. 1. P e r c e n t a g e of r a d i o a c t i v i t y r e t a i n e d in various c o m p a r t m e n t s of t h e in vivo a n d in vitro labeled m e t a c e r c a r i a as d e t e c t e d b y radioassay.

lustrate the percentage of radioactivity retained among various compartments o f the intact in vivo and in vitro labeled metacercariae. Figure 1 summarizes the results. The microautoradiograph of in vivo labeled metacercariae showed that the Ag ° grains were evenly distributed in the b o d y parenchyma, as well as in the inner cyst walls. A 9 × 4 p m rectangularly-shaped aggregate of Ag ° grains was present on the o u t e r p o r t i o n of the inner cyst wall where the tail had been attached (Figs. 2 and 3). The outer cyst wall contained a higher c o n c e n t r a t i o n of Ag ° grains which appeared on both sides of the wall. Th e autoradiograph developed f r om various c o m p a r t m e n t s of the in vivo labeled metacercaria were satisfactory. There was an Ag ° focus on the developed film corresponding to each marita or e m p t y cyst wall. The Ag ° foci f r o m maritas were smaller than those of e m p t y cyst walls. In vitro labeled metacercariae

The average radioactivity of an in vitro labeled metacercaria w i t h o u t

239

Fig. 2. Metacercaria of F. hepatica with the outer cyst wall removed. T points where the tail of cercaria had been attached. The excretory granules (E) and the gut (G) are visible through the inner cyst wall. 400)<. the o u ter cyst wall was determined by radioassay as 190 -+ 14 cpm/metacercaria. The 7SSe-M distribution among various c o m p a r t m e n t s is shown in Tables I and II, and Fig. 1. Microautoradiographs of in vitro labeled metacercariae d e m o n s t r a t e d a significant c o n c e n t r a t i o n of Ag ° grains on the inner cyst walls (Fig. 4). Th e autoradiograph developed f r om various c o m p a r t m e n t s o f t he metacercariae showed larger Ag ° foci f or the o u t e r cyst walls and the e m p t y cyst walls. The size o f the Ag ° focus varied with the activity retained within each c o m p a r t m e n t ; the higher the activity, the larger the size. Only one o f the 10 maritas excysted from in vitro labeled metacercariae was represented by a focus with a density comparable to the Ag ° loci representing maritas excysted f r om in vivo labeled metacercariae. F o u r of these 10 maritas were each represented by a m u c h less dense Ag ° focus than those representing maritas f r om in vivo labeled metacercariae. The remaining five maritas had no Ag ° foci.

The uptake and distribution o f 7SSe-M by L. columella Radioassay of the shells from labeled snails did n o t dem onst rat e any accumulation of 7SSe f r om 7SSe-M. Table III shows the results o f the uptake

240

Fig. 3. Microautoradiograph of an in vivo labeled metacercaria. The Ag° grains are evenly distributed in the marita within the cyst wall. A focal Ag° grain aggregate (arrow) is on the outer periphery of the inner cyst wall. Compare the location of this Ag° grain aggregate to the area T in (1). 400x. a n d d i s t r i b u t i o n o f 7SSe-M a m o n g v a r i o u s s o f t tissues o f t h e labeled snails. I t a p p e a r s t h a t t h e r a d i o a c t i v i t y o f t h e labeled snails c o n c e n t r a t e d m a i n l y in t h e m u s c u l a r tissues (feet, h e a d s a n d m a n t l e s ) a n d digestive glands. T h e snail feces c o n t a i n e d m a n y e n c y s t e d m e t a c e r c a r i a e a n d o t h e r debris. R a d i o assays o f t h e s e m e t a c e r c a r i a e s h o w e d t h a t t h e y h a d a r a d i o a c t i v i t y o f 63 +- 28 c p m / m e t a c e r c a r i a . T h e n u m b e r s o f m e t a c e r c a r i a e in t h e feces o n l y slightly i n f l u e n c e d t h e p e r c e n t a g e o f ZSSe-M u p t a k e a n d d i s t r i b u t i o n a m o n g v a r i o u s o r g a n s in e a c h snail. T h e a u t o r a d i o g r a m d e v e l o p e d f r o m t h e liver, m a n t l e , f o o t , h e a d , h e r m a p h r o d i t i c o r g a n , G.I. t r a c t , h e a r t a n d feces o f each o f t h r e e r a d i o l a b e l e d snails c o n t a i n e d b l a c k Ag ° p a t c h e s . T h e size a n d s h a p e o f t h e s e Ag ° p a t c h e s c o r r e s p o n d t o t h e w h o l e a r e a o f t h e l a b e l e d squashes. A l t h o u g h n o activity was m e a s u r e d b y r a d i o a s s a y in t h e h e a r t o f Snail

