a cytological study of chilomonas paramecium with particular reference to the so-called trichocysts

380 J. PROTOZOOL. 9(4), 380-395 (1962).

A Cytological Study of Chilomonas paramecium with Particular Reference to the So-called Trichocysts”

EVERETT

Department of Zoology, University

SYNOPSIS. Chilomonas paramecium has been studied by light and electron microscopy with special attention to the so-called trichocysts. The electron miscroscope reveals that the anatomy of these bodies is unlike that of the classical trichocysts of ciliates. Since these structures can be extruded from the body of the organism they have been called ejectisomes. The ejectisomes have a regular geometrical shape and a complex internal structure. They consist of two unequal components which are enclosed by a thin membrane. Mor-

ESPITE the many morphological studies made on D flagellates at submicroscopic levels (2,3,5,6,20,2 1 , 44,45), few reports have been presented concerning the cytoarchitecture of organisms of the family Cryptomo- nadidae( 12) . The organism in question, Chilomonas paramecium, is a member of this family and has been the subject of innumerable cytological, biochemical and physiological investigations (23,2 5,26,2 7,34,3 7,38). In many of the anatomical studies made with the light microscope investigators have called attention to certain inclusion bodies which are found closely associated with the vestibule as well as in the peripheral cytoplasm. These structures have been named trichocysts and a few authors have suggested that they might be homologous to protrichocysts (cf. 33) . Since Jakus (30) presented her now classical electron micrographs of extruded trichocysts of Paramecium there has been much discussion concerning the supramolecu- lar structure, function and origin of this interesting organelle ( 15,29,46,5 1 ) .

Observations made with the electron microscope show that the so-called trichocysts of Chilomonas paramecium possess a structural organization unlike the trichocysts of Paramecium. Accordingly, the present communication describes these bodies and other fine structural features of this organism.

MATERIALS ASD METHODS

The Organisms were obtained from Carolina Biological Supply Company and were subsequently cultured in either a wheat infusion, hay infusion or a medium consisting of 4 grains of polished rice per 50 cc of distilled water.

For general cytological analysis Chilomonas was fixed in Champy’s fluid and subsequently impregnated with a 2% osmium tetroxide solution according to the method of Kola- chev (cf. 9) . Some were fixed in Bouin’s and Maximow’s

*This investigation was supported by a grant (RG 8776) from the Sational Institutes of Health, United States Public Health Service. I wish t o thank Miss Mathilda Boulanger for her able technical assistance.

ANDERSON

of Massachusetts, Amherst, Mass

phological polarity is established by an anterior smaller unit and a posterior larger unit. When the ejectisomes are found in the peripheral cytoplasm or in a perivestibular position the smaller unit is always oriented toward the surface. A suggestion is made that the ejectisomes are derived from the vesicular component of the Golgi complex. An interpretation is offered concerning a mechanism by which ejectisomes may be extruded from the body.

solution and others in a 10% aqueous solution of acrolein for 10 minutes(35). The acrolein fixative gave excellent preserva- tion of protoplasmic structure with little or no distortion. Paraffin sections as well as whole mounts were stained with either Heidenhain’s hematoxylin or Mallory’s triple.

Procedure for electron microscopy was as follows: organisms from the various culturing media were concentrated by centrifugation and fixed in a 2% solution of osmium tetroxide buffered to pH 8.5 corresponding to the procedure of Pa- lade(40); others were fixed with a 2% solution of osmium tetroxide in acetate veronal buffer (pH 8.5) prepared according to the method of Kellenberger et 02. without the versene test(31). The fixed cells were mixed with a 1% agar solution which, after gelation, was cut into small blocks. These blocks were rapidly dehydrated in acetone or a graded series of alcohols, infiltrated and embedded in Epon(36). Electron staining with phosphotungstic acid was applied by adding 0.5% phosphotungstic acid to the 70% alcohol used in dehy- dration. The majority of the sections were stained by float- ing the grids on a solution of uranyl acetate. Thin sections were obtained with a Porter-Blum ultramicrotome and observed in an RCA EMU 3E Electron Microscope.

