International Journal
for Parasitology.
1976. Vol. 6. pp. 271-278. Pergnmon Press. Printed in Great Britain.
ULTRASTRUCTURAL
OBSERVATIONS OF IN A LIQUID MEDIUM: THE KINETOPLAST-MITOCHONDRION IN DIVISION FORMS
TRYPANUSOMA
CRUZI
JOSE A.
Center of Microbiology
O’DALY and A. BRETARA
and Cell Biology, Instituto Venezolano de Investigaciones Caracas, Venezuela
Cientificas,
Apartado
1827,
(Received 3 September 1975) Abstract-@DALY J. A. and BRETARAA. 1976. Ultrastructuml observations of Tr~pu~oso~a cruzi in a liquid medium: the kinetoplast-mitochondrion in division forms. ~~~e~~uri~~~~Journal for Parasitology 6: 271-278. Morphogenesis of the kinetoplast-mitochondrion complex in T. cruzi epimastigotes obtained from a liquid culture medium is described. A crystal-like pattern of channels between mitochondria and kinetoplasts is described. A new structure at the flagellar end in epimastigotes is reported. INDEX KEY WORDS: kinetoplast-mitochondria
Trypanosoma cruzi; in vitro cultivation; new liquid medium; ultrastructure; relationship; crystal-like array; new flagellar structure.
INTRODUCTION THE VARIATIONSin size and shape of kinetoplasts in T. ctwzi (Brack, 1968) are thought to be due to different arrangements of the DNA-loops in one layer fepimastigote) or in 34 layers (trypomastigote) respectively. A basket-like structure in the kinetoplast of trypomastigotes (Meyer, 1968, 1969) has been described as characteristic for metacyclics. Inoki, Ozeki & Kambara (1971) reported the transition of kinetoplast DNA from compact coiled fibrils (epimastigote) to uncoiled fibrils (trypomastigote). On the other hand, Sanabria (1966) and Maria, Tafuri & Brener (1972) suggested a pro-
gressive mitochondrial development from intracellular amastigote to bloodstream trypomastigote to vector epimastigote. Vickerman (1974) has shown localized dissection of m~tochondria by cytoplasmic channels containing endoplasmic reticulum. T. cruzi has been cultured in a liquid medium @‘Daly, 197.5) in which trypanosomes present a cycle from epimastigotes (exponential phase) to trypomastigotes (stationary phase of growth) in each subculture. In this paper, attention will be focused on changes of the kinetoplast-mitochondrion complex in epimastigote division and growth. A new crystal-like structure related with mitochondria development is described. A particular formation at the tip of the epimastigote flagellum is reported. EATERIES
AND ~THODS
Trypanosuma crazi (strains Y, Ma and FL) were grown in a synthetic medium @‘Daly, 1975) containing 5%
(V/V) IPT-FCS (Immunoprecipitin tested fetal calf serum (GIBCO). Tissue culture disposable flasks (250 ml Falcon plastics, Oxnard, U.S.A.) containing 50 ml of culture medium with parasites at the exponential phase of growth were fixed in 25% glu~mldehyde by mixing equal volumes of medium and 5 % glutaraldehyde (V/V) at pH 72 and kept for 2 h at 4°C. The trypanosomes were pelleted by centrifuging at 950 g for 30 min at 4”C, washed 3 times in O-1 M-sodium phosphate buffer containing 0.18 M-sucrose at pH 7.2 and post-fixed overnight in lo/, (V/V) osmium tetroxide in 0.1 M-sodium uhosuhate buffer. ‘After serial dehydrations in graded alcohols, the material was embedded in Maraglas. Ultrathin sections were made by a Porter-Blum MT-2 Ultramicrotome, stained with lead citrate and many1 acetate, and examined with a JEOL 100-B electronmicroscope at 80 k.V. RESULTS Figure 1 shows a parasite after nuclear division its
