- Email: [email protected]
In vivo incorporation of tritiated cytidine and tritiated thymidine by the cestode, Hymenolepis microstoma
EXPERIMENTAL
PARASITOLOGY
In
14, 316-322
(1963)
Viva Incorporation of Tritiated Cytidine Tritiated Thymidine by the Cestode, Hymenolepis microstoma James
Department
of Zoology
A.
Dvorak
and
and Entomology,
(Submitted
Arthur
W.
The University
for publication,
21 February
and
Jones
of Tennessee, Knoxville 1963)
The mouse bile duct cestode, Hymenolefiis microstoma, was exposed in vivo in separate experiments to tritiated cytidine and tritiated thymidine. Nucleolar and nuclear labeling by cytidine and nuclear labeling by thymidine, as revealed by autoradiographic methods, occurred in both host and cestode tissues. As expected, only synthetically active nuclei were labeled: those of the neck, developing organs, and embryos of the cestode. Bile duct epithelium and pancreatic acinar cells, autoradiographed as a control, showed the same degree and localization of cytidine labeling as the active ce!ls of the cestode. The assimilation of cytidine and thymidine by absorption from the fluid contents of the bile duct implies that cestodes can absorb such materials from their immediate environment.
and Taylor histochemical techniques using radionuclides Read (1956), Daugherty (1956), Goodchild and Wells (1957), Kent should prove useful in the search for physio(1957), Roberts (1961), and others have logical facts about cestodes. Among the funbegun to study the biochemistry of cestodes, damental processes demonstrable by radioproceeding by methods of quantitative anal- nuclide histochemistry is the incorporation ysis for such substances as lipids, proteins, of exogenously furnished nucleosides into amino acids, and carbohydrates. The use of both ribose nucleic acid (RNA) and deoxyradionuclides in the biochemical analysis of ribose nucleic acid (DNA). It is well known cestodes (Read, 1950; Phifer, 1960) has also that cytidine, the nucleoside derivative of the proven to be a profitable approach. In addi- pyrimidine base cytosine, is utilized by many tion, some histochemical studies have been organisms in the formation of RNA. Such made (Hedrick, 1958 ; Heyneman and Voge, synthesis appears to occur in the nucleus 1957; and others). Both normal and experi- usually at the region of the nucleolus. Since mentally treated worms have been studied. It cytidine is also an essential in the structure is hoped that this line of research will con- of the DNA molecule, it may eventually be tinue to yield information on various aspects incorporated into the chromosomes during of cestode nutrition, both as to what materials DNA synthesis. Also, cytidine incorporated are utilized and as to the pathways by which in RNA may be found throughout the cytoplasm, some time after its initial localization the cestode obtains materials from its host. We believe that in addition to the biochem- in the nucleolus. Thymidine, the nucleoside ical and histochemical work mentioned above, derivative of the pyrimidine base thymine, one of the four essential bases in the DNA 1 This study was supported in part by Research molecule, is incorporated only into the chroContract AT(40-1) 1749 with the US. Atomic Energy mosomes themselves. When fully incorporated Commission. 316
RADIONUCLIDES
into the living cell, thymine, therefore, occupies chromosomal locations. Both cytidine and thymidine are taken up by active cells, especially those cells that are premitotic or engaged in active synthesis. The radioactive forms of these nucleosides, tritiated cytidine and tritiated thymidine, can be used, because of the above-mentioned behavior, to indicate by radioactivity (or labeling) the sites in cells or tissues where RNA or DNA synthesis is going on. In cestodes, the cells or tissues most active in growth are known to be those of the neck region, the developing organs, and embryos. We expected to be able to label these regions. Two difficulties involved in studying the absorption of materials by cestodes are competition for the materials by the host, if the experiment is performed in ~i’uo, and doubt as to the condition of the worm, if the worm is placed with the experimental material in vitro. The presence of uncontrolled contaminants in the milieu of most cestodes (the gut contents of the host) further complicates absorption experiments. We think we avoided these difficulties by performing our experiments in a living host and by using a cestode the environment of which is ‘Lcontrolled” to an unusual degree. The bile duct tapeworm of mice (Hymenolepis microstoma) can be kept within its normal habitat by ligation and can be subjected to measured quantities of absorbable material within the ligated bile duct. Competition by the host tissues for the material (in this case cytidine or thymidine) can not be controlled, of course, but can at least be observed; the extent of labeling of adjacent host tissue indicates relative degrees of absorption by the host. MATERIALS