OBSERVATIONS

General Cytological Features Chilomonas paramecium is a saprozoic colorless

flagellate approximately 35 p long. It is slightly oval with an obliquely truncated anterior end and a somewhat pointed and slightly bent posterior end. The organelles, as well as inclusion bodies, of a typical vegetative organism are depicted in Fig. 1 which is slightly modified from Hollande(25). The two flagella ( F ) are approximately of equal length and each is attached to a basal body (B) . From one of the basal bodies arises a long slender structure which proceeds posteriorly almost the entire length of the body and is called by light microscopists the rhizoplast (R). In many preparations this structure appears to come into direct contact with the surface of the nuclear membrane. At the upper right of the figure is the contractile vacuole (CV) surrounding an inpocketing of the

CYTOLOGICAL STUDY OF Chilomonas paramecium 381

Fig. 1. Diagrammatic drawing of Chilomonas paramecium (redrawn and modified from Hollande 2 5 ) . F, flagella; T, trichocysts ; AM, amphosome ; N, nucleus ; NCL, nucleolus ; PM, paramylum bodies ; P, inpocketing of plasma membrane ; CV, contractile vacuole ; B, basal body ; PB, parabasal body ; V, vestibule; R, rhizoplast; C, cortical area.

plasma membrane (P) . According to Hollande’s de- scription, this inpocketing of the plasma membrane is the contractile vacuole. Further details of this region (see below) will demonstrate that this modified area of the plasma membrane is not the contractile vacuole proper. Opposite and below the contractile vacuole is a dark structure known as the amphosome (AM). The dual parabasal bodies are found in a lateral position (PB). In the cytoplasm surrounding the vestibule (V) are a number of inclusion bodies which lie nearly parallel to the longitudinal axis of the organism’s body and have been referred to as trichocysts (T). They are densely stained with Heidenhain’s hematoxylin in material fixed in Bouin’s and Maxi- mow’s solutions; they are osmiophilic in organisms treated according to the method of Kolachev; and they are fuchsinophilic when stained with Mallory’s triple stain after fixation in 10% aqueous acrolein.

The general cytoplasm appears alveolar in both liv- ing and fixed organisms. Such an appearance of the cytoplasm in Ckilomonas is a pseudoalveolation due

to the many highly refractile inclusions known as paramylum bodies (PM). The nucleus (N) , with its prominent nucleolus (NCL) and many scattered chromatin bodies, is spherical and is usually located in the middle of the body.

In light microscopic preparations of sectioned material one gets the impression that there is a definite cortical area which appears to be structurally organ- ized as shown a t C.

Fine Structure

Flagella, rhizoplast, contractile vacuole and amphosome. The structure of the flagella of Chilomonas is that usually found, i.e., the characteristic 9 plus 2 pattern. The entire set of filaments is enclosed by a flagellar membrane which is continuous with the plasma membrane. The constituent filaments of the flagella are anchored to basal bodies (Fig. 2 BB also insert BB) which are hollow cylinders formed by the outer 9 peripheral filaments which are triple and similar in this respect to those shown by Noirot- TimothCe (39, Barnes (4) and Gibbons ( 19). The basal bodies of Ckilomonas are joined by dense connectives (Fig. 2 DC). Arising from the base of one of the basal bodies is a structure called by classical proto- zoologists the rhizoplast. This structure is composed of many fine parallel filaments which together show cross striation (Fig. 2 (insert) SF and Figs. 6, 7, 9, and 12, SF). This filamentous structure should now be referred to as a flagellar rootlet since, on the basis of comparative morphology, its origin is similar to many flagellar-ciliary rootlets of protozoan and metazoan cells. Sometimes the filaments of the flagellar rootlet come into direct contact with the external nuclear envelope, and the nucleus appears indented. That this structure does not terminate here, but rather somewhere in the posterior portion of the organism among other organelles and inclusion bodies, is borne out in light microscope preparations as well as in the electron micrographs (Fig. 17, SF). Perhaps the orientation of organisms in certain preparations of light microscopists was instrumental in their suggesting that the flagellar rootlet (rhizoplast) connected the basal body with the nucleus and that it was this basal body which initiated mitosis (cf. 28).