241

Fig. 4. Microautoradiograph of three in vitro labeled metacercariae Ag ° grains are significantly concentrated in the cyst walls.

of

F. hepatica.

The

T A B L E III U p t a k e a n d d i s t r i b u t i o n o f 7SSe-M a m o n g v a r i o u s o r g a n s o f t h e l a b e l e d snails Snail

Liver

Gut

Heart

Hermaphroditic organs

Foot, head and mantle

Feces

cpm %

cpm

%

cpm

cpm

0 113 467

270 1666 1075

5 13 7

2015 4920 5403

7SSe-M r e t a i n e d cpm 1 2 3

1869 3158 4514

% 36 24 28 (30+6)*

cpm 968 1784 1638

% 19 14 10 (14-+4)

-1 3 (2-+1)

(8-+4)

% 39 38 34 (37-+3)

-1419 2899

% -11 18 (14-+5)

* M e a n -+ s . d .

1, the Ag ° patch in its autoradiogram was significant. A possible reason for the inconsistency is that the radioactivity was too low to be detected by the Tober 7-counter.

Retention of 7SSe by maritas migrating in mice Of the mice t h a t had been inoculated with either in vivo or in vitro radiolabeled maritas, two from each group exhibited lesions of migratory

242 tracts; the third mouse in each group did n o t have any lesions in the liver. The liver o f the c ont r ol mouse showed obvious migratory tracts. There were three small Ag ° foci on each autoradiogram of liver from mice given in vivo labeled metacercariae, indicating the presence of maritas. The size of these Ag ° foci was consistent and comparable to the size of the Ag ° focus in the autoradiogram made from an in vivo labeled marita which was ex cys t e d in vitro. Although the Ag ° focus of the in vitro labeled marita e x c y s t e d in vitro was quite large, there were no Ag ° foci present on the autoradiograms of any o f the liver squashes f r om mice dosed with in vitro labeled metacercariae. The autoradiograms f r om cont r ol mouse liver squashes were also negative, as ex p ected . DISCUSSION Radiolabeled larvae o f F. hepatica were first studied by Nansen and Frandsen (1974) and Nansen and Christensen (1976a,b), who obtained radiolabeled eggs by inoculating 7SSe-M into the peritoneal cavity of mice carrying a p a t ent infection of F. hepatica. After hatching from these radiolabeled parasite eggs, each miracidium retained 73% of the label of the egg, a p p r o x i m a t e l y 2--6 cpm. In the present e x p e r i m e n t radiolabeled metacercariae and maritas were obtained. Both techniques of radiolabeling metacercariae of F. hepatica were successful and the radioactivity of 77 + 36 and 190 + 14 cpm per metacercariae when labeled in vivo and in vitro respectively, were satisfactory. Both in vivo and in vitro labeled metacercariae and the maritas derived f r om t hem appeared normal and their motility and infectivity for mice did n o t differ from t hat of unlabeled metacercariae and maritas. After inoculating bot h types of radiolabeled metacercariae into the mouse, it appeared t ha t 7SSe-M was retained in the maritas from in vivo labeled metacercariae and was suitable for autoradiography. The 7SSe-M o f the marita from in vitro labeled metacercariae was n o t retained after the marita entered the mouse liver. Hence, utilization of in vitro labeled metacercariae in studying host/parasite relationship is n o t feasible. It was shown by microautoradiographs that 7SSe-M was evenly distributed in the b o d y of maritas excysted f r om in vivo labeled metacercariae. It is assumed th at 7SSe-M was i ncor por at ed into the h e m o l y m p h o f snails and was transferred to all organs and tissues. By ingesting labeled snail tissues, the rediae and cercariae consumed 7SSe-M and i n c o r p o r a t e d it into their tissues. Studies by Dixon (1966) and K~bie et al. (1977) showed that the comp o n en ts of o u t e r and inner cyst walls o f metacercariae are tanned protein, m u c o p r o t e i n , m uc opol ys acchar i de and neutral proteins. Since there was a high rate o f p r o d u c t i o n of protein c o m p o n e n t s in cystogenic cells making cyst walls, one would postulate t hat cystogenic cells rapidly accumulate and incorporate the elements which constitute cyst walls. The results of