kinetoplasts with tightly packed DNA fibrils, its mitochondria with abundant cristae and a clear matrix. (Figs. 2 & 3.) Figures 4-6 present trypanosomes starting division as indicated by the presence of two flagella. The kinetoplast DNA appears as a triple row of filaments (Fig. 4), but some sections show at the periphery of the kinetoplast 6 rows of filaments separated by a groove. This structure looks like a Y, the single and double branches of the Y having 3 rows of filaments each. (Figs. 5 & 6). A common point where filaments of the longer branch unite to filaments belonging to shorter branches is clearly seen. (Fig. 6.) The mitochondria and the DNA duplicate synchronously (Fig. 7). Once kinetoplast division is 271
Josh A. 0’Dlzi.u and
A. BRETA~A
l.J.1’. VOI h. 1976
FIG. 1. Longitudinal section of an epimastigote after nuclear(N) division and kinetoplast (I() migration. Electron-dense granules fg) of various sizes can be seen throughout the cytoplasm. The peripheral nucleoli. mitochondrial system (m) is already organized. n FIG. 2. Higher magnification of the upper kinetoplast from Fig. I. The DNA is tightly packed: b, basal body: k, kinetoplast. FIG. 3. Cross section of a kinetoplast (k) in resting epimastigote showing the DNA tibrils tightly packed.
I.J.P. VOL.
6. 1976
The kinetoplast-mitochondrion
in T. cruzi division forms
FIG. 4. Section showing an epimastigote with two flagella (f), polyribosomes and electron dense granules (g) in the cytoplasm. The kinetoplast DNA has changed its appearance into three parallel rows with abundant puffs at the periphery; b, basal body; k, kinetoplast; m, mitochondria. FIG. 5. Kinetoplast (k) cross section showing six parallel rows separated at the middle by a groove (arrow); b, basal body. FIG. 6. Kinetoplast (k) cross section showing DNA in the process of division. At the arrow the junction point between the newly formed branches and the old branch of the kinetoplast is clearly seen. b, basal body. Note the mit~hondrial enlargement around the small branches of the kinetoplast. FIG. 7. Kinetoplast (k) cross section showing the division process almost completed. The DNA rows are separated in most of the trajectory and two mitochondria surround each bundle of 3 parallel filaments.
273
Jo&
274
A.
O’DALY
finished a system of channels can be observed connetting the two kinetoplasts with the newly developing mitochondria (Fig. 8.). Numerous plate-like
FIG. 8. Cross section of a parasite after separation bound
channels
provide
and
A.
BRETARA
I.J.P.
VOL.
6.
1976
cristae are seen in the kinetoplast and mitochondrial matrix. Figure 9 shows a kinetoplast with many vesicles
of kinetoplasts (k). Numerous double membrane communication (arrows) between the kinetoplast and the mitochondrial (m) system in formation. f, flagellum: PC, Dlate-like cristae.
I.J.P.
VOL.
6. 1976
The kinetoplast-mitochondrion
in T. cruzi division forms
275
FIG. 9. Cross section of kinetoplast with abundant single membrane-bound vesicles (v) of clear matrix. The vesicles have cristae inside (c). Some of the vesicles communicate (double arrow) with the channel structure in formation (see Fig. 8). Electron dense material (Ed) non-membrane bound can be seen in
the kinetoplast matrix, in the periphery of the channel system and outside in the cytoplasm. Note the lack of continuity of the kinetoplast outer membrane (arrows) at the place of contact with the channel system. containing cristae and electron-dense matrix similar to the kinetoplast. All the vesicles together with the kinetoplast are surrounded by a membrane. Some of the vesicles are continuous with the channel system. Electron-dense granules, non-membrane bound can be distinguished in the kinetoplast matrix, around the channel system and parasite cytoplasm. The channel system can be observed as a symmetrical array around a vesicle, each channel formed by a double membrane (Fig. 10). In sections with few peripheral mitochondria (Fig. ll), the outer channeis of the structure appear as tubular formations in continuity with the peripheral mitochondria. As the kinetoplasts migrate from each other the channel system disappears progressively (Fig. 12), and only few double membranes around electrontransparent spaces are observed as the parasite ends division. At this stage the abundant electron-dense material is noteworthy surrounding the periphery of the channels (Fig. 13). In adult epimastigotes as well as in trypomastigotes the system of channels has not been observed. At the exponential phase of growth in the liquid medium (Figs. 14 & 15) numerous colonies of
epimastigotes, with radially arranged parasite bodies can be observed (O’Daly, 1975) the flagella being intermingled at the center (Fig. 15). Some epimastigotes present a cytoplasmic expansion at the flagellar tip (Figs. 16 & 17). This structure- is formed by an electron-dense material containing a vesicle or expansion at right angles to the flagellar axis. Around the flagellar end a coiled structure of IO-12 turns is clearly seen (Fig. 17). DISCUSSION In this paper, we have correlated changes in kinetoplast morphology with the parasite division process in epimastigote forms when cultured in a liquid medium (O’Daly, 1975). The figures suggest that kinetoplasts are responsible for the mitochondria formation in the parasites (Brack, 1968; Vickerman, 1974). This process is accomplished probably by the system of channels here described, structures that disappear once the parasite division is completed. The system of channels has continuity with kinetoplasts and with the newly formed mitochondria, but not with the endoplasmic reticulum. Mit~hondria-like cristae can be seen in the system
Josh A. O’DALY and A.
BR~TA~A
I.J.Y. VOL. 6.
FIG. 10. Cross section of channel system showing a crystal-like array. All thedouble r~lembran~ bound channels have the same diameter. Near the center a vesicle (v) of clear matrix can be observed. FIG. I I. Cross section of channel system beginning to be disorganized, with clear spaces in between the channels. The peripheral channels (arrow) start to fuse to each other. Communication with the mitocholldriai (m) system is apparent (double arrow). FIG. 12. Cross section showing two kinetoplasts (k) separated from each other with DNA tightly packed. The system of channels shows clear spaces containing cytoplasm with polyribosomes. Electron dense granules (g) can be observed in the cytoplasm. N, nucleus; b, basal body; f, flagellum. Frc. 13. Cross section of a parasite showing the final stage of the channel system with abundant electron dense material (arrows) at their surface. Vesicles of various sizes (v) and electron dense granules (g) with dense material inside can be seen in the cytoplasm. N, nucleus: n, nucleolus; m, mitochondria.
1976
I.J.P. VOL.6. 1976
The kinetoplast-mitochondrion
in T.
crud
division forms
FIG. 14. Low power view of epimastigotes showing abundant electron dense granules (g) in their cytoplasm. Membrane structures (mb) can be observed around the parasites, free in the medium. These membranes increase in quantity as the stationary phase of growth is approached. FIG. 15. Colony of epimastigotes showing the parasites organized radially, with the flagella intermingled at the center. Some of the flagella have a coiled structure around its axis (arrows). FIG 16. Cross section of epimastigote with the coil structure at the tip of the flagellum (arrows). FIG 17. At high magnification the coil structure can be observed at the epimastigote flagellar end. An expansion(e) can be seen at right angles to the flagellar axis. This expansion shows band-like structures inside. From the tip of the flagellum upwards, a coil (arrows), like a cord, turns around the flagellum.
278
Josi A.
O’DALY
of channels, suggesting for them a role in respiratory phenomena. Numerous electron-dense granules, apparently related to this structure can be seen in the parasite cytoplasm, but its significance has yet to be ascertained. Under the Iight inverted phase microscope it is possible to detect sometimes an epimastigote stuck to the bottom of the plastic culture flask by means of an expansion at the tip of the flagellum, which might well correspond to the structure described in Figs. 16 & 17. These epimastigotes could use this structure for adhesion
to cell surfaces.