In addition to the flagellar rootlet there is usually found a filamentous structure arising a t about the middle of the other basal body (Fig. 2, BF). Where this structure terminates has been difficult to ascer- tain. Similar structures have been observed by Faw- cett and Porter(l7) and Rhodin and Dalhamn(50) to which Gibbons(l9) has given the term basal foot.

The elements of the contractile vacuole are usually seen surrounding an inpocketing of the plasma membrane (Fig. 13 P). In certain planes of sectioning

382 CYTOLOGICAL STUDY OF Chilomonas paramecium

Fig. 2 . .4n electron micrograph illustrating the major fea- (BB) , with their connectives (DC), basal filament (BF), tures of the anterior portion of the organism. The ejecti- Golgi complexes ( G C ) , and a large, somewhat modified mito- domes occupying a perivestibular position (EJI) , smaller ones chondrion (M). Note in the insert the striated flagellar root- in the cytoplasm ( E J Y ) , as well as an extruded form of this let (SF) and a basal body labeled BB. Fig. 2 X 18,000; body (EJ,) are demonstrated. Also shown are basal bodies insert X 8,000.


Fig. 3. A longitudinal section of the upper portion of the Note also ejectisomes IEJI), a mitochondrion (M) and nu- organism illustrating the vestibule (V) which is bounded, cleus (N) with its double nuclear envelope (NE). Insert in its upper region, by a striated plasma membrane (SV) shows the amphosome (AM). Fig. 3 X 24,000; insert X and in its lower region by a smooth plasma membrane (SM). 20,000.

.-

384 CYTOLOGICAL STUDY OP Chilomonas paramecium

there is usually associated with this inpocketing a mitochondrion (Fig. 13, MI). This mitochondrion is somewhat modified, but examination of many micrographs shows that the general structure is unquestion- ably that of this organelle. The contractile vacuole proper (Figs. 4, 13 and 14, CV) is bounded by a thin membrane having evaginations (Fig. 14, EV) . Some of the evaginations are smooth surfaced while others appear to have attached dense particles. The contractile vacuole is almost always seen to be surrounded by a multitude of vesicles of varying diameters.

Mast and Doyle(38) first called attention to certain ellipsoid bodies, to which Hollande( 25) later applied the term amphosome, located between the nucleus and the anterior end of the body. In electron micrographs these bodies are dense, spherical homogeneous structures which are sometimes found bounded by a thin membrane (Insert, Fig. 3, A M ) . The function of such bodies is unknown. Mast and Doyle(38) speculated that, “It may possibly be that these bodies are vestigial eyespots for nearly all of the closely related organisms have eyespots and many of them have two.” Hollande ( 2 5 ) suggested that they may be physiologically analogous to pyrenoids.

Vestibule, so-called trichocysts and Golgi complex. A longitudinal section of the organism shows the vestibule to be funnel-shaped (Fig. 3, V) . The plasma membrane constituting the limits of the upper portion of the vestibule is, in certain regions, finely striated (Fig. 3, SI’) whereas that of the lower part is thin with no evidence of a surface structure (Fig. 3, SM). When the vestibule is viewed in transverse section the plasma membrane at certain levels of sectioning appears to overlap (Fig. 6, F) whereas at other levels this overlapping is not evident. There is, however, a structure adjacent to the plasma membrane in this region which appears as a fiber (Fig. 5 , F) . The iden- tity of this structure is unknown.