243

radioassays in the present work showed that the cyst walls contained 95% o f the activity present in each in vivo labeled metacercaria. This finding supports the hypothesis that 7SSe-M was incorporated into cystogenic cells of the cercariae and was utilized in the making of cyst walls. Verification of this will be provided in future studies if the microautoradiographs of cercariae show ~ 95% of the Ag ° grains located over the cystogenic cells. There is little information available on the subject of tail shedding in cercariae of F. hepatica. The study of Dixon and Mercer (1967) indicated that muscular movement of metacercariae separates the tail and outer cyst wall. Before shedding, the proximal end of the tail becomes included within the secretion which constitutes the outer cyst wall. The ultrastructure study of cercariae and metacercariae of F. hepatica (K~bie et al., 1977) showed that the tail remains attached until the outer cyst wall is fully formed. The ultrastructure o f the "scar" in the outer cyst wall after the tail has been shed appears to be a short stub of the proximal end of the tail. In the microautoradiographs of in vivo labeled metacercariae, the high concentration o f Ag ° grains over the outer periphery of inner cyst walls may be due to the retention of 7SSe-M in the tail plus the activity from the tanned protein which covered it. From the microautoradiograph there did not appear to be higher concentration of Ag ° grains over the inner cyst wall than over the marita b o d y of in vivo labeled metacercariae as was shown by radioassay. The higher radioactivity on the inner cyst wall could be explained by this high concentration of Ag ° grains over the outer periphery of the inner cyst wall and not by the high concentration of Ag ° grains on the inner cyst wall. Autoradiography supported the radioassay finding that 0.4% of the 7SSe-M was retained in t h e maritas excysted from in vitro labeled metacercariae. Not all maritas, however, showed radioactivity in the autoradiographic and microautoradiographic studies. To investigate the loss of radioactivity further, mice were infected with in vitro labeled metacercariae. The maritas were shown to have lost all the radioactivity, suggesting that 7SSe-M was attached to, or had diffused into, the outer periphery of the marita tegument. As the secretory products of tegument cells changed bodies after maritas entered into the liver (Bennett and Threadgold, 1975), the turnover of the tegument causes the loss of the label. It was shown by Threadgotd (1963) and by BjSrkman and Thorsell (1964), that F. hepatica tegument ultrastructurally resembles that of cestodes, which can absorb low molecular weight metabolites. Later, Isseroff and Read (1969) ligated the m o u t h of adult F. hepatica and demonstrated that low molecular weight amino acids, including methionine, enter the fluke by diffusion. Our studies provide evidence of 7SSe-M transferring through the tegument since: (1) Radioactivity of the in vitro labeled metacercariae increased as a function of longer incubation times (Han, 1983).

244

(2) The o u te r cyst wall of in vitro labeled metacercariae contained 92% o f the total radioactivity, and 7.6% of the total radioactivity was retained in the inner cyst wall. These observations suggest that absorption/diffusion and a t t a c h m e n t played an i m p o r t a n t role in labeling metacercariae in vitro. It was n o t surprising t hat the o u t e r cyst wall contained 92% of the 75Se-M in the in vitro labeled metacercariae. The out er layer of the metacercarial cyst wall is made up of a thick spongy meshwork of filaments, com posed o f cigar-shaped tanned protein. The porous structure of this layer protects the metacercaria against desiccation by absorbing and storing water. It is possible for a large p r o p o r t i o n of 7SSe-M to be retained here. Au to r ad io gr aphy of snail tissues showed that 7SSe-M was i ncorporat ed into all soft tissues of the infective snails; this is consistent with the finding o f Knight et al. (1968) who used 7SSe-M as the labeling agent for B. glabrata. It was observed by Barber (1962) that rediae and cercariae could be f o u n d in almost any organ of the snail e x c e p t for the heart and the lung cavity, although th e y tend to c o n c e n t r a t e in the foot, mantle, visceral cavity and digestive gland. Fr om the radioassays of the tissues of labeled snails in the present work it is interesting to not e t hat rediae and cercariae tend to c o n c e n t r a t e in the organs containing a majority of the label, i.e., foot, mantle and digestive gland. Little or no radioactivity was present in the snail heart as evidenced by radioassaying and autoradiography, and no stages of F. hepatica have ever been f o u n d in the heart (Barber, 1962). ACKNOWLEDGEMENTS I am grateful to Dr. J.R. Georgi and Dr. F. Lengemann for their guidance and for providing the laboratory space and instruments during the course o f this experiment, and in preparation of the manuscript. Thanks are also due to Mr. N. Baldwin for the supply of F. hepatica metacercariae and snail host, L. columella; Drs. H. Erb, M. Frongillo, M. Georgi, and J.M. King for their valuable criticism o f the text; and Ms. L. H enry for typing the manuscript. This work was s uppor t ed by the U.S. D e p a r t m e n t o f Agriculture, Section 1433, formula funds.

