In vitro uptake of tritiated glucose, tyrosine and leucine by adult Alaria marcianae (La Rue) (Trematoda)

Camp. Biochem. Physiol., 1971, Vol. MA, pp. 987 to 997. Pergamon Press. Printed in Great Britain

IN VITRO UPTAKE OF TRITIATED GLUCOSE, TYROSINE AND LEUCINE BY ADULT ALARIA MARCIANAE (LA RUE) (TREMATODA) IFTIKHAR

BHATTI*

and ALLEN

D. JOHNSON

Department of Biology, University of South Dakota, Vermillion, South Dakota 57069 (Receiwed 11 May 1971) Abstract-l. Autoradiographic studies demonstrated the in vitro uptake of tritiated glucose, tyrosine and leucine by the tegument covering and lateral to the holdfast organ of adult Alaria murciunae (La Rue, 1917). 2. The holdfast organ tegumental surface layer (“cuticle”) showed a regional difference in uptake for all three radiocompounds; the surface layer in the posterior two-thirds exhibited more absorption than that in the anterior one-third. 3. In liquid scintillation counting, a positive correlation was found between the specific activity (dis/min per pg of protein) and the duration of exposure (min) of the flukes to the radiocompounds. INTRODUCTION

ADULT strigeoid flukes (superfamily Strigeoidea) possess a unique structure called the holdfast organ (adhesive organ, organe tribocytique). Dubois (1957) considered this structure of major importance in determining their host specificity because of its participation in an unusual mode of feeding (enzyme secretion for extracorporeal digestion). This view has been recently questioned by Johnson et al. (1971). Another possible function of the holdfast organ, which may play a role in determining specificity, is the absorption of nutrients. Studies so far have only provided indirect evidence for such a function. Thus, all in situ studies have revealed a close association between the holdfast organ and host tissue (Augustine & Uribe, 1927; Erasmus & ohman, 1963; ohman, 1965, 1966a, b; Bogitsh, 1966; Erasmus, 1969b; Johnson et al., 1971), and a chambered surface bearing microvilli has been observed on the holdfast organ tegument of certain strigeoids (Erasmus & Ohman, 1965 ; Erasmus, 1969b, 1970a). Histochemical staining methods have demonstrated acid phosphatase activity in the holdfast organ tegument (see Johnson et al., 1971, for review). Since this enzyme is found in lysosomes which in turn may be involved in endocytosis, its presence might indicate nutrient uptake by such a process (Novikoff, 1963; de Duve & Wattiaux, 1966). The purpose of this study was to determine if tritiated glucose, tyrosine and leucine are taken up in vitro by the holdfast organ tegument of the diplostomatid * Present address: Department of Biology, Buena Vista College, Storm Lake, Iowa. 987 33

strigeoid Alaria marcianae (La Rue, 1917). A method devised for high-resolution autoradiography of small water-soluble compounds was employed (Wilske & Ross, 1965). Liquid scintillation counting was used to determine the relative concentration of the tritiated compounds in worms exposed for different time periods. MATERIALS

AND

METHODS

Mesocercariae of A. marcianae were collected from naturally infected leopard frogs, Rana pipiens (Steinhilber & Co., Oshkosh, Wis.). Two dogs were injected intraperitoneally with the larvae (2500 each) and killed 30 days after infection. Immediately after the animals were killed, adult worms from the small intestines were removed to Hedon-Fleig’s saline (Dawes, 1954) at 37°C. Autoradiographic