AND METHODS
Tritiated cytidine experiment. Mature male Rockland strain white mice were infected with 5 cysts each of H. microstoma. The cestodes were allowed to develop for 10 days. Each mouse was then anesthetized with so-
IN A CESTODE
317
dium nembutal (7.5 mg/lOO gm body weight; Amos and Wakefield, 1958), and a partial hepatectomy was performed. In this operation the left and median lobes of the liver were removed to facilitate visualization and to further operations on the infected bile duct. The bile duct once exposed was ligated at its point of bifurcation at the base of the liver and at its junction with the duodenum. The duodenum was also ligated on both sides of the bile duct-duodenum junction. Tritiated cytidine (New England Nuclear Corp., 577 Albany St., Boston 18, Mass.) having a specific activity of 7.81 curies per millimole in the concentration of 10 PC per cubic centimeter was then injected into the lumen of the bile duct. The vehicle consisted of 0.1 cc of sterile 0.85% saline. The abdominal incision was then closed to prevent drying or marked temperature changes of the tissues. After 30 minutes the ligated bile duct with some associated duodenal and pancreatic tissue was removed and fixed in toto in Carnoy’s 6-3 (absolute ethanol-glacial acetic acid). Overnight refrigeration of the tissue blocks was followed by dehydration, clearing, and paraffin embedding. Serial sections cut at 10 lo were mounted on slides coated with potassiumchrome alum (Boyd, 1955) and allowed to dry overnight. Following hydration, the sections were treated with 5% trichloroacetic acid at 4°C for 5 minutes to remove any unincorporated tritiated cytidine. The slides were then coated with Kodak NTB-2 liquid emulsion, dried, and stored over a desiccant (silica gel) in a light-tight box at 4°C. Exposure time was 14 days. The emulsion was developed in Kodak D-19 (4 minutes), rinsed in water ( 15 seconds), and fixed (5 minutes), all at 17°C. The sections were stained with Toluidine Blue 0. Control sections were treated with RNase, which removed incorporated label to the level of background. Tritiated thymidine experiment. Procedures identical to the above were followed, with one deletion and one addition. No hepatectomy
318
DVORAK AND JONES
lack of labeling (Fig. 1). The scolex is not an active site of nucleic acid synthesis compared to other portions of the cestode; however, various structural features of this organ could probably be labeled with increased incubation time, indicating maintenance and repair. The embryonic neck region of the cestode (a region directly behind the scolex consisting of parenchyma and undifferentiated cells) is intensely labeled (Fig. 2). Some of these cells are thought to give rise eventually to the cestode’s reproductive system, although little is known about the mode of differentiation of these cells. The use of radionuclides might be a rewarding approach to this histogenetic problem. Farther back, where the sex organs have begun to differentiate within the parenchyma-filled cavity of the cestode, the parenchymal labeling is less pronounced (Fig. 3). In the same region, however, the ovarian tissue is heavily labeled. This tissue contains the developing oocytes. The presence of heavy labeling is taken to indicate rapid synthesis RESULTS of nucleic acid. Figures 4-9 show some of Both cytidine and thymidine were absorbed these oocytes at various stages of developand presumably utilized by both host and ment, the nucleoli of these cells being very parasite. Labeling occurred in definite parts prominently labeled. of the cestode and in various tissues of the Among the tissues of the host, pancreatic host. acinar cells show nuclear labeling (Fig. 11) . We believe that both cytidine and thymiAs the cytoplasm of these cells, although dine were utilized, for the following reasons. known to be rich in RNA, is not labeled it The thymidine used (see above) was known can be assumed that the labeled cytosine in to be tritiated in the base methyl group. this case is restricted to nuclear RNA (i.e., Therefore, preabsorptive hydrolysis, even if nucleolus) and has not “spilled over” into the it occurred, could not separate the label from cytoplasm. The results obtained in the panthe base, and labeled cell elements must have creatic acinar tissue of the mouse are similar been sites