In the cytoplasm are found dense, smooth-membrane-limited bodies which show a regular geometrical shape and a rather complex internal structure. These structures have been called trichocysts. They are usually found just beneath the plasma membrane in a perivestibular position or at the periphery of the cytoplasm. They are not, however, limited to these positions, for smaller ones are frequently seen closely associated with the Golgi complex (Figs. 4 and 6, EJ). There appears to be no modification of the limiting membrane enclosing these structures nor is there any unusual structural modification of the plasma membrane adjacent to them. Dragesco(l3) and Kriiger (32) reported that these bodies were of two morphologically distinct types. From the present study only one morphological type which varies in size is demon- strable. Figure 2 is an excellent example illustrating

the larger bodies in the perivestibular cytoplasm. When the plane of section is longitudinal or tangential the most frequently encountered profile is that of two triangles whose apices are opposed (Figs. 2, 5, and 9 , EJ,) . As observed here, these structures display morphological polarity which is established by a small wing shaped anterior unit (Figs. 2 and 5 9 ) . This uni t sits a t an angle within the V-shaped area of the external portion of the larger wing-shaped unit. Thus these bodies are arranged with their longitudinal axes transverse to the long axis of the organism, with the smaller unit always directed towards the surface. In longitudinal sections these bodies have a laminated internal structure ordered in parallel array (Figs. 4 and 5 ) . According to the level at which the plane of section is made, the arrangement of the lamellae appears to display a circular or a semi-circular pattern (Figs. 4 and 14).

In certain profiles another interesting feature is noted. Sometimes this structure appears solid (Figs. 4 and 5, TS). High magnification electron micrographs, however, leave no doubt that this component is tubular; in Fig. 4 (T) it appears to be attached to the smaller unit (also see insert), A cross section of the tubular structure as found in the larger unit is shown in Fig. 4 (TC). In this connection attention should be directed to the tubular structures of varying diameters found within the vestibule (Figs. 9 and 10, EJ4) and immediately in contact with or in the vicin- ity of the organism (Figs. 2 , 11 EJ4; see also insert, Fig. 11, EJ4). These structures show a close structural similarity to the tubular component seen within the bodies. The long, extracyotplasmic, tubular structures are here interpreted as the extruded forms of these bodies. At high magnification they are devoid of any surface architecture and show a relatively lucid interior. This observation is contrary to that of Dragesco( 13) who, from an electron microscope observation of whole mounted specimens of Chilomonas, reported that they showed a longitudinal striation.

In Fig. 12 (EJ3) are two of the smaller bodies located in the peripheral cytoplasm which were perhaps fixed before they could be extruded from the body (see also lower right insert). The appearance of the raised plasma membrane immediately over such bodies gives the impression that some force from within was being exerted at that spot. Also located at the periphery are many prominent vacuoles with no apparent internal structure (Fig. 12, PV). Where the vacuoles are found the associated mitochondria (M) (to be discussed below) are indented a t those positions. Such notching of the mitochondria suggests that this organelle encountered an obstacle more rigid than a vacuole. The possibility exists that these vacuoles harbored the dense bodies; this would also account


Fig. 4. Tangential section showing contractile vacuole (CV) , magnification. Also illustrated in the micrograph are Golgi ejectisomes (EJ & EJI) with tubular component (TC, TS, T). complexes (GC), the vestibule (V) and nucleus (N). Fig. In the insert the tubular component is shown at higher 4 X 20,000; insert X 40,000.

386 CYTQLOGICAL STUDY OF Chilomonas paramecium

Fig. 5. Enlarged area showing a cross-section of the vesti- aspects of the tubular component (TS) and a portion of the bule (V), an adjacent fibrous Structure (F), ejectisomes with anterior component ( 9 ) of one ejectisome. Fig. 5 X 70,000.


Figs. 6-8. Enlarged areas showing 'ejectisomes (EJ,) sur- demonstrated are filaments of the flagellar rootlet (Figs. 6 rounding the vestibule (V) with its adjacent fibrous com- & 7, SF), endoplasmic reticulum (ER) a mitochondrion (M) ponent (F), and Golgi complexes ( G C ) . Note dense Golgi and a paramylum body (PB). Figs. 6 & 7 X 20,000; Fig. 8 vesicles at GCI (Figs. 6 & 7 ) and small ejectisomes at EJ. Also X 24,000.


