THE ULTRASTRUCTURE OF Tritrichomonas 7 1

REFEREXCES

1. Dunn. F. W,. Ravel. 1. M., 8: Shive, W. 1956. Inhibition studies with peptides of thienylalanine and phenylalanine. J . Biol. Ch~iiz. 219, 809-22.

2. Eichel, H. T., 8: Roth, J. S. 1953. The effect of X-radia- tion on nuclease- activity and respiration of Trtuahyincna g e l &

3 . Kaufniau. S. 1959. Studies on the mechanism of the en- zymatic conversion of phenylalanine to tyrosine. 1. Biol. Cheiii. 234, 2677-82.

4. Kidder, G. W., 8: Dewey, V. C. 1949. Studies on the bio- chemistry of Trtrahyi~iena. XI. Components of factor I1 of known chemical nature. Arch. Biochein. 20, 433-43.

5 . Kihara, H., 8: Snell, E. E. 1955. Peptides and bacterial :ro\vth. VII. Relation to inhibitions by thienylalanine, ethio- nine and canavanine. J . Biol. Chem. 212, 8.1-94.

6. Roka, L., Konig, G., 8: Rubner. H. 1958. Diefermenta-

IT. Rlol. B d . 10.1, 351-8.

tive oxydation von p-hydroxyphenylbrenztraubensaure zu ho- mogentisinsaure. Zeit. physiolog. Chem. 313, 87-96.

7. Roth, J. S., & Eichel, H. J . 1955. The effect of S-radia- tion on enzyme systems of Tetrahvwzcna pyrifornzis. Riol. Bull. 108, 308-17.

8 . Roth. J. S., Eichel, H. J., 8: Ginter, E. 1 9 3 . The osida- tion of amino acids by Tetrahymena p.wifor.irtis W. d r r h . Biorhriii. and Biophys. 48, 112-9.

9. Tokuyama, K. 1959. Studies on homopentisicasc. 111. Kinetic studies on the enzyme action. J . of Biorheni. 16, 1.7.59- 68.

10. Viswanatha, T., 8: Liener, I. E. 1956. Isolation and properties of a proteinase from Tetrehymena pyr.i,forinis IV. I r r h Biocheni. and Biophys. 61, 410-21.

11. Zannoni, V. G., 8: La Du, B. N. 1959. The tyrosine oxi- dation system of liver. IV, Studies on the inhibition of p- h~droxyphenylpyruvic acid oxidase by excess substrate. J . Biol. Cheiii. 234, 2925-31.

J . PKOTOZOOL. 8(1), 71-75 (1961)

The Ultrastructure of Tritrichomo nas with Special Reference to the Blepharoplast Complex*

EVERETT ANDERSON and H. W. BEAMS

Depavtinent of Zoology, State Unirwvsity of Iowa, Iowa Ci ty , Iowa

SYTOPSIS. Electron micrographs of sections through the is more slender than the costa. I t is attached to the base of blepharoplast complex reveal it to be composed of four kineto- the kinetosome of an anteriorly directed flagellum and subse- somes. In many of the sections the kinetosomes contain densc quently proceeds posteriorly to the leveI of the nucIeus. The particles approsimately 180-200 H in diameter. The costa is costa and parabasal fiber probably serve as anchoring or sup- a striated fiber which extends posteriorly almost the entire portive structures. The size of the costa may reflect its role lcnpth of the body. I ts slender proximal portion appears to as a supporting structure for the most highly differentiated be attached to the base of the kinetosome of the recurrent locomotor organelle, the undulating membrane. ilaaellum. The parabasal fiber is similarly constructed, but

S a previous paper ( I ) the ultramicroscopic struc- I[ ture of Tiitrichomonas muris was described. Elec- tron micrographs demonstrated that the blepharoplast complex is composed of four kinetosomes and that the costa and parabasal fiber are associated with this complex. The question of the relationship of the costa and parabasal fiber to the kinetosomes comprising the blepharoplast complex was left unanswered; the pres- ent report attempts to answer this question.