of thymidine incorporation. Al- to those obtained with the rat by Fitzgerald though we cannot be certain of cytidine be- and Vinijchaikul (1959). The epithelial cells havior, not knowing the exact location of the of the bile duct lumen (Fig. 10) present a tritium substitution, our use of RNase picture similar to that of host acinar cells (above) to remove labeled RNA from con- and the presumably active tissues of the tapetrol sections convinced us that the nucleoside worm. had been incorporated. Thy&dine experiment. Figure 12 is a longiCytidine experinzent. The scolex, a holdfast tudinal section of the scolex and part of the organ formed during an intermediate stage embryonic neck region of a cestode. The in the cestode’s life cycle, shows a complete scolex is again unlabeled. The labeled area
was performed. Since the thymidine experiment was performed later than the cytidine, experience gained during the latter made hepatectomy unnecessary. An additional control was required. Because the adjacent host tissues do not contain rapidly proliferating cells, we could not use them as indicators of uptake of thymidine. Therefore a separate experiment was performed. About 1 cm of the duodenum of a mouse was tied off and the same amount of radionuclide as given below was injected and allowed to remain for 30 minutes. The duodenal tissue was then treated in the same way as the cestodes and adjacent bile duct tissue. Tritiated thymidine (New England Nuclear Corp.) having a specific activity of 6.60 curies per millimole was used in the concentration of 10 uc per cubic centimeter. The vehicle consisted of 50 ul of sterile 0.85rjc saline. Control sections were treated with DNase, which removed incorporated label to the level of background.
RADIONUCLIDES
IN A CESTODE
319
FIG. 1. Scolex, unlabeled after tritiated cytidine exposure. (Scale on Fig. 1 for Figs. l-11 equals 10 CL.) FIG. 2. Portion of embryonic neck region with heavy tritiated cytidine labeling. FIG. 3. Portion of mature segment with diffuse tritiated cytidine labeling. FIGS. 4-9. Developing oocytes showing labeled nucleoli. FIG. 10 Bile duct epithelial cells labeled after exposure to tritiated cytidine. FIG. 11. Pancreatic acinar cells labeled after tritiated cytidine exposure.
320
DVORAK
AND
JONES
FIG. 12. Longitudinal section of scolex (unlabeled) and neck region (labeled) of a cestode after tritial ted thymidine exposure (scale equals 50 p) FIG. 13. Cross section of embryonic neck region showing medullary labeling after tritiated thymid ine exposure. (Scale on Fig. 13 for Figs. 13 and 14 equals 50 F). FIG. 14. Oblique section through scolex (unlabeled) after tritiated thymidine exposure. FIGS. 15 and 16. Selected labeled areas of worm shown in Fig. 12. (Scale on Fig, 15 for Figs, 1.5 and 16 equals 10 p) .
RADIONUCLIDES
IN A CESTODE
321
tion of these radioactive substances in metabolically or mitotically active nuclei fulfilled expectations, the experiments can be considered effective in demonstrating that cestode cells, like those of other organisms, (Bowen, 1952) utilize the above nucleosides. Two questions remain to be considered, one concerning competition for materials and the possibility of indirect uptake by the parasite, the other concerning general theories of cestode nutrition. First, is it reasonable to conclude that the compounds were used directly by the cestode and were not first absorbed competitively by the host and then, perhaps in altered form, transferred to the parasite? Cestodes may absorb some materials directly from the host tissue with which they are in contact, and it is possible that the cytidine and thymidine followed such a devious route into the cestode. If the latter were the case, one would expect to find evidence, for example, of difference either in distribution or in degree between labeling of host and labeling of parasite tissues. The autoradiographs indicate that no such difference exists, active cells in both mouse and cestode being comparably labeled. Therefore there is no reason to believe that either organism affected the uptake of the compounds by the other during the 30-minute period involved, or that consecutive synthesis and transfer are evident. In a sense the second question, whether this experiment sheds any light on cestode nutrition in general, has just been answered. We believe we have shown that a cestode normally not in contact with intestinal contents, and usually restricted to the relatively limited environment of the bile duct lumen, can absorb at least two essential nucleosides DISCUSSION from its natural environment and can incorporate these into cellular components. This Since the results generally confirmed the fact implies that other diffusible substances hypothesis that RNA and DNA labeling may be readily obtained by cestodes, not would occur after treatment of the cestodes only by contact with the host tissue, but also in vivo with tritiated cytidine and tritiated directlv from the fluids surroundingv the oarathymidine, respectively, and since the localiza.~~~ , I