technique

Flukes were used as soon as possible after removal from the host. Ten adults at a time were placed in fine-mesh (No. 100) stainless steel wire baskets. They were exposed for periods of 1, 2, 5, 10, 20 and 30 min in 2 ml of Hedon-Fleig’s saline (37°C) containing a tritiated compound with an activity of 100 PC/ml. The compounds used were n-glucose-6-T (sp. act. 500 mc/mM, Nuclear Chicago), L-leucine-4,5-T (sp. act. 1000 mc/mM, Amersham/ Searle) and L-tyrosine-T(G) (sp. act. 434 mc/mM, Amersham/Searle). Following exposure the parasites were quickly rinsed in two changes of the saline and immediately quenched in liquid nitrogen (- 180°C). They were then transferred to the specimen chamber ( - 70°C) of a cryosorption pump (Stumpf & Roth, 1964, 1967). This system was initially pumped down with a two-stage mechanical pump. The specimens were freeze-dried for 4 days, with a gradual return to room temperature during the last 24 hr. One group of freeze-dried flukes was fixed in osmium tetroxide vapors and a second in Carnoy’s fluid, both at room temperature. In the case of 3H-glucose, a third group was fixed in formaldehyde vapors at 60°C. Fixation in all instances was carried out for 24 hr. Specimens fixed in osmium tetroxide and paraformaldehyde were embedded in Epon 812 (Luft, 1961) containing lyb silicone fluid 200 (Dow-Corning, 350 cstks) (Stirling & Kinter, 1967). Sections 2 p thick were cut with glass knives on a rotary microtome, floated on water on glass slides and dried at 40°C. Parasites fixed in Camoy’s fluid were embedded in paraffin (Tissuemat, Fisher Scientific) and sections 4 p thick mounted on albuminized slides. Epon and paraffin sections covered with Kodak NTB-2 nuclear emulsion were exposed for 5, 12 and 28 days at -10°C. After exposure the slides were developed in Dektol, rinsed in water and fixed in Kodak fixer. Slides were stained with O*OS% toluidine blue (pH 5), dehydrated in ethanol, cleared in xylene and mounted in Permount. Grain counts were made with an ocular grid (O-1 mme) at oil immersion from slides exposed for 5 days. Twenty areas, each 50 p2, were counted in the designated tissue region. Background was determined from counts in areas adjacent to labelled sections and from areas in control sections. For liquid scintillation counting, flukes were exposed and quenched as given above. Five frozen worms for each exposure period were transferred to 1.0 N sodium hydroxide (2 ml) for 24 hr. From each sample 0.5 ml was removed and mixed with 20 ml of scintillation fluid (Xylene fluid; water tolerance 0.5 ml water/20 ml solvent; composition, PPO 5 g, POPOP 0.1 g, xylene 500 ml and ethanol 500 ml). The protein content of 1 ml of the remaining 1.5 ml was determined by the method of Nakai & Chi Le (1970). RESULTS

Since the adult worms and the holdfast organ and associated gland cells have been described (see Johnson, 1968; Johnson et al., 1971), only a brief description will be given here.

UPTAKE OF TBITIATBD COMPOUNDS BY ALARIA

MARCIANAE

989

The elongate holdfast organ is situated on the ventral surface of the forebody posterior to the acetabulum. The tegument on its ventral surface displays a distinct border which probably corresponds to the chambered border of other strigeoids (Erasmus, 196915,197Oa). Small bands of muscles are present under the tegumental surface layer (“cuticle”). A series of excretory spaces, which are connected to larger channels extending into the holdfast organ, lie under the muscle bands. The gland cells (presumably modified tegumentary cells) are located under these spaces throughout the entire length of the holdfast organ but form a more dense mass anteriorly (anterior mass of holdfast organ gland cells). Autoradiography

Except for the 1-min period with SH-leucine, autoradiographs of adult worms showed an uptake of tritiated glucose, tyrosine and leucine by the holdfast organ tegument in all exposure periods (1, 2, 5, 10, 20 and 30 min) (Table 1). In addition, a regional difference in uptake for all three compounds by the holdfast organ tegumental surface layer was found for all periods; the surface layer in the posterior two-thirds exhibiting more absorption than that in the anterior one-third (Table 1). These differences were significant at the 5 per cent level (Student’s t-test). The anterior one-third is that region essentially consisting of the anterior mass of holdfast organ gland cells. From mean grain counts, the tegumental surface layer of both regions exhibited an increase in uptake with each succeeding period, except for the 30-rain period with SH-tyrosine (Table 1). The ventral forebody tegument lateral to the holdfast organ showed an uptake of all three radiocompounds for all periods, with the exception of the 1-min period with tyrosine and the l- and 2-min periods with leucine (Table 1; counts from ventral areas just lateral to the holdfast organ). For all three compounds there was a gradual decrease in radioactivity of the surface layer going from the holdfast organ to the lateral margin of the scoop-shaped forebody. There was no absorption of the compounds by the rest of the forebody tegument, the hindbody tegument or the cecal gastrodermis. Although 3H-glucose uptake was essentially the same for osmium tetroxide and formaldehyde fixed sections, less tissue shrinkage and better staining contrast was achieved with the former. Thus, all grain counts and descriptions are from osmium tetroxide fixed specimens. Glucose