For details of the methods used the reader is re- ferred to Anderson and Beams( 1) .

OBSERVATIONS AND DISCUSSION

The morphological details of T . muris obtained with the light microscope need not concern us here since they have been extensively studied by others( 9,lO. 20). It is only necessary to state, for the sake of clarity, that the blepharoplast complex after treat- ment with Heidenhain's hematoxylin appears as a densely stained body located at the anterior end.

'kSupported by grants (RG-4706 and 5479) from the Na- tional Institutes of Health, United States Public Health Service and the Xational Science Foundation (G 9879).

Electron micrographs of sections through the bleph- aroplast complex at different angles are illustrated in Figs. 1-7. In most of these sections the fine structure of kinetosomes (AK and R K ) is similar to that pre- viously described for this organism, other flagellates and ciliates (see references in 1). Also shown within the kinetosomes are dense particles (180-200 -1 i n di- ameter) (Figs. 1, 2 and 6, DG) first described in the flagellate, T . m u i s , by Anderson and Beams( 1 ) and in the ciliate, Stentor polymorphus, by Randall and Jackson( 1 5 ) . The latter authors stated that these particles were particularly noticeable in conjuqating and dividing organism. In the same publication they also presented some preliminary data which suggested that the kinetosomes of Stentor contain a Feulgen positive material indicating the presence of deosyribo- nucleoprotein. This work has now been extended by Randall( 16) to include the kinetosomes of the larvae of Cionn intestinalis and those in the gills of larvae of ..lnzbystonza opacum. Might it not be that the parti- cles seen in the kinetosomes represent sections of highly polymerized deosyribonucleoprotein like that found in maturing sperm(3)? It is interesting to

72 THE ULTRASTRUCTURE OF Tritrichomonas

point out that Samuels(l7) in his study of abnormal mitosis and morphogenesis in T. batrachorum stated that (‘. , . the blepharoplast is brlieved to be a group of kinetosomes, each acting as a cytogene for a specific organelle.”

The costa is a striated fiber (Figs. 3-6, C) which . extends posteriorly almost the entire length of the body. I t is broadest near the middle, tapering to a

slender distal portion and to a less slender proximal end. In Fig. 3 the proximal portion of the costa is seen closely associated with a kinetosome (AK) of the complex. Figs. 4 and 5 show a longitudinal section of a kinetosome (AR) from an anteriorly directed flagellum in addition to a cross section of the kineto- some of the recurrent flagellum (RE;); the latter bears a close association with the costa (C). In Fig.

Figs. 1-5. Tangential sections of the blepharoplast complex showing kinetosomes of anterior flagella (AK), recurrent flagel- lum (RE;) and dense granules within kinetosomes (Figs. 1, 2

and 4, DG). The costa (Figs. 3-5, C ) appears to be attached to the kinetosome of the recurrent flagellum (Fig. 4, RK) by short fibrils (SF) . Figs. 1-4, X 50,000; Fig. 5 , X 30,000.

Fig. 6. Micrograph showing tangential section of kinetosomes axostyle (AX) are also illustrated. X 60,000. Fig. 7. Section showing kinetosomes of anteriorly directed flagella (AK), one of which has attached to its base the striated parabasal fiber (PF). Note Golgi complex (GC), axostyle (Ax), and nucleus t N ) . X 50,000.

of the three anteriorly directed flagella (AK) , a longitudinal section of the kinetosome (RK) of the recurrent flagellum with dense particles (DG) and attached costa ( C ) . The undulating membrane (UM), a cross section of component filaments (FR) +f the recurrent flagellum, nucleus ( N ) and a portion of thc

74 THE ULTRASTRUCTURE OF Tritrichomonas

4 the costa appears to be intimately associated with the kinetosome (RK) by short fibrils (SF) . Of spe- cial interest is Fig. 6 which illustrates, in tangential section, the entire blepharoplast complex showing ki- netosomes of the three anteriorly directed flagella (AK) and a longitudinal section of the kinetosome (RK) of the recurrent flagellum. The fibrils of the recurrent flagellum ( F R ) comprise a portion of the outer margin of the undulating membrane (UM) . At the base of the kinetosome of the recurrent flagellum (RE;) is seen the attached costa (C) . Also illustrated in Figs. 6 and 7 are portions of the axostyle (AX) (1) .