Figs. 9-11. Tangential sections showing the distribution of are the filaments of the flagellar rootlet (SF), Golgi com- ejectisomes (EJI). Note the extruded forms (EJ,) within the plex ( G C ) , a mitochondrion (M, Fig. 11) and paramylum vestibule (V) as well as at the surface of the organism (Fig. bodies (PB, Figs. 9 & 11). Fig. 9 X 15,000; Fig. 10 X 15,000; 11). Insert shows enlarged ejectisome (EJ,). Alsodemonstrated Fig. 11 X 24,000; Insert X 40,000.


Fig. 12. A section through the middle of the body showing the lower left, peripheral vacuoles (PV) and two ejectisomes the nucleus (N), nucleolus (NCL), mitochondrion (M), fila- (EJs) a t the surface of the organism. The lower one is en- ments of flagellar rootlet (SF), Golgi complex (GC), endo- larged in the insert at bottom right. Fig. 12 X 24,000; left plasmic reticulum (ER), paramylum body (PB), with a fila- insert X 20,000; right insert X 30,000. mentous component (FPB) shown in the insert located at


Figs. 13 and 14. Section showing inpocketing of plasma (EJI) , mitochondrion (MI, and Golgi complex (GC) with membrane (P) which is surrounded by a modified mito- dense Golgi vesicles (GC,). Fig. 13 X 24,000; Fig. 14 X chondrion (MI, Fig. 13). Note various aspects of the con- 30,M)O. tractile vacuole (CV) with evagjnations (EV) , ejectisomes


for the structure of the peripheral cytoplasmic area in light microscope preparation schematically illustrated in Fig. 1 (C) .

A study of the two-dimensional figures of these

dense bodies allows one to suggest a form for each unit from which a reasonable three-dimensional model of the two major components may be constructed. Such efforts are depicted in Fig. 18. As shown in this dia-

Figs. 15-17. Sections showing various profiles of modified at M in Fig. 17. An ejectisome is labeled EJ*, flagellar root- mitochondria. Note the extraordinarily branched mitochondrion let SF and a parmylum body PB. Fig. 15-17 X 24,000.


C

Fig. 18. A schematic proposal of the components of an ejectisome. The “trapezoidal” smaller anterior unit is shown a t A and the larger “hexagonal” portion illustrated a t B. These two structures are seen in position as indicated by the model at C which is similar to the ejectisomes observed in the electron micrograph in Fig. 5 .

gram at A one can imagine that the smaller anterior unit is trapezoidal. Such a trapezoid may be folded so that its lateral edges appose one another resulting in a semi-tubular structure. The larger unit is viewed as being a hexagon as shown at B whose lateral edges may be curved so as to come in apposition with one another. The two units may be put together resulting in the figure illustrated in C .

The many prominent Golgi complexes are situated in the anterior end of the body (Figs. 2, 4, 6-8, 9 and 14). Each consists of a system of closely stacked smooth membranes ordered in parallel array and some show a curvilinear pattern. A few of the smooth membranes have dilated extremities. Associated with these membranes is a vast assemblage of smooth membrane vesicles. In some preparations these vesicles appear empty but a large number show dense interiors (Figs. 6, 7 , and 14 GC,). The dense and apparently empty vesicular components of the Golgi complex are interpreted as early stages of the formation of the dense bodies just described.

Nucleus, endoplasmic reticulum, mitochondria and paramylum bodies. The double nuclear envelope is, (Fig. 3, YE) for the most part, continuous; however, it is occasionally interrupted by pores. In addition to the large nucleolus (Fig. 21, NCL) and the many chromatin bodies, the nucleoplasm consists of a par- ticulate matter primarily in the form of rod-like particles.

The endoplasmic reticulum is in the form of long slender membranes whose surfaces are studded with many ribonucleoprotein particles (RNP) (Figs. 7 and 12, ER). Other elements of the endoplasmic reticulum consist of small cisternae with similar characteristics. The cytoplasm teems with many single and clumped RNP particles.