After 1 min sH-glucose was present in the tegument of the entire holdfast organ. It was concentrated primarily in the surface layer although some radioactivity was observed in the subtegument (Fig. 1; Table 1). The latter area is defined here to include the holdfast organ gland cells, the muscle bands and possibly cytoplasmic processes from parenchymal cells. After 2 min radioglucose was equally distributed in the surface layer and the subtegument. However, it was concentrated around the excretory spaces in the subtegument (Fig. 2). Radioglucose after 5, 10 and 20 min was found throughout the entire holdfast organ

1 2 5 10 20 30

1 2 5 10 20 30

Tyrosine

Leucine 360 4.45 4.15 3.50

f f f +

0,462 0.504 0.460 0.879

0 0

0t + 0460 f 0.542 + 0.632 + 0.472 ? 0464

+ O-542* f 0.646 I!I0.646 &O-721 f 0.533 AZ0.698

Region lateral to the holdfast organ

2.15 6.30 4.50 6.75 6.00

COMPOUNDSBY

0 + 0.408 + 0.559 I!IO-512 ? 0.478 AI0545

0 0 5.70f0.696 6.45 z!z0.612 8.50 + 0.508 940 + 0.652

2.90 7.75 8.05 9.25 8.75

4.70 + 0.581 7.30 + 0.701 IO*80 rt O-589 12.75 + 0.903 14.15 k 0.669 15.20 + 0.768

Anterior one-third

0.585 0.562 0,621 0,653 0.543 0.721

+ 0,623 + 0.685 + 0.988 -f.0688 f 0.793 + 0.605

f f f f + +

0 6.00 I!I0.502 11*35+0.603 15.05 + 1.134 17.20 f 0.599 18.35 + 0.732

7.20 12.15 13.60 16.50 19.70 11.60

10.15 14.80 18.20 20.15 23.05 24.20

Posterior two-thirds

Holdfast organ

Tegumental surface layer Forebody

4.00 7.50 7.00 7.25 8.05 8.75

TRITIATED

ADULT

A. marcianae

3.15 6.05 7.95 9.35

2.75 525 7.95 9.45 10.15

0 0 + 0.536 + 0.672 + 0.682 + 0.597

0 f 0.692 +_0469 f 0.493 + 0.682 *o-575

3.25 f 0.482 6.10 + 0.584 9.90 + 0.654 12.35 f 0.873 13.89 + 0.731 16-15 kO.829

Anterior one-third

7-45 12.65 16.85 20.10

3.75 9.15 11.70 15.80 18.50 18.75

5.30 13.65 18.85 19.90 23.50 24.75

0 0 + 0.482 + 0.837 + 0.936 I! 0.985

+ 0.568 f 0.872 i 0.637 f 0594 + 0.629 f 0.736

+ 0.702 +I0.589 + 0,625 f 0.843 rt0.783 + 0.693

Posterior two-thirds

Holdfast organ

Subtegument

Mean grain counts (less background) for 50 $ of tissue

VITRO UPTAKEOF

* Two standard errors of the mean. I Grain count was comparable to background.