The parabasal fiber (Fig. 7, P F ) is also a striated structure, but is more slender than the costa. It is attached to the base of a kinetosome of an anteriorly directed flagellum (AK) and subsequently proceeds posteriorly to the level of the nucleus ( N ) . Some dis- tance away from the point of attachment of the para- basal fiber. and parallel to it, is the agranular double membraned system that constitutes the Golgi complex (GC) . Anderson and Beams( 1) have pointed out the reniar1;able similarity between the parabasal body of T . nzrais and the Golgi complex of other organisms. Its position within the organism suggests that it may be similar to the localized type of Golgi material seen a t one side of the nucleus in many types of metazoan cells ( 2 1 ) . The apparent intimate anatomical relation- ship which exists between the Golgi complex of T . mrris and the striated parabasal fiber is thought to be coincidental rather than a dynamic one ( 7 ) .

Since most of the locomotor organelles of tritricho- monads are associated with the blepharoplast com- plex, the cytology of this region has been of interest to students of this group for many years (see Kirby, 9 ) . Because of its small size, and the limited resolving power of the light microscope, it is not surprising that the organization and relationship of components com- prising this region could not be determined. Light microscopists suggested that the blepharoplast complex served as an attachment point for the three anterior flagella. recurrent flagellum, accessory filament, undu- lating membrane, axostyle, costa, parabasal filament and parabasal body(9.10,19,20). Evidence presented here and previously( 1) suggests that one might view the blepharoplast complex as a unit consisting of four closely associated kinetosomes, two of which have at- tached “rootlets,” i.e., costa and parabasal fiber. Light microscope preparations of T . horninis made by Wenrich (20) apparently bear out this relationship. In this study LVenrich presented line drawings of ele- ments of the mastigont, the surrounding cytoplasm of which had been destroyed presumably during prepara- tion. I n his Figs. 4 and 5 the blepharoplast complex and associated organelles are shown. Although the

costa is not labeled in his drawing, its presence is obvious.

I n publications dealing with fine structure of loco- motor organelles of certain ciliates a kinetodesmal sys- tem, composed of striated fibers which arise from the bases of kinetosomes, has been demonstrated( 12.13. 18). Metz, Pitelka and Westfall( 1 2 ) have suggested that kinetodesma may serve in the coordination of ciliary activity. Not only have striated fiber systems been found associated with kinetosomes of protozoa. but they have also been demonstrated in a variety of metazoan ciliated epithelial tissues(2.5,22). Further- more, a striated rootlet has been demonstrated in cer- tain highly specialized organs, eg . , the “ear” of an insect, Locusta migmtoria migratoriodes( 6 ) . From an evolutionary point of view the previously mentioned morphological relationships would appear to be signi- ficant, since they bear on the problem of whether these structures have arisen independently in different groups of organisms (8) .

It is of particular interest to point out that the costa and parabasal fiber share two common characteristics with the kinetodesma of Paranzeciuwz( 18) and Tetra- hyrneizu( 13) viz., locus of attachment and periodic structure. On the other hand. when the kinetodesma of Stentor( 15) are compared with the costa and para- basal fiber they are found to possess only one of the aforementioned characteristics, namely, point of at- tachment. As previously mentioned, the kinetodesmal system has been implicated as serving in the coordina- tion of ciliary activity; a concept of great importance. I n this connection Pitelka and Schooley’s( 14) state- ment that, “. . . this function has not been definitely proved and may quite conceivably reside elsewhere in the cell cortex,” seems worthy of consideration. Fur- thermore, i t is desirable to call attention to the fact that the well known kinetodesmal system was origi- nally described in apostomous ciliates( 11) and to our knowledge no papers dealing with the fine structure of this group have appeared. Therefore, it would seem reasonable to seek a definition of the kinetodesma with respect to fine structure in this group of ciliates Further studies may reveal invaluable morphological clues as to whether the kinetodesma in the classical sense are homologous to components so identified re- cently by electron microscopists (4,i 2.13 , I 5.18).