Mitochondria, exhibiting a matrix of high density, are numerous and show an internal morphology similar to that frequently demonstrated in most metazoan(41) and a few protozoan(24) mitochondria. I t should be

pointed out that the mitochondria1 membranes of Chilomonas do not have the configuration characteristic of certain chrysomonads (49), amebae (42) and ciliates( 51). Many of the extraordinarily long mitochondria assume a peripheral position and come in direct contact with the plasma membrane (Fig. 2 , M) ; some are spectacularly branched as illustrated in Fig. 17. A large number of the mitochondria are either encircled by or caught between paramylum bodies (to be discussed below). In addition many mitochondria show some interesting modifications. Such variations show the organelle to have several lamellae (Figs. 15 and 16) and others show the organelle assuming a circular pattern (Fig. 15). In this connection, it should be pointed out that in 1930 Hall(22) made a study of Chilomonas as well as other flagellates and found one to three globular osmiophilic bodies which he designated as Golgi material. It is conceivable that what Hall observed were modified mitochondria as illustrated in this study. What this lamellar elabo- ration of mitochondria means in terms of function is not clear. I t may, however, be a method of membrane formation making available more active surface area for particular metabolic processes. The reader is referred to the papers of Fawcett(l8) and Robertson (48) for a general discussion of cytoplasmic membranes.

The large oval paramylum bodies are thought to be carbohydrate reserves with approximately equal parts of amylopectin and amylose (cf. 26, 2 7 ) . In the electron micrographs these structures are surrounded by a thin membrane and show a clear internal area and an outer region which appears relatively homogeneous (Figs. 7, 9, 12, and 17, PB) ; however, in certain pic- tures this outer area sometimes consists of a fibrous element (Fig. 12 (left insert) FPB). A second membrane also bounds these structures and its external surface is studded with RNP particles (Fig. 12, ER) . Such a characteristic would classify this membrane as an element of the endoplasmic reticulum.

DISCUSSION

In organisms of the order Cryptomonadina certain unique, densely staining, inclusion bodies have long been described within the cytoplasm( 8,47). These bodies have been called “kugelige Kornchen” (43), “Tapetenkorner oder Trickocysten”( 7 ) or its Eng- lish equivalent trichocysts( 13,25,32). According to Jakus(30), Ellis (1769) was the first to observe in the ciliate Paramecium peculiar structures which had been extruded from the cortical cytoplasm. He compared these structures with the ciliated combs of the ctenophoran Beroe. Allman( 1) later called these structures trichocysts, a term derived from the Greek thrix which means hair and kystis meaning bladder. Since these early times, there has been much interest


concerning the structural organization of this organelle. With recent technical advances for the study of fine structure, many investigators have seized the oppor- tunity to study the detailed anatomy of this interesting structure. Our knowledge was increased considerably when Jakus(30) published her elegant studies of “resting” and extruded forms of trichocysts of Paramecium. In the resting condition the author found that the body was uniformly dense and the tip was found lying within a less dense cap. The extruded form consisted of a sharply pointed tip and a cross-striated shaft. From the studies dealing with trichocysts, substantial agreement has been reached, despite the fact that certain details of structure vary from species to species (cf. 51).

From a light microscopic study made by Visscher #( 53) on the gymnostomatous ciliate, Dileptus, he suggested that the name trichocysts should be discon- tinued since these bodies had no structure when dis- charged. He recommended the term toxicysts (poison- sac). Many investigators disagreed with Visscher but his terminology persisted. Dragesco( 11,12) made an electron microscope study of whole mounts of twenty-four species of gymnostomes (genera included Spathidium, LoxophylZum, Dileptus, Lionotus, Lacry- maria, Didinium, Chaenea, Bryophyllum, Prorodon and Pseudoprorodon) and suggested that the toxic trichocysts of these organisms were similar to the complex nematocysts of peridinian flagellates. Observa- tions made by FaurC-Fremiet( 16) of sectioned material of some of the above listed genera essentially parallel those of Dragesco. Dumont(l4) in a recent electron microscope observation of Dileptus anser depicted an interesting structure, for which he retained the name trichocysts, and described it as consisting of two portions, a head and a body. The body was found to contain an amorphous material and a thread with a 540 W periodicity which is attached to the trichocyst head.