1 2 5 10 20 30

Time (min)

Glucose

Tritiated compound

TABLE ~-IN

Abbreviations (all figures): ES, excretory space; F, forebody: HO, holdfast organ; ST, subtegument; T, tegumental surface layer; I’, vitellaria; VC, vitelline cells. FIGS. l-9. Autoradiographs of A. marcianae. FIGS. 1-6, 8 and 9 from Epon sections, osmium tetroxide fixation; Fro. 7 from paraffin section, Carnoy’s fluid fixation. 1. Posterior holdfast organ region, 1 min, H-glucose. Note higher radioactivity in tegumental surface layer than in subtegument. x 500. 2. Posterior Kate concentration of radioglucose holdfast organ region, 2 min, 3H-glucose. x 500. 3. Posterior holdfast organ region, 5 min, around excretory spaces. 3H-glucose. Note radioactivity throughout entire holdfast organ with highest x 200. 4. Posterior holdfast organ activity in and around excretory spaces. region, 10 min, rH-glucose. Note radioactivity in excretory space. x 500. 5. Entire worm, 60 min, 3H-glucose. Note high radioactivity in vitelline cells. x 20. 6. Holdfast organ, 60 min, 3H-glucose. Note high radioactivity in vitelline cells. Note incorporated radioglucose x 500. 7. Holdfast organ, 20 min, sH-glucose. x 500. 8. Posterior holdfast organ region, 1 min, in and around vitelline cells. 3H-tyrosine. Note higher activity in tegumental surface layer than in subtegument. x 500. 9. Posterior holdfast organ region, 5 min, 3H-tyrosine. Note higher radioactivity around excretory spaces. x 500.

FIGS. 10 and 12-14 from I’pon FIGS. 10-15. Autoradiographs of d. nzarciunae. sections, osmium tetroxide fixation ; FIG. 11 and 13 from paraffin sections, Carnoy’s fluid fixation. 10. Posterior holdfast organ region, 20 min, “H-tyrosine. x 500. 11. Holdfast organ vitellaria, Note radiotyrosine in excretory spaces. 20 min, 3H-tyrosine. Note incorporated 3H-tyrosine in vitelline cells. x 500. 12. Posterior holdfast organ region, 2 min, 3H-leucine. Note slight radioactivity in tegumental surface layer. x 500. 13. Posterior region of holdfast organ, 5 min, 3H-leucine. Note radioleucine in tegumental surface layer and subtegument. x 500. 14. Posterior holdfast organ region, 20 min, 3H-leucine. Note radioleucine in excretory space. x 500. 15. Holdfast organ vitellaria, 60 min, “Hx 500. leucine. Note absence of incorporated radioleucine in vitelline cells.

UPTAKE OF TRITIATBD COMPOUNDS BY ALARIA

MARCIANAE

991

(Fig. 3) and was now also detected in the excretory spaces (Fig. 4). The distribution after 30 min was similar to that after 20 min but a definite accumulation was evident in certain vitelline cells (Figs. 5 and 6). Radioglucose absorption by the tegument lateral to the holdfast organ for the l- and 2-min periods was similar to that for the same periods by the anterior holdfast organ region (Table 1). However, less radioactivity was present after the 5, lo,20 and 30-min periods. Tyrosine After 1 min 3H-tyrosine was found only in the tegumental surface and subtegument in the posterior region of the holdfast organ (Fig. 8). More radioactivity was present in the surface layer than in the subtegument (Table 1). After 2 min and all subsequent periods radiotyrosine was detected in the surface layer and subtegument of both regions (Table 1). After 5 min it was observed concentrated around the excretory spaces (Fig. 9). Tritiated tyrosine after 10, 20 and 30 min was present throughout the holdfast organ and was also detected in the excretory spaces (Fig. 10). Uptake of 3H-tyrosine by the tegument lateral to the holdfast organ after 2 and 5 min was similar to that for the same time periods by the anterior holdfast organ region (Table 1). Less radioa~vity was evident after lo,20 and 30 min than in the anterior region. Leucine

After 2 min 3H-leucine was seen only in the tegumental surface layer in the posterior region of the holdfast organ (Fig. 12; Table 1). After 5 min it was found in the surface layer and subtegument of the entire holdfast organ. The former area showed more radioactivity than the subte~ment~ area (Table 1). Radioleucine was not observed concentrated around the excretory space in any of the exposure periods (Fig. 13). After 10, 20 and 30 min it was found throughout the holdfast organ and was now also in the excretory spaces (Fig. 14). The uptake of tritiated leucine by the tegument lateral to the holdfast organ after 5 min was less than that for the same period in the anterior holdfast organ region (Table 1). There was no increase in the radioactivity in subsequent exposure periods. Inc~por ffled rud~oco~~~n~