REFERENCES Barber, I.W., 1962. The pathology produced in the snail Lymnaea columella by the larval stages of F. hepatica. Ph.D. Thesis, University of California, Berkeley, CA. Bennett, C.E. and Threadgold, L.T., 1975. Fasciola hepatica: development of tegument during migration in mouse. Exp. Parasitol., 38: 38--55. Bj6rkman, N. and Thorsell, W., 1964. On the fine structure and resorptive function of the cuticle of the liver fluke, F. hepatica. Exp. Cell Res., 33: 319--329. Bj6rkman, N., Thorsell, W. and Lienert, E., 1963. Studies on the action of some enzymes on the cuticle of F. hepatica. Exp. Basle, 19: 3--4. Christensen, N.O., 1977. A method for the in vivo labeling of S. mansoni and S. mansoni and S. intercalatum cercariae with radioselenium. Zeitschr. Parasitenk., 54: 275-288.

245 Dixon, K.E., 1966. A morphological and histochemical study of the cystogenic cells of the cercariae of F. hepatica. Parasitology, 56: 287--297. Dixon, K.E. and Mercer, E.H., 1967. The formation of the cyst wall of the metacercaria of F. hepatica. Zeitschr. Zellforsh. Mikrosk, Anat., 77: 345--360. Gong, J.K., Shipman, W.H., Weiss, H.V. and Cohn, S.H., 1957. Uptake of fission products and neutron induced radionuclides in the clam. Proc. Soc. Exp. Biol. Med., 95: 451-454. Han, H., 1983. Studies of the early migration of radiolabeled F. hepatica in mice and of the comparative hepatic pathology due to fascioliasis in rabbits, mice and rats. Ph.D. Thesis, Cornell University, Ithaca, NY. Isseroff, H. and Read, C.P., .1969. Studies on membrane transport -- VI. Absorption of amino acid by Fascioliid trematodes. Comp. Biochem. Physiol., 30: 1153--1159. Johnson, C.R., Angel, C.R. and Erickson, D.G., 1962. The uptake, distribution and excretion of four radionuclides in Australorbis glabratus. Am. J. Trop. Med. Hyg., 11: 855--860. Knight, W.B., Liard, F., Ritchie, L.S., Pellegrino, J. and Chiriboga, J., 1968. Labeling of B. glabrata and cercariae of S. mansoni with radioselenium. Exp. Parasitol., 22: 309--315. K~bie, M., Nansen, P. and Christensen, N., 1977. Stereoscan studies of rediae, cercariae, cysts, excysted metacercariae and migratory stages of F. hepatica. Zeitschr. Parasitenk., 54: 289--297. Nansen, P. and Christensen, N., 1976a. A technique for in vivo labeling of F. hepatica miracidia with radioselenium. Zeitschr. Parasitenk., 49: 73--80. Nansen, P. and Christensen, N., 1976b. A study on snail location by F. hepatica using radioisotopically labeled miracidia. Parasitology, 72 : 163--171. Nansen, P. and Frandsen, F., 1974. Studies on F. hepatica miracidia labeled with radioselenium. Acta Vet. Scand., 15: 144--146. Threadgold, L.T., 1963. The tegument and associated structures of F. hepatica. Q. J. Microsc. Sci.~ 104: 505--512.