(arrow) begins directly behind the scolex and extends throughout the entire neck region, becoming somewhat diffuse as organs begin to develop within the parenchyma. Figure 13 shows the embryonic neck region in cross section. The labeled medullary area (arrow) is typical of all sections examined. The most heavily labeled cells occupy a position contiguous with but interior to the body muscles. This was also observed in the cytidine experiment. In no instances were the cortical cells of the cuticle observed to be labeled. Although there is little doubt that these cells are responsible for the absorption of nutrients, they are evidently not sites of active nucleic acid synthesis. In an experiment where unincorporated nucleoside is allowed to remain in the tissue or one in which the worm is allowed a longer access to the isotope, these cells would probably be labeled. In Fig. 14 (an oblique section through the scolex) , as in Figs. 1 and 12, the organ is unlabeled. Figures 15 and 16 are enlargements of selected labeled areas of Fig. 12. A remarkable difference between the heavily labeled nuclei directly beneath the body muscles and the unlabeled subcuticular nuclei is evident. The adjacent host tissues in this experiment are not actively growing tissues like the labeled portions of the cestode. Therefore we did not obtain significant labeling in these tissues. Separately treated duodenal tissues, however, assimilated the radionuclide in the same manner and to the same degree as the cestode; crypt cell nuclei of the duodenum exhibited a picture similar to that seen in the medullary cells of the cestode. (The crypt cells are rapidly reproducing portions of the duodenal epithelium.)
322
DVORAK
sites. Berntzen (1962) has demonstrated the growth and development of a cestode in vitro, a demonstration, of course, that all essential materials can be absorbed from a liquid medium, without any contact whatsoever between the worm and the tissues of the host. Our work provides evidence that in nature, in viva, that is, the same kind of absorption required by Berntzen’s experimental results can occur. REFERENCES AMOS, D. B., AND WAKEFIELD, J. D. 1958. Growth of mouse ascites tumor cells in diffusion chambers. I. Studies of growth rate of cells and of the rate of entry of antibody. Journal of the National Cancer Institute 21, 657-670. BERNTZEN, A. K. 1962. In vitro cultivation of tapeworms. II. Growth and maintenance of Hymenolepis nana (Cestoda: Cyclophyllidea) . Journal of Parasitology 48, 785-797. BOWEN, V. T. (Ed.). 1952. The chemistry and physiology of the nucleus (Symposium). Experimental Cell Research 3, (Suppl. 2). BOYD, G. A. 1955. “Autoradiography in Biology and Medicine.” Academic Press, New York. DAUGHERTY, J, W., AND TAYLOR, D. 1956. Regional distribution of glycogen in the rat cestode, Hymenolepis diminuta. Experimental Parasitology 5, 376-390. FITZGERALD, P. J., AND VINIJCHAIKUL, K. 1959.
AND
JONES
Nucleic acid metabolism of pancreatic cells as revealed by cytidine-Hs and thymidine-H”. Laboratory Investigation 8, 319-329. GOODCHILD, CHAUNCEY G., AND WELLS, 0. C. 1957. Amino acids in larval and adult tapeworms (Hymenolepis diminuta) and in the tissues of their rat and beetle hosts. Experimental Parasitology 6, 575-585. H~DRICK, R. M. 1958. Comparative histochemical studies on cestodes. II. The destribution of fat substances in Hymenolepis diminuta and Raillietina cesticillus. Journal of Parasitology 44, 75-84. HEYNEMAN, D., AND VOGE, M. 1957. Glycopen distribution in cysticercoids of three hymenolepidid cestodes. Journal of Parasitology 43, 527-531. KENT, H. N. 1957. Biochemical studies on the proteins of Hymenolepis diminuta. Experimental Parasitology 6, 351-357. PHIFER, K. 1960. Permeation membrane transport in animal parasites: Further observations on the uptake of glucose by Hymenolepis diminuta. 46, 137-144. Journal of Parasitology READ, C. P. 1950. The acquisition of isotopically labeled inorganic phosphate by the tapeworm Hymenolepis diminuta, with some remarks on the host-parasite relationship. Journal of Parasitology 36, 34-40. ROBERTS, L. S. 1961. The influence of population density on patterns and physiology of growth in Hymenolepis diminuta (Cestoda: Cyclophyllidea) in the definitive host. Experimental Parasitology 11, 332-371.