In paraffin sections incorporated 3H-glucose was found after 10 and 20 min in and around the holdfast organ vitelline cells (Fig. 7). After 30 min it was also observed in the vitelline reserve which is located in the anterior part of the hindbody. Incorporated tritiated tyrosine was observed after 20 and 30 min in the vitelline cells of the holdfast organ (Fig. 11). No assimilated 3H-leucine was detected in paraffin sections (Fig. 15).

992

~~IKHA~B~A~I

AND ALLEN D.JOHNSON

Liquid sci~t~~lati~ counting

In liquid scintillation counting a direct correlation was found between the specific activity (dis/min per pg of protein) and the exposure time (min) for all three radiocompounds (Table 2). The rate of sH-glucose uptake (mpc/min) for the first minute was higher than the average rate for the other exposure periods (Table Z), possibly indicating a rapid initial uptake. The rate decreased with each successive time period. In the case of 3H-tryosine and 3H-leucine, the average rate of uptake showed considerable variation from one period to another (Table 2). In worms exposed for 1 min, scintillation counting revealed an uptake of 3H-leucine, although as noted above autoradiographs for this time period were negative. Liquid scintillation counting was also carried out with freeze-dried worms fixed in Carnoy’s fluid and dehydrated in ethanol. The results confirmed the presence of incorporated 3H-glucose after 10 min and incorporated 3H-tyrosine after 20 min, with an increase in radioactivity in succeeding periods for both compounds. DISCUSSION

This study is the first report of absorption of small water-soluble compounds by the tegument of an adult strigeoid. However, there is both direct and indirect evidence for uptake of such compounds in other digenetic trematodes. Those studies furnishing indirect evidence are reviewed by Nollen (1968). Direct evidence for uptake was provided by Nollen (1968) with ~~ilop~t~a~rn~ megal~~ (tritiated glucose, tyrosine, leucine, thymidine) and by Parkening & Johnson (1969) with Haematoloechus medioplexus (3H-glucose) and a Gorgoderina sp. (3H-glucose). These authors used autoradiographic methods similar to the one utilized in this study. The results here with adult A. mawianae demonstrated an in vitro uptake of 3H-glucose, 3H-tyrosine and aH-leucine by the holdfast organ tegument and to a lesser extent by the forebody tegument lateral to the holdfast organ Furthermore, a regional difference in uptake of these compounds by the tegumental surface layer of the holdfast organ was observed; the surface layer in the posterior twothirds exhibited more absorption than that in the anterior one-third. In relation to this, a regional difference in non-specific esterase activity is known to occur in the holdfast organ of A. marcianae (Johnson et al., 1971). However, in contrast to absorption, the esterase activity is limited to the anterior region (anterior mass of holdfast organ gland cehs). Erasmus & ljhman (1965) and Erasmus (1967b, 1969a, b, c, 1970a, b) in electron microscope studies of strigeoids have revealed the existence of regional differentiation of the tegument. In strigeoids with lappets three main areas of specialization are recognized ; the general body tegument, the holdfast organ tegument and the lappet tegument. Apparently, in A. marcianae differentiation is extended further to include two major areas of tegumental specialization within the holdfast organ. From histological and histochemical studies (Johnson et al., 1971)

%-LIQUID

72 96 252 984 1848 1896

2652 1452 3264 4836

1: 20 30

1 2 5 10 20 30

192 360

1992 2976 6252 8916 10,464 11,796

Total (dpm) *

174 168 170 170 172 174

170 172 172 172

170 168

172 170 170 174 172 172

Total pg protein t

0.41 0.57 1.48 5.79 lo,74 10.89

15.42 8.54 18.98 28.12

1.13 2.14

[email protected] 17.50 36.77 51.24 60.84 68.58

dis/min per pg

O-032 0.043 o-113 0443 O-832 0.854

0.654 I.194 I.470 2.178

0.086 0.162

O-897 l-340 2.816 4.016 4.713 5,313

A. pnarcianat?

O-032 o-021 O-024 O-066 0.039 o-002

0.108 O-164 O-027 O-069

O-086 0.076

O-897 0443 0.492 o-240 0.068 0.060

Average rate of uptake (m~/min)

IN ADULT

Total uptake (m&S

STUDIES ON THE UPTAKE OF TRITIATRD COMPOUNDS

1 2

5 10 20 30

:.