In any case, i t is difficult to understand how the cross-striated costa and parabasal fiber of the flagella of T. muris could play an active role in flagellar ac- tivity, since all flagella are moving continuously and such a structural system is not associated with every kinetosome of the complex. There is no evidence at present for either a coordinative or a contractile func- tion for the costa and parabasal fiber of T . muris. One might speculate that the striated fibers observed

SILVERLINE AND FIBRILLAR SYSTEMS 75

in this organism serve as anchoring or supportive structures. The size of the costa may reflect its role as a reinforcing structure for the most highly differen- tiated locomotor organelle, i.e., the undulating mem- brane.

1. Anderson, E., 8 Beams, H. W. 1959. The cytology of Tritric-howinitas as revealed by the electron microscope. J . Morphol. 104, 2@i-36.

2 . Bradfield, J . R . G. 1955. Fiber patterns in animal flagella and cilia. S ~ J J J ~ . Soc. Erp t l . Biol. 9, 306-34.

3 . Dass, C. M . S. , 8 Kis, H. 1958. Submicroscopic organi- zation of the nucleus during spermiogenesis in the grasshopper. J . Rio9hys. Hiorhem. Cgtol . 4, 129-32.

4. Ehcrt, C. F., & Powers, E. L. 1959. The cell surface of Paraineriirin. Intt‘rnatl. Rrv. C y f o l . 8, 97-134.

5 . Fawcett, D. W. 1958. Structural specialization of the cell surface. In S. L. Palay, ed., Frontiers i)z Cytology, Yale Univ. Press, New Haven, Conn., 19-41.

6. Gray, E. G., & Pumphery, R. J . 1958. Ultra-structure of the insect ear. Natirre 181, 618.

7. Grimstone, A. 1’. 1959. Cytoplasmic membranes and the nuclear memhrane in the flagellate Trichonynipha. J . Biopiiys. i9iochrni. Cv to l . 6 , 369-78.

8. __ 1959 Cytology, homology and phylogeny-.% note on “organic design.” A ~ i i . Naturalist 93 , 273-81.

9. Kirby, H . 1944. Some observations on cytology and morphogenesis in flagellates. J . Morphol . 75, 361-421.

10. Kirby, H., 8 Honigberg, B. M. 1949. Flagellates of the caecum of ground squirrels. Univ . Calif. Pub. 2001. 53, 315-66.

11. Lwoff, A. 1950. Probleiits of Morphoganesis of Ciliates, John Wile?: and Sons, New York.

1 2 . Metz, C. B., Pitelka, D. R., & Westfall, J. .\. 1953. The fibrillar systems of ciliates as revealed by the electron micro- scope. I. ~ a r f l ~ 7 ~ e ~ i z i ? n . B i d . B d 1 . 104, 408-25.

13. Metz, C. B., 8 Westfall, J. A. 19.54. The fibrillar sys- tems of ciliates as revealed by the electron microscope. 11. Tr t rahymena . Biol. Birll. 107, 1015-?2.

14. Pitelka, D. R., 8 Schooley, C. N. 1958. The fine struc- ture of the flagellar apparatus in Ti irhonyt t tpha . J . J fo rpko l . 102, 199-246.

15. Randall, J. T., 8 Jackson, S. F. 1958.‘ Fine structure and function in Stentor po1yiIinrphiis. J . Biophys. Hiocherri. Cylol. 4, SO7-30.

16. Randall, J. T. 1959. The nature and significance o l kinetosomes. J . Protosool. (Suppl.) 6 , 30.

17. Samuels, R. 1959. Studies of Trifrichoirrontzs hncira- choriini. 3 . Abnormal mitosis and morphogenesis. Trari\. .-I J J J . Microsfop. Soc. 78. 49-65.