PARASITOLOGY
In
14, 316-322
(1963)
Viva Incorporation of Tritiated Cytidine Tritiated Thymidine by the Cestode, Hymenolepis microstoma James
Department
of Zoology
A.
Dvorak
and
and Entomology,
(Submitted
Arthur
W.
The University
for publication,
21 February
and
Jones
of Tennessee, Knoxville 1963)
The mouse bile duct cestode, Hymenolefiis microstoma, was exposed in vivo in separate experiments to tritiated cytidine and tritiated thymidine. Nucleolar and nuclear labeling by cytidine and nuclear labeling by thymidine, as revealed by autoradiographic methods, occurred in both host and cestode tissues. As expected, only synthetically active nuclei were labeled: those of the neck, developing organs, and embryos of the cestode. Bile duct epithelium and pancreatic acinar cells, autoradiographed as a control, showed the same degree and localization of cytidine labeling as the active ce!ls of the cestode. The assimilation of cytidine and thymidine by absorption from the fluid contents of the bile duct implies that cestodes can absorb such materials from their immediate environment.
and Taylor histochemical techniques using radionuclides Read (1956), Daugherty (1956), Goodchild and Wells (1957), Kent should prove useful in the search for physio(1957), Roberts (1961), and others have logical facts about cestodes. Among the funbegun to study the biochemistry of cestodes, damental processes demonstrable by radioproceeding by methods of quantitative anal- nuclide histochemistry is the incorporation ysis for such substances as lipids, proteins, of exogenously furnished nucleosides into amino acids, and carbohydrates. The use of both ribose nucleic acid (RNA) and deoxyradionuclides in the biochemical analysis of ribose nucleic acid (DNA). It is well known cestodes (Read, 1950; Phifer, 1960) has also that cytidine, the nucleoside derivative of the proven to be a profitable approach. In addi- pyrimidine base cytosine, is utilized by many tion, some histochemical studies have been organisms in the formation of RNA. Such made (Hedrick, 1958 ; Heyneman and Voge, synthesis appears to occur in the nucleus 1957; and others). Both normal and experi- usually at the region of the nucleolus. Since mentally treated worms have been studied. It cytidine is also an essential in the structure is hoped that this line of research will con- of the DNA molecule, it may eventually be tinue to yield information on various aspects incorporated into the chromosomes during of cestode nutrition, both as to what materials DNA synthesis. Also, cytidine incorporated are utilized and as to the pathways by which in RNA may be found throughout the cytoplasm, some time after its initial localization the cestode obtains materials from its host. We believe that in addition to the biochem- in the nucleolus. Thymidine, the nucleoside ical and histochemical work mentioned above, derivative of the pyrimidine base thymine, one of the four essential bases in the DNA 1 This study was supported in part by Research molecule, is incorporated only into the chroContract AT(40-1) 1749 with the US. Atomic Energy mosomes themselves. When fully incorporated Commission. 316
RADIONUCLIDES
into the living cell, thymine, therefore, occupies chromosomal locations. Both cytidine and thymidine are taken up by active cells, especially those cells that are premitotic or engaged in active synthesis. The radioactive forms of these nucleosides, tritiated cytidine and tritiated thymidine, can be used, because of the above-mentioned behavior, to indicate by radioactivity (or labeling) the sites in cells or tissues where RNA or DNA synthesis is going on. In cestodes, the cells or tissues most active in growth are known to be those of the neck region, the developing organs, and embryos. We expected to be able to label these regions. Two difficulties involved in studying the absorption of materials by cestodes are competition for the materials by the host, if the experiment is performed in ~i’uo, and doubt as to the condition of the worm, if the worm is placed with the experimental material in vitro. The presence of uncontrolled contaminants in the milieu of most cestodes (the gut contents of the host) further complicates absorption experiments. We think we avoided these difficulties by performing our experiments in a living host and by using a cestode the environment of which is ‘Lcontrolled” to an unusual degree. The bile duct tapeworm of mice (Hymenolepis microstoma) can be kept within its normal habitat by ligation and can be subjected to measured quantities of absorbable material within the ligated bile duct. Competition by the host tissues for the material (in this case cytidine or thymidine) can not be controlled, of course, but can at least be observed; the extent of labeling of adjacent host tissue indicates relative degrees of absorption by the host. MATERIALS
AND METHODS
Tritiated cytidine experiment. Mature male Rockland strain white mice were infected with 5 cysts each of H. microstoma. The cestodes were allowed to develop for 10 days. Each mouse was then anesthetized with so-
IN A CESTODE
317