Time (min)

SCINTILLATION

* Average of three I-min counts less background radiation multiplied by four. t Determined by method of Nakai & Chi Le (1970). $ Based on millimicrocurie = 3.7 x 10 dps.

Leucine

Tyrosine

Glucose

Tritiated compound

TABLE

994

IFTIKHAR BHATTI .~ND ALLEN

D.JOHNSON

and the results of this study, it is suggested that there is (a) an anterior region primarily concerned with enzyme secretion for extracorporeal digestion and (b) a posterior region primarily involved with nutrient absorption. Electron microscope studies would be of interest here to determine possible differences in the fine structure of the tegument in these two areas. In this study more 3H-glucose was taken up by the holdfast organ tegument than the two amino acids (Tables 1 and 2). Nollen (1968) with P. megulurus also noted more absorption of 3H-glucose than of two amino acids (3H-tyrosine and 3H-leucine). On the other hand, he found that the amino acids were absorbed more rapidly by the cecal gastrodermis than the 3H-glucose. In this study no absorption of radioglucose by the gastrodermis ws observed. Parkening & Johnson (1969) also did not observe uptake of sH-glucose by the gastrodermis of H. medioplexus and a Gorgoderina sp. Incorporated triated glucose and tyrosine were found here only in and around the vitellaria of the holdfast organ of A. marcianae. In other digenetic trematodes incorporated labelled glucose and tyrosine have been reported in the vitellaria and also in a number of other tissues (Burton, 1962, 1963; Pantelouris, 1965; Thorsell et al., 1966; Fripp, 1967; Nollen, 1968; Parkening & Johnson, 1969). It may be that the maximum exposure period (30 min) used in this study was too short for transport and incorporation of the compounds in tissues outside of the holdfast organ. Light and electron microscope studies of adult strigeoids in situ have revealed an intimate association between the holdfast organ and the host mucosal tissue (Augustine& Uribe, 1927; Erasmus& ijhman, 1963,196s; ijhman, 1965,1966a, b; Erasmus, 1967b, 1970a; Johnson et al., 1971). Thus, the excretory spaces in the holdfast organ are brought into close association with the host tissue. It has been suggested that this system may function in the transport of soluble material, in addition to involvement in excretion and in the expansion of the holdfast organ (Erasmus & ijhman, 1963; ohman, 1966a, b; Erasmus, 1967a, 1970a). The only support for such a role is the results of a study on Cyathocotyle bushiensis by Erasmus (1967a). He found that the lipid droplets present in the cytoplasm of the epithelial cells lining the spaces became associated with the outer plasma membrane lamellae and later were released into the lumen of the spaces. In the present investigation 3H-glucose and 3H-tyrosine were found concentrated around the excretory spaces in the holdfast organ after 2 and 5 min, respectively. In the succeeding periods they were also detected in the lumen of the spaces. Leucine was not observed concentrated around the spaces but it was present in the lumen after 10 min. These results then would support the view that the reserve excretory system may play a role in nutrient transport. In liquid scintillation counting a direct correlation was found between specific activity (dis/min per pg of protein) and the duration of exposure (min) of the flukes with all three radiocompounds. Parkening & Johnson (1969) did not find this correlation in either starved or unstarved adult H. medioplexus nor in unstarved adults of a Gorgoderina sp. They stated that this was possibly due to the worms

UPTAKEOF TRITIATEDCOMPOUNDS BY

ALARIA MARCIANAE

995

being of different ages, size and recovered from several different frogs. In this study the adults used were of the same age and of approximately the same size. SUMMARY