As previously stated, electron microscopic observations on structures designated as trichocysts in flagellates are meager. In the dinoflagellate, Uxyrrhis marina, Dragesco( lo) observed that the trichocysts have certain similarities to those found in Paramecium. They could be extruded from the body and, when extruded, consisted of a long thread with a 600 A periodicity. The so-called trichocysts of Chilomoms paramecium have been studied by more investigators. That these structures are irritable systems was pointed out as early as 1889 by Butschli(8) and subsequently confirmed by the works of others (cf. 28, 33) . Dra- gesco (1 3 ) investigated the extruded structures with dark-field illumination, phase contrast and electron microscopes. He stated that the extruded form consisted of two distinct parts each of which is attached t o the other. As mentioned above, he suggested that

a longitudinal striation could be observed in his electron micrographs.

The present investigation has, from sectioned material, clarified morphologically the characteristic features of these dense cytoplasmic bodies found in Chilomonas paramecium. On the basis of their submicroscopic anatomy and also their origin (to be discussed below) the term trichocysts does not seem to identify them properly. It is probably best to dis- continue the term trichocyst, and, in light of the thought that these bodies are extruded from the body of the organism, it is here proposed that the name ejectisome be given to these structures.

An unequivocal statement cannot be made with respect to the origin of the ejectisomes in Chilomonas. In the electron micrographs, however, it was observed that small ejectisomes were closely associated with the vesicular component of the Golgi complex. The morphological observations suggest that one might think of the genesis of these structures as commencing within the vesicular component of the Golgi complex. One can imagine that the ejectisome begins within a simple vesicle which accumulates the synthetic product which subsequently condenses and gradually forms a small, dense, fully formed body. During further differentia- tion they acquire their “mature” form and assume a definite orientation in certain regions of the body. If the hypothesis set forth is true, namely that the ejectisomes are derived from the vesicles of the Golgi complex, one must assume that the vesicles of the complex continue to replenish themselves perhaps from the membranous portion. In this connection, the author concurs with the view advanced by Grimstone( 2 1) from his experimental investigations of Trichonympha that the Golgi complex should be viewed ‘( . . . not as a static structure but as a dynamic steady-state system -it is the site of a flux of membranes” Readers are referred to the papers of PaIay(41) and Smith and Littau (52) for morphological evidence which suggests that the Golgi complex is involved in the production of various structural entities.

From this study the conclusion is made that these bodies are extruded from the organism. This conclusion is drawn in spite of the fact that sections which would show some of the more crucial stages in such a process have not been obtained. Such an achievement would be difficult, since the process is obviously a rather rapid one and many different kinds of stimuli initiate the process. I t is further thought that each unit is extruded from the body, perhaps not simultane- ously, but one after the other. I t is difficult to suggest a mechanism for how this process is accomplished. One can envision, however, that the lamellae of the ejectisomes are folded in telescopic fashion and when everted manifest themselves in a tubular form. The varying diameters shown by the tubules could be ex-


plained on the basis of the two constituent unequal components. The “trapezoidal” and “hexagonal” components could also explain the two parts as depicted in Figs. 2 and 3 of Dragesco’s paper( 13). Such an hypothesis must, before it can be substantiated, await an experimental analysis.

Nothing can be said about the function of the ejectisomes. I t is hoped that some of the morphological information presented in this study may stimulate further investigations and lead eventually to an under- standing of the function of these interesting structures.

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UTILIZATION OF GLUCOSE BY Astasia longa 395

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Utilization of Glucose by Astasia Zonga*