dium nembutal (7.5 mg/lOO gm body weight; Amos and Wakefield, 1958), and a partial hepatectomy was performed. In this operation the left and median lobes of the liver were removed to facilitate visualization and to further operations on the infected bile duct. The bile duct once exposed was ligated at its point of bifurcation at the base of the liver and at its junction with the duodenum. The duodenum was also ligated on both sides of the bile duct-duodenum junction. Tritiated cytidine (New England Nuclear Corp., 577 Albany St., Boston 18, Mass.) having a specific activity of 7.81 curies per millimole in the concentration of 10 PC per cubic centimeter was then injected into the lumen of the bile duct. The vehicle consisted of 0.1 cc of sterile 0.85% saline. The abdominal incision was then closed to prevent drying or marked temperature changes of the tissues. After 30 minutes the ligated bile duct with some associated duodenal and pancreatic tissue was removed and fixed in toto in Carnoy’s 6-3 (absolute ethanol-glacial acetic acid). Overnight refrigeration of the tissue blocks was followed by dehydration, clearing, and paraffin embedding. Serial sections cut at 10 lo were mounted on slides coated with potassiumchrome alum (Boyd, 1955) and allowed to dry overnight. Following hydration, the sections were treated with 5% trichloroacetic acid at 4°C for 5 minutes to remove any unincorporated tritiated cytidine. The slides were then coated with Kodak NTB-2 liquid emulsion, dried, and stored over a desiccant (silica gel) in a light-tight box at 4°C. Exposure time was 14 days. The emulsion was developed in Kodak D-19 (4 minutes), rinsed in water ( 15 seconds), and fixed (5 minutes), all at 17°C. The sections were stained with Toluidine Blue 0. Control sections were treated with RNase, which removed incorporated label to the level of background. Tritiated thymidine experiment. Procedures identical to the above were followed, with one deletion and one addition. No hepatectomy
318
DVORAK AND JONES
lack of labeling (Fig. 1). The scolex is not an active site of nucleic acid synthesis compared to other portions of the cestode; however, various structural features of this organ could probably be labeled with increased incubation time, indicating maintenance and repair. The embryonic neck region of the cestode (a region directly behind the scolex consisting of parenchyma and undifferentiated cells) is intensely labeled (Fig. 2). Some of these cells are thought to give rise eventually to the cestode’s reproductive system, although little is known about the mode of differentiation of these cells. The use of radionuclides might be a rewarding approach to this histogenetic problem. Farther back, where the sex organs have begun to differentiate within the parenchyma-filled cavity of the cestode, the parenchymal labeling is less pronounced (Fig. 3). In the same region, however, the ovarian tissue is heavily labeled. This tissue contains the developing oocytes. The presence of heavy labeling is taken to indicate rapid synthesis RESULTS of nucleic acid. Figures 4-9 show some of Both cytidine and thymidine were absorbed these oocytes at various stages of developand presumably utilized by both host and ment, the nucleoli of these cells being very parasite. Labeling occurred in definite parts prominently labeled. of the cestode and in various tissues of the Among the tissues of the host, pancreatic host. acinar cells show nuclear labeling (Fig. 11) . We believe that both cytidine and thymiAs the cytoplasm of these cells, although dine were utilized, for the following reasons. known to be rich in RNA, is not labeled it The thymidine used (see above) was known can be assumed that the labeled cytosine in to be tritiated in the base methyl group. this case is restricted to nuclear RNA (i.e., Therefore, preabsorptive hydrolysis, even if nucleolus) and has not “spilled over” into the it occurred, could not separate the label from cytoplasm. The results obtained in the panthe base, and labeled cell elements must have creatic acinar tissue of the mouse are similar been sites of thymidine incorporation. Al- to those obtained with the rat by Fitzgerald though we cannot be certain of cytidine be- and Vinijchaikul (1959). The epithelial cells havior, not knowing the exact location of the of the bile duct lumen (Fig. 10) present a tritium substitution, our use of RNase picture similar to that of host acinar cells (above) to remove labeled RNA from con- and the presumably active tissues of the tapetrol sections convinced us that the nucleoside worm. had been incorporated. Thy&dine experiment. Figure 12 is a longiCytidine experinzent. The scolex, a holdfast tudinal section of the scolex and part of the organ formed during an intermediate stage embryonic neck region of a cestode. The in the cestode’s life cycle, shows a complete scolex is again unlabeled. The labeled area
was performed. Since the thymidine experiment was performed later than the cytidine, experience gained during the latter made hepatectomy unnecessary. An additional control was required. Because the adjacent host tissues do not contain rapidly proliferating cells, we could not use them as indicators of uptake of thymidine. Therefore a separate experiment was performed. About 1 cm of the duodenum of a mouse was tied off and the same amount of radionuclide as given below was injected and allowed to remain for 30 minutes. The duodenal tissue was then treated in the same way as the cestodes and adjacent bile duct tissue. Tritiated thymidine (New England Nuclear Corp.) having a specific activity of 6.60 curies per millimole was used in the concentration of 10 uc per cubic centimeter. The vehicle consisted of 50 ul of sterile 0.85rjc saline. Control sections were treated with DNase, which removed incorporated label to the level of background.