Autoradiographic studies demonstrated the in vitro uptake of tritiated glucose, tyrosine and leucine by the tegument covering and lateral to the holdfast organ of adult A. marciunae (La Rue, 1917). Freeze-dried worms were embedded in Epon-812 to detect free radiocompounds and in paraffin to detect those incorporated. Absorption was evident for 3H-glucose and 3H-tyrosine after 1 min and for 3H-leucine after 2 min. Incorporated 3H-glucose and 3H-tyrosine were observed after 10 and 20 min, respectively. The tegumental surface layer (“cuticle”) of the holdfast organ showed a regional difference in uptake for all three radiocompounds; the surface layer in the posterior two-thirds exhibiting more absorption than that in the anterior one-third. It is suggested that the posterior region may be primarily concerned with nutrient absorption and anterior region mainly involved with enzyme secretion for extracorporeal digestion. Tritiated glucose and tyrosine were found concentrated around the excretory spaces in the holdfast organ after 2 and 5 min, respectively. In subsequent periods these compounds were observed in the lumen of the spaces. Leucine was detected in the lumen of the spaces after 10 min. These results support the view that the reserve excretory system may play a role in nutrient transport. In liquid scintillation counting, a positive correlation was found between specific activity (dis/min per pg of protein) and the duration of exposure (min). The average rate of uptake &c/min) decreased with each successive period with 3H-glucose, but considerable variation occurred with 3H-tyrosine and 3H-leucine. REFERENCES AUGUSTINED. L. & URIBE C. (1927) Alaria arisaemoides, n.sp., a trematode from Vulpes fulva. Parasit. 19, 236-244. BOGIT~H B. J. (1966) Histochemical observations on Posthodiplostomum minimum-II. Esterases in subcuticular cells, holdfast organ cells, and the nervous system. Exp. Parasit. 19, 64-70. BURTON P. R. (1962) In vitro uptake of radioglucose by a frog lung-fluke and correlation with the histochemical identification of glycogen. J. Pan&t. 48,874-882. BURTON P. R. (1963) A histochemical study of vitelline cells, egg capsules, and Mehlis’ gland in the frog lung-fluke, Haematoloechus medioplexus. J. exp. Zool. 154, 247-257. DAWES B. (1954) Maintenance in vitro of Fasciola hepatica. Nature, Land. 174, 654-655. DUBOIS G. (1957) La spCcifitCde fait chez les strigeida (Trematoda). In Premier Symposium SW la Sp&&itC Parasitaire des Parasites de Vert&rks (Edited by ATTINGERP.), pp. 213-228. Pub. Univ. Neuchltel. DE DUVE C. & WATTIAUX R. (1966) Functions of lysosomes. A. Rev. Physiol. 28,435-492. ERASMUS D. A. (1967a) Ultrastructural observations on the reserve bladder system of Cyathocotyle bushiensis Khan, 1962 (Trematoda: Strigeoidea) with special reference to lipid excretion. J. Parasit. 53, 525-536. ERASMUS D. A. (1967b) The host-parasite interface of Cyathocotyle bushiensis Khan, 1962 (Trematoda: Strigeoidea)-II. Electron microscope studies of the tegument. J. Parasit. 53, 703-714.

