Extrusive Organelles in Protists*

Extrusive Organelles in Protists' KLAUS HAUSMANN Lehrstuhl fur Zellenlehre, Universitat Heidelberg, Heidelberg, West Germany I. Introduction . . . . . . , . . A. Definition of Extrusive Organelles . . . B. Historical Background . . . . . . 11. Methods . . . . . . . . . A. Preparation of Material . . . . . . B. Light Microscopy . . . . . . . C. Electron Microscopy . . . . . . 111. Characterization and Distribution of Extrusomes IV. Fine Structure, Extrusion Merhanism, Function, and Origin of the Different Types of Extrusomes . . A. Spindle Trichocysts . . . . . . B. Mucocysts (Including Muciferous Bodies, . . Kinetocysts, and Clathrocysts) . . . . C. Toxicysts (Including Cyrtocysts, Pexicysts, and Haptocysts) . . . . . . . . D. Rhabdocysts . . . . . . . . E. Ejectisomes (Taeniobolocysts) . . . . F. Discobolocysts . . . . . . . G . Nematocysts (Cnidocysts) . . . . . H. Polar Filament of Cnidosporidians . . . I. Varia . . . . . . . . . V. Conclusions . . . . . . . . . References . . . . . . . . .

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