RADIONUCLIDES
IN A CESTODE
319
FIG. 1. Scolex, unlabeled after tritiated cytidine exposure. (Scale on Fig. 1 for Figs. l-11 equals 10 CL.) FIG. 2. Portion of embryonic neck region with heavy tritiated cytidine labeling. FIG. 3. Portion of mature segment with diffuse tritiated cytidine labeling. FIGS. 4-9. Developing oocytes showing labeled nucleoli. FIG. 10 Bile duct epithelial cells labeled after exposure to tritiated cytidine. FIG. 11. Pancreatic acinar cells labeled after tritiated cytidine exposure.
320
DVORAK
AND
JONES
FIG. 12. Longitudinal section of scolex (unlabeled) and neck region (labeled) of a cestode after tritial ted thymidine exposure (scale equals 50 p) FIG. 13. Cross section of embryonic neck region showing medullary labeling after tritiated thymid ine exposure. (Scale on Fig. 13 for Figs. 13 and 14 equals 50 F). FIG. 14. Oblique section through scolex (unlabeled) after tritiated thymidine exposure. FIGS. 15 and 16. Selected labeled areas of worm shown in Fig. 12. (Scale on Fig, 15 for Figs, 1.5 and 16 equals 10 p) .
RADIONUCLIDES
IN A CESTODE
321
tion of these radioactive substances in metabolically or mitotically active nuclei fulfilled expectations, the experiments can be considered effective in demonstrating that cestode cells, like those of other organisms, (Bowen, 1952) utilize the above nucleosides. Two questions remain to be considered, one concerning competition for materials and the possibility of indirect uptake by the parasite, the other concerning general theories of cestode nutrition. First, is it reasonable to conclude that the compounds were used directly by the cestode and were not first absorbed competitively by the host and then, perhaps in altered form, transferred to the parasite? Cestodes may absorb some materials directly from the host tissue with which they are in contact, and it is possible that the cytidine and thymidine followed such a devious route into the cestode. If the latter were the case, one would expect to find evidence, for example, of difference either in distribution or in degree between labeling of host and labeling of parasite tissues. The autoradiographs indicate that no such difference exists, active cells in both mouse and cestode being comparably labeled. Therefore there is no reason to believe that either organism affected the uptake of the compounds by the other during the 30-minute period involved, or that consecutive synthesis and transfer are evident. In a sense the second question, whether this experiment sheds any light on cestode nutrition in general, has just been answered. We believe we have shown that a cestode normally not in contact with intestinal contents, and usually restricted to the relatively limited environment of the bile duct lumen, can absorb at least two essential nucleosides DISCUSSION from its natural environment and can incorporate these into cellular components. This Since the results generally confirmed the fact implies that other diffusible substances hypothesis that RNA and DNA labeling may be readily obtained by cestodes, not would occur after treatment of the cestodes only by contact with the host tissue, but also in vivo with tritiated cytidine and tritiated directlv from the fluids surroundingv the oarathymidine, respectively, and since the localiza.~~~ , I