996

IFTIKHAR BHATTI ANDALLEN D. JOHNSON

ERASMUSD. A. (1969a) Studies on the host-parasite interface of strigeoid trematodes-IV. The ultrastructure of the lappets of Apatemon gracilis minor Yamaguti, 1933. Parasitology 59, 193-201. ERASMUSD. A. (1969b) Studies on the host-parasite interface of strigeoid trematodes-V. Regional differentiation of the adhesive organ of Apatemon gracilis minor Yamaguti, 1933. Parasitology 59, 245-256. ERASMUSD. A. (1969~) Studies of the host-parasite interface of strigeoid trematodes-VI. Ultrastructural observation on the lappets of Diplostomum phoxini Faust, 1918. Z. F. Parasitk. 32, 48-58. ERASMUSD. A. (1970a) The host-parasite interface of strigeoid trematodes-VII. Ultrastructural observations on the adhesive organ of Diplostomum phoxini Faust, 1918. Z. F. Parasitk. 33, 211-224. ERASMUSD. A. (1970b) The host-parasite interface of strigeoid trematodes-IS. A probe and transmission electron microscope study of the tegument of Diplostomum phoxini Faust, 1918. Parasitology 61, 3541. ERASMUSD. A. & OHMAN C. (1963) The structure and function of the adhesive organ in strigeoid trematodes. Ann. N. Y. Acad. Sci. 133, 7-35. ERASMUSD. A. & Z)HMANC. (1965) Electron microscope studies of the gland cells and host parasite interface of the adhesive organ of Cyathocotyle bushiensis Khan, 1962. r. Parasit. 51,761-769. FRIPP P. J. (1967) The sites of (I-14C) glucose assimilation in Schistosoma haematobium. Camp. Biochem. Physiol. 23, 893-898. JOHNSON A. D. (1968) Life history of Alaria marcianae (La Rue, 1917) Walton, 1949 (Trematoda: Diplostomatidae). J. Parasit. 54, 324-332. JOHNSONA. D., BHATTI I. & KANEMOTON. (1971) Structure and function of the holdfast organ and lappets of Alaria marcianae (La Rue, 1917) (Trematoda: Diplostomatidae). r. Parasit. 51, 235-243. LUFT J. H. (1961) Improvement in epoxy resin embedding methods. J. biophys. biochem. Cytol. 9, 409-414. NAKAI S. & CHI LE A. (1970) Spectrophotometric determination of protein and fat in milk simultaneously. J. Dairy Sci. 53, 276-278. NOLLEN P. M. (1968) Uptake and incorporation of glucose, tyrosine, leucine, and thymidine by adult Philophthalmus megalurus (Cort, 1914) (Trematoda), as determined by autoradiography. r. Parasit. 54, 295-304. NOVIKOFFA. B. (1963) Lysosomes in the physiology and pathology of cells: contributions of staining methods. In Lysosomes (Edited by DE REUCK A. V. S. & CAMERONM. P.) pp. 36-73. Little, Brown & Co., Boston. OHMANC. (1965) The structure and function of the adhesive organ in strigeoid trematodesII. Diplostomum spathaceum Braun, 1893. Parasitology 55, 481-502. OHMAN C. (1966a) The structure and function of the adhesive organ in strigeoid trematodes-III. Apatemon gracilis minor Yamaguti, 1933. Parasitology 56, 209226. OHMAN C. (1966b) The structure and function of the adhesive organ in strigeoid trematodes-IV. Holostephanus luehei Szidat, 1936. Parasitology 56, 481-491. PANTELOURISE. M. (1965) Utilization of methionine by the liver fluke, Fasciola hepatica. Res. Vet. Sci. 6, 334-336. PARKENINGT. A. & JOHNSONA. D. (1969) Glucose uptake in Haematoloechus medioplexus and Gorgoderina trematodes. Exp. Parasit. 25, 358-367. STIRLING C. E. & KINTER W. B. (1967) High-resolution radioautography of galactose-SH accumulation in rings of hamster intestine. J. cell Biol. 35, 585-604. STUMPF W. E. & ROTH L. J. (1964) Vacuum freeze drying of frozen sections for drymounting, high-resolution autoradiography. Stain Tech. 39, 219-223.

UPTAKEOF TRITIATEIICOMPOUNDS BY ALARIA

MARCIANAE

997

STUMPFW. E. & ROTH L. J. (1967) Freeze-drying of small tissue samples and thin frozen sections below -60°C. A simple method of cryosorption pumping. J. Histochem. Cytochm. 15, 243-251. THORSELL W., BJ~~RKMAN N. & APPELGRENL. E. (1966) Radioautographic studies on the ovary and vitelline glands of the liver fluke, Fasciola hepatica L. after short in vitro incubation with some amino acids. 2. F. Pam&k. 28, 108-l 15. WILSKJZK. R. & Ross R. (1965) Autoradiographic localization of lipid and water-soluble compounds: a new approach. J. Histochem. Cytochem. 13, 38-43. Key Word IndeaLAlaria autoradiography.

marcianae; trematode absorption; glucose; tyrosine; leucine;