SUE-NING C. BARKYt

Department of Zoology, University o f Maryland, College Park, Haryland

‘SYNOPSIS. A mutant strain of Astasia longa utilized glucose for growth whereas the parent (J) strain did not. The optimal p H for growth of the mutant with glucose (sole carbon source) was near neutrality; the optimal glucose concen- tration 0.02 M. Cell-free extracts or cell homogenates pro- .duced Cl4OZ when incubated in the presence of C”-labeled glucose. On the other hand, after incubation with C14-labeled glucose, intact parent cells and their respiratory COZ showed no radioactivity while the mutant-strain cells and COZ pro- duced were active. Dissimilation of g l~cose-1-C~~ and glucose- ~ 6 - c ’ ~ yielded the same amount of radioactivity in metabolic

stasia longa strain J, a colorless phytoflagellate, is A an “acetate flagellate”(9). Its minimal nutri- tional requirements are now thought to be an organic carbon source, an inorganic nitrogen source, other inorganic salts to supply essential elements, thiamine and vitamin BIZ ( 2 1 ) . As a general rule acetate flagellates are unable to utilize glucose. Euglena gracilis var. bacillaris, grown in darkness, is an exception( 16) , as confirmed by Cramer & Myers(5); they found, however, that another strain of E . gracilis, the Vischer strain, could not use glucose. No experimental evi- *dence has been reported to explain this difference between the two strains of E. gracilis; it has been suggested (9) that inability of acetate flagellates to utilize glucose might be due to lack of hexokinase or phosphoglucomutase, or to a difference in the permeability characteristics of the cell membrane( 5,9,14,16).

Observations during our routine maintenance of stock cultures of different strains of A . longa suggested that one strain might be able to utilize glucose. After a glucose metabolism had been demonstrated in one strain, information was sought on the pathway by which glucose is metabolized.

*This paper is part of a dissertation submitted in partiaI fulfillment of the requirements for the Ph.D. in the Depart- ment of Zoology, University of Maryland, College Park, Maryland.

This investigation was aided by grant 5028 from the Division of Research Grants, U. S. Public Health Service.

t Present address: Dept. of Histology and Embryology, Baltimore College of Dental Surgery, Dental School, Univer- sity of Maryland, Baltimore 1, Maryland.

COX in cell-free extracts of both strains. Of five enzymes as- sayed, hexokinase, phosphoglucomutase, and lactic dehydrogenase were present whereas glucose-6-POi dehydrogenase and glucose dehydrogenase were absent in cell homogenates of both strains. Presumably these two strains of A . longa differ in permeability of the plasma membrane. Further tracer and enzyme studies indicated that the Embden-Meyerhof scheme is the principal pathway of glucose catabolism ; the hexose mono-phosphate shunt and the direct oxidative pathway were either not operating or quantitatively insignificant.

MATERIALS AND METHODS

The two strains of A . longa studied were strain J, the parent strain, and a mutant strain originally designated strain 460(20) derived from a parent-strain cell subjected to an X-ray dose of 20,000 r. Both strains were axenic.

Before experimenting with strain 460, new clones were established in the complete medium described below, by spread- ing a drop of culture containing about 100 cells over an agar plate (2% agar in complete medium) ; single cells were trans- ferred with a wire loop from the agar to tubes of complete medium. Yields of the newly-established clones varied considerably (technique described later). A high-yielding clone was arbitrarily selected for study (strain 460-17). It was un- necessary to establish a new clone from the parent strain since when this was done previously all new clones grew simi- larly(20).

In the minimal medium (MM) used (Table 1) all chemi- cals were reagent grade as elsewhere. The complete medium (CM) was M M plus (per liter medium): Difco Tryptone, 3.0 g ; N-2-Case, 3.0 g ; glucose, 2.0 g ; sucrose, 2.0 g ; Difco yeast extract, 2.5 g ; hydrolyzed yeast nucleic acid, 0.1 g ; inosi- tol, 10 mg; choline, 2 mg; nicotinamide, 2 mg; Ca pantothe-

TABLE 1. Composition of minimal medium

Organic carbon source 0.01 M

CaClZ-2H,O MnClz.4Hz0 CuS04*5H,0 HJQ NazMo0,.2H,0 Thiamine.HC1 Vitamin BIZ Distilled HzO to

~- 0.1 g 0.1 g 0.3 g 0.005 g 0.005 g 0.004 g

0.1 mg 0.057 mg 0.045 mg 1.0 mg

1.00 hter

0.002 g

0.15 qg

a cytological study of chilomonas paramecium with particular reference to the so-called trichocysts

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