(arrow) begins directly behind the scolex and extends throughout the entire neck region, becoming somewhat diffuse as organs begin to develop within the parenchyma. Figure 13 shows the embryonic neck region in cross section. The labeled medullary area (arrow) is typical of all sections examined. The most heavily labeled cells occupy a position contiguous with but interior to the body muscles. This was also observed in the cytidine experiment. In no instances were the cortical cells of the cuticle observed to be labeled. Although there is little doubt that these cells are responsible for the absorption of nutrients, they are evidently not sites of active nucleic acid synthesis. In an experiment where unincorporated nucleoside is allowed to remain in the tissue or one in which the worm is allowed a longer access to the isotope, these cells would probably be labeled. In Fig. 14 (an oblique section through the scolex) , as in Figs. 1 and 12, the organ is unlabeled. Figures 15 and 16 are enlargements of selected labeled areas of Fig. 12. A remarkable difference between the heavily labeled nuclei directly beneath the body muscles and the unlabeled subcuticular nuclei is evident. The adjacent host tissues in this experiment are not actively growing tissues like the labeled portions of the cestode. Therefore we did not obtain significant labeling in these tissues. Separately treated duodenal tissues, however, assimilated the radionuclide in the same manner and to the same degree as the cestode; crypt cell nuclei of the duodenum exhibited a picture similar to that seen in the medullary cells of the cestode. (The crypt cells are rapidly reproducing portions of the duodenal epithelium.)
322
DVORAK
sites. Berntzen (1962) has demonstrated the growth and development of a cestode in vitro, a demonstration, of course, that all essential materials can be absorbed from a liquid medium, without any contact whatsoever between the worm and the tissues of the host. Our work provides evidence that in nature, in viva, that is, the same kind of absorption required by Berntzen’s experimental results can occur. REFERENCES AMOS, D. B., AND WAKEFIELD, J. D. 1958. Growth of mouse ascites tumor cells in diffusion chambers. I. Studies of growth rate of cells and of the rate of entry of antibody. Journal of the National Cancer Institute 21, 657-670. BERNTZEN, A. K. 1962. In vitro cultivation of tapeworms. II. Growth and maintenance of Hymenolepis nana (Cestoda: Cyclophyllidea) . Journal of Parasitology 48, 785-797. BOWEN, V. T. (Ed.). 1952. The chemistry and physiology of the nucleus (Symposium). Experimental Cell Research 3, (Suppl. 2). BOYD, G. A. 1955. “Autoradiography in Biology and Medicine.” Academic Press, New York. DAUGHERTY, J, W., AND TAYLOR, D. 1956. Regional distribution of glycogen in the rat cestode, Hymenolepis diminuta. Experimental Parasitology 5, 376-390. FITZGERALD, P. J., AND VINIJCHAIKUL, K. 1959.
AND
JONES
Nucleic acid metabolism of pancreatic cells as revealed by cytidine-Hs and thymidine-H”. Laboratory Investigation 8, 319-329. GOODCHILD, CHAUNCEY G., AND WELLS, 0. C. 1957. Amino acids in larval and adult tapeworms (Hymenolepis diminuta) and in the tissues of their rat and beetle hosts. Experimental Parasitology 6, 575-585. H~DRICK, R. M. 1958. Comparative histochemical studies on cestodes. II. The destribution of fat substances in Hymenolepis diminuta and Raillietina cesticillus. Journal of Parasitology 44, 75-84. HEYNEMAN, D., AND VOGE, M. 1957. Glycopen distribution in cysticercoids of three hymenolepidid cestodes. Journal of Parasitology 43, 527-531. KENT, H. N. 1957. Biochemical studies on the proteins of Hymenolepis diminuta. Experimental Parasitology 6, 351-357. PHIFER, K. 1960. Permeation membrane transport in animal parasites: Further observations on the uptake of glucose by Hymenolepis diminuta. 46, 137-144. Journal of Parasitology READ, C. P. 1950. The acquisition of isotopically labeled inorganic phosphate by the tapeworm Hymenolepis diminuta, with some remarks on the host-parasite relationship. Journal of Parasitology 36, 34-40. ROBERTS, L. S. 1961. The influence of population density on patterns and physiology of growth in Hymenolepis diminuta (Cestoda: Cyclophyllidea) in the definitive host. Experimental Parasitology 11, 332-371.