~ckn~w~edge~en~s-The authors wish to thank Miss Mina Marin for secretarial help with the manuscript. This investigation was supported in part by a research grant from the Consejo National de investigaciones Gent&as y Tecnologicas (CONICIT).
REFERENCES UnterBRACK C. 1968. Electronenmikroskopische suchungen zum Lebenszyklus von Trypanosoma cruzi. Acta Tropica 25: 289-356.
and A. BRETA&A
I.J.P. vol.. 6. 1976
iNOKlS., OZEKI Y. & KAM~AR~H. 1971. Ultra-structural changes in the kinetoplast of Trypunusoma crazi during transition of form in vitro. &ken Journal 14: 37750. MARIA T. A., TAFUR~W. & BRENERZ. 1972. The fine structure of different bloodstream forms of Tr~lpanosoma cntzi. Annals of Tropical sifology 66: 423-431.
Medicine
and Para-
MEYERH. 1968. The tine structure of the flagellum and kinetoplast-chondriome of Trypanosoma (Schizotrypanum) cruzi in tissue culture. Journal of Profozoology 15 (3): 614-621, MEYERH. 1969. Further studies on the fine structure of the kinetoplast-chondriome of Trypa~usoma (Schizotrypanum) cruzi in thin sections of infected tissue cultures. Revbfa Do tnsf~tata de Medic&a Tropical De Scia Pa&o II (1): 48-56.
O-DALY J. A. 1975. A new liquid medium for T~~~Q!xP soma (Schizotrypanum) cruzi. Journal of Protozoolagy 22: 265-270.
SANABRIAA. 1966. Ultrastructure of Trypa~asama crazi in the rectus of Rhodnius prolixus. Experimental Parasitology 19: 276-299. VICKERMANK. 1914. Trvpanosomiasis and Leishmaniasis wirh Special Reference‘k
Chagas’ Disease, pp. 225-254.
Ciba Foundation Symposium 20 (new series). Elsevier Excerpta Medica Foundation, Amsterdam.
for Parasitology.
1976. Vol. 6. pp. 271-278. Pergnmon Press. Printed in Great Britain.
ULTRASTRUCTURAL
OBSERVATIONS OF IN A LIQUID MEDIUM: THE KINETOPLAST-MITOCHONDRION IN DIVISION FORMS
TRYPANUSOMA
CRUZI
JOSE A.
Center of Microbiology
O’DALY and A. BRETARA
and Cell Biology, Instituto Venezolano de Investigaciones Caracas, Venezuela
Cientificas,
Apartado
1827,
(Received 3 September 1975) Abstract-@DALY J. A. and BRETARAA. 1976. Ultrastructuml observations of Tr~pu~oso~a cruzi in a liquid medium: the kinetoplast-mitochondrion in division forms. ~~~e~~uri~~~~Journal for Parasitology 6: 271-278. Morphogenesis of the kinetoplast-mitochondrion complex in T. cruzi epimastigotes obtained from a liquid culture medium is described. A crystal-like pattern of channels between mitochondria and kinetoplasts is described. A new structure at the flagellar end in epimastigotes is reported. INDEX KEY WORDS: kinetoplast-mitochondria
Trypanosoma cruzi; in vitro cultivation; new liquid medium; ultrastructure; relationship; crystal-like array; new flagellar structure.
INTRODUCTION THE VARIATIONSin size and shape of kinetoplasts in T. ctwzi (Brack, 1968) are thought to be due to different arrangements of the DNA-loops in one layer fepimastigote) or in 34 layers (trypomastigote) respectively. A basket-like structure in the kinetoplast of trypomastigotes (Meyer, 1968, 1969) has been described as characteristic for metacyclics. Inoki, Ozeki & Kambara (1971) reported the transition of kinetoplast DNA from compact coiled fibrils (epimastigote) to uncoiled fibrils (trypomastigote). On the other hand, Sanabria (1966) and Maria, Tafuri & Brener (1972) suggested a pro-
gressive mitochondrial development from intracellular amastigote to bloodstream trypomastigote to vector epimastigote. Vickerman (1974) has shown localized dissection of m~tochondria by cytoplasmic channels containing endoplasmic reticulum. T. cruzi has been cultured in a liquid medium @‘Daly, 197.5) in which trypanosomes present a cycle from epimastigotes (exponential phase) to trypomastigotes (stationary phase of growth) in each subculture. In this paper, attention will be focused on changes of the kinetoplast-mitochondrion complex in epimastigote division and growth. A new crystal-like structure related with mitochondria development is described. A particular formation at the tip of the epimastigote flagellum is reported. EATERIES
AND ~THODS
Trypanosuma crazi (strains Y, Ma and FL) were grown in a synthetic medium @‘Daly, 1975) containing 5%
(V/V) IPT-FCS (Immunoprecipitin tested fetal calf serum (GIBCO). Tissue culture disposable flasks (250 ml Falcon plastics, Oxnard, U.S.A.) containing 50 ml of culture medium with parasites at the exponential phase of growth were fixed in 25% glu~mldehyde by mixing equal volumes of medium and 5 % glutaraldehyde (V/V) at pH 72 and kept for 2 h at 4°C. The trypanosomes were pelleted by centrifuging at 950 g for 30 min at 4”C, washed 3 times in O-1 M-sodium phosphate buffer containing 0.18 M-sucrose at pH 7.2 and post-fixed overnight in lo/, (V/V) osmium tetroxide in 0.1 M-sodium uhosuhate buffer. ‘After serial dehydrations in graded alcohols, the material was embedded in Maraglas. Ultrathin sections were made by a Porter-Blum MT-2 Ultramicrotome, stained with lead citrate and many1 acetate, and examined with a JEOL 100-B electronmicroscope at 80 k.V. RESULTS Figure 1 shows a parasite after nuclear division its
kinetoplasts with tightly packed DNA fibrils, its mitochondria with abundant cristae and a clear matrix. (Figs. 2 & 3.) Figures 4-6 present trypanosomes starting division as indicated by the presence of two flagella. The kinetoplast DNA appears as a triple row of filaments (Fig. 4), but some sections show at the periphery of the kinetoplast 6 rows of filaments separated by a groove. This structure looks like a Y, the single and double branches of the Y having 3 rows of filaments each. (Figs. 5 & 6). A common point where filaments of the longer branch unite to filaments belonging to shorter branches is clearly seen. (Fig. 6.) The mitochondria and the DNA duplicate synchronously (Fig. 7). Once kinetoplast division is 271
Josh A. 0’Dlzi.u and
A. BRETA~A
l.J.1’. VOI h. 1976
FIG. 1. Longitudinal section of an epimastigote after nuclear(N) division and kinetoplast (I() migration. Electron-dense granules fg) of various sizes can be seen throughout the cytoplasm. The peripheral nucleoli. mitochondrial system (m) is already organized. n FIG. 2. Higher magnification of the upper kinetoplast from Fig. I. The DNA is tightly packed: b, basal body: k, kinetoplast. FIG. 3. Cross section of a kinetoplast (k) in resting epimastigote showing the DNA tibrils tightly packed.
I.J.P. VOL.
6. 1976
The kinetoplast-mitochondrion
in T. cruzi division forms
FIG. 4. Section showing an epimastigote with two flagella (f), polyribosomes and electron dense granules (g) in the cytoplasm. The kinetoplast DNA has changed its appearance into three parallel rows with abundant puffs at the periphery; b, basal body; k, kinetoplast; m, mitochondria. FIG. 5. Kinetoplast (k) cross section showing six parallel rows separated at the middle by a groove (arrow); b, basal body. FIG. 6. Kinetoplast (k) cross section showing DNA in the process of division. At the arrow the junction point between the newly formed branches and the old branch of the kinetoplast is clearly seen. b, basal body. Note the mit~hondrial enlargement around the small branches of the kinetoplast. FIG. 7. Kinetoplast (k) cross section showing the division process almost completed. The DNA rows are separated in most of the trajectory and two mitochondria surround each bundle of 3 parallel filaments.
273
Jo&
274
A.
O’DALY
finished a system of channels can be observed connetting the two kinetoplasts with the newly developing mitochondria (Fig. 8.). Numerous plate-like
FIG. 8. Cross section of a parasite after separation bound
channels
provide
and
A.
BRETARA
I.J.P.
VOL.
6.
1976
cristae are seen in the kinetoplast and mitochondrial matrix. Figure 9 shows a kinetoplast with many vesicles
of kinetoplasts (k). Numerous double membrane communication (arrows) between the kinetoplast and the mitochondrial (m) system in formation. f, flagellum: PC, Dlate-like cristae.
I.J.P.
VOL.
6. 1976
The kinetoplast-mitochondrion
in T. cruzi division forms
275
FIG. 9. Cross section of kinetoplast with abundant single membrane-bound vesicles (v) of clear matrix. The vesicles have cristae inside (c). Some of the vesicles communicate (double arrow) with the channel structure in formation (see Fig. 8). Electron dense material (Ed) non-membrane bound can be seen in
the kinetoplast matrix, in the periphery of the channel system and outside in the cytoplasm. Note the lack of continuity of the kinetoplast outer membrane (arrows) at the place of contact with the channel system. containing cristae and electron-dense matrix similar to the kinetoplast. All the vesicles together with the kinetoplast are surrounded by a membrane. Some of the vesicles are continuous with the channel system. Electron-dense granules, non-membrane bound can be distinguished in the kinetoplast matrix, around the channel system and parasite cytoplasm. The channel system can be observed as a symmetrical array around a vesicle, each channel formed by a double membrane (Fig. 10). In sections with few peripheral mitochondria (Fig. ll), the outer channeis of the structure appear as tubular formations in continuity with the peripheral mitochondria. As the kinetoplasts migrate from each other the channel system disappears progressively (Fig. 12), and only few double membranes around electrontransparent spaces are observed as the parasite ends division. At this stage the abundant electron-dense material is noteworthy surrounding the periphery of the channels (Fig. 13). In adult epimastigotes as well as in trypomastigotes the system of channels has not been observed. At the exponential phase of growth in the liquid medium (Figs. 14 & 15) numerous colonies of
epimastigotes, with radially arranged parasite bodies can be observed (O’Daly, 1975) the flagella being intermingled at the center (Fig. 15). Some epimastigotes present a cytoplasmic expansion at the flagellar tip (Figs. 16 & 17). This structure- is formed by an electron-dense material containing a vesicle or expansion at right angles to the flagellar axis. Around the flagellar end a coiled structure of IO-12 turns is clearly seen (Fig. 17). DISCUSSION In this paper, we have correlated changes in kinetoplast morphology with the parasite division process in epimastigote forms when cultured in a liquid medium (O’Daly, 1975). The figures suggest that kinetoplasts are responsible for the mitochondria formation in the parasites (Brack, 1968; Vickerman, 1974). This process is accomplished probably by the system of channels here described, structures that disappear once the parasite division is completed. The system of channels has continuity with kinetoplasts and with the newly formed mitochondria, but not with the endoplasmic reticulum. Mit~hondria-like cristae can be seen in the system
Josh A. O’DALY and A.
BR~TA~A
I.J.Y. VOL. 6.
FIG. 10. Cross section of channel system showing a crystal-like array. All thedouble r~lembran~ bound channels have the same diameter. Near the center a vesicle (v) of clear matrix can be observed. FIG. I I. Cross section of channel system beginning to be disorganized, with clear spaces in between the channels. The peripheral channels (arrow) start to fuse to each other. Communication with the mitocholldriai (m) system is apparent (double arrow). FIG. 12. Cross section showing two kinetoplasts (k) separated from each other with DNA tightly packed. The system of channels shows clear spaces containing cytoplasm with polyribosomes. Electron dense granules (g) can be observed in the cytoplasm. N, nucleus; b, basal body; f, flagellum. Frc. 13. Cross section of a parasite showing the final stage of the channel system with abundant electron dense material (arrows) at their surface. Vesicles of various sizes (v) and electron dense granules (g) with dense material inside can be seen in the cytoplasm. N, nucleus: n, nucleolus; m, mitochondria.
1976
I.J.P. VOL.6. 1976
The kinetoplast-mitochondrion
in T.
crud
division forms
FIG. 14. Low power view of epimastigotes showing abundant electron dense granules (g) in their cytoplasm. Membrane structures (mb) can be observed around the parasites, free in the medium. These membranes increase in quantity as the stationary phase of growth is approached. FIG. 15. Colony of epimastigotes showing the parasites organized radially, with the flagella intermingled at the center. Some of the flagella have a coiled structure around its axis (arrows). FIG 16. Cross section of epimastigote with the coil structure at the tip of the flagellum (arrows). FIG 17. At high magnification the coil structure can be observed at the epimastigote flagellar end. An expansion(e) can be seen at right angles to the flagellar axis. This expansion shows band-like structures inside. From the tip of the flagellum upwards, a coil (arrows), like a cord, turns around the flagellum.
278
Josi A.
O’DALY
of channels, suggesting for them a role in respiratory phenomena. Numerous electron-dense granules, apparently related to this structure can be seen in the parasite cytoplasm, but its significance has yet to be ascertained. Under the Iight inverted phase microscope it is possible to detect sometimes an epimastigote stuck to the bottom of the plastic culture flask by means of an expansion at the tip of the flagellum, which might well correspond to the structure described in Figs. 16 & 17. These epimastigotes could use this structure for adhesion
to cell surfaces.
~ckn~w~edge~en~s-The authors wish to thank Miss Mina Marin for secretarial help with the manuscript. This investigation was supported in part by a research grant from the Consejo National de investigaciones Gent&as y Tecnologicas (CONICIT).
REFERENCES UnterBRACK C. 1968. Electronenmikroskopische suchungen zum Lebenszyklus von Trypanosoma cruzi. Acta Tropica 25: 289-356.
and A. BRETA&A
I.J.P. vol.. 6. 1976
iNOKlS., OZEKI Y. & KAM~AR~H. 1971. Ultra-structural changes in the kinetoplast of Trypunusoma crazi during transition of form in vitro. &ken Journal 14: 37750. MARIA T. A., TAFUR~W. & BRENERZ. 1972. The fine structure of different bloodstream forms of Tr~lpanosoma cntzi. Annals of Tropical sifology 66: 423-431.
Medicine
and Para-
MEYERH. 1968. The tine structure of the flagellum and kinetoplast-chondriome of Trypanosoma (Schizotrypanum) cruzi in tissue culture. Journal of Profozoology 15 (3): 614-621, MEYERH. 1969. Further studies on the fine structure of the kinetoplast-chondriome of Trypa~usoma (Schizotrypanum) cruzi in thin sections of infected tissue cultures. Revbfa Do tnsf~tata de Medic&a Tropical De Scia Pa&o II (1): 48-56.
O-DALY J. A. 1975. A new liquid medium for T~~~Q!xP soma (Schizotrypanum) cruzi. Journal of Protozoolagy 22: 265-270.
SANABRIAA. 1966. Ultrastructure of Trypa~asama crazi in the rectus of Rhodnius prolixus. Experimental Parasitology 19: 276-299. VICKERMANK. 1914. Trvpanosomiasis and Leishmaniasis wirh Special Reference‘k
Chagas’ Disease, pp. 225-254.
Ciba Foundation Symposium 20 (new series). Elsevier Excerpta Medica Foundation, Amsterdam.