IS. Sedar, .4. W., 8 Porter, K. R . 1955. The fine structure of corlical components of Parawzeciiriii i i t ~ i l t i i ~ ~ i c r o ~ ~ ~ r ~ l e ~ ~ t ~ i ~ i ~ . . I . BioQhys. Biockeiii. Cytol . 1, S83-604.

19. Wenrich, D. H . 1921. The structure and division oi 7 r i , . h - O I I Z O J Z ~ S nizwis Hartman. J. Morphol . 36, 119--*

20. -- 1944. Morphology of the intestinal trichumonad flap4lates in mail and of similar forms in monkeys. cats. dogs and rats. J . Morphol . 71, 189-211.

21. Wilson, E. B. 1947. T h e C f l l i ~ i D P W ~ O ~ J J I I ’ J ~ ~ a n d HI,- rrdi ty . The Macmillan Co., New York.

2 2 . Wislocki, G., & Ladman, A. J . 19.58. The fine structure of the mammalian choroid plexus. In , Ciba Fozindation .YJin- posiutrf on the Cerebrospinal Fluid 5 5 - 7 5 .

J . PROTOZOOL. 8(1 ) , 7 5 4 9 (1961).

Fine Structure of the Silverline and Fibrillar Systems of Three Tetrahymenid Ciliates7k

DOROTHY R. PITELKA Depavfnterzf of Zoology and its C a m u Rerearch Genrtics Laboratorv,

Univevsi fy of California, Bprkelry

SYSOPSIS. Application of fragmentation and thin-sectioning trchniques to TrtrahVnrfrzu pqrifoimi.r, Co~pidiiiiiz cai?tpylilnz and Glaucoma f h o f t o n i has permitted an analysis of the ultra- structure of their silverline and fibrillar systems. The classical silverline system consists of a mosaic of flat, membrane-bound blisters whose rims represent the sites of selective silver deposi- tion. Cilia and protrichocysts emerge between adjacent blisters. The pellicle consists of the membranes outlining the blisters, overlain by a continuous outer membrane that covers the whole cell and cilia. Fibrillar structures, which are not argentophilic, include: (1) tapering, striated kinetodesmal fibers arising singly from the kinetosomcs, passing to the right and anteriad, and overlapping to form a loose bundle accompanying each

4EATI;RE of the organization of ciliate proto-

troversy than their so-called fibrillar systems. During most of the first half of this century, detailed and

No zoa has given rise to more speculation and con-

kinety ; ( 2 ) a longitudinal fibril band immediately beneath the peI!icle at the right of each kinety, consisting of overlapping individual fibrils; (3) a transverse band of fibrils arising at the left side of each kinetosome and passing to the left under the pellicle; and (4) a set of postciliary fibrils arisina at the right posterior edge of each kinetosome and passing posteriad under the pellicle. The fibrils of sets ( 2 ) , ( 3 ) , and (1) all are about 20 mp in diameter and appear tubular in cross-section; they arc very unlike the heavier, solid kinetodesmal fibers. None of the Gbril sets directly interconnect. although trans- verse and postciliary fibrils end in the vicinity of the lonpi- tudinal fibril band.

often conilicting descriptions of their morphology and topography, and attempts to identify their functions by experiment or by logical inference, multiplied to form a large fraction of the protozoological literature.

* P a r t of this work was performed while the author was a Public Health Service Research Fellow of the National Cancer Institute, working a t the Laboratoire d’Evolution des Etres Organists, Paris; to Pr . P.-P. Grasse, director of the laboratory, the author is deeply indebted for his kindness and hospitality.

Grateful appreciation is also due to Pr. E. FaurC-Fremiet for stimulating consultation and for the culture of Colpidizim used in the study. For technical assistance during parts of the work, the author is grateful to Caroline N. Schooley, Jane A Westfall and Carolyn G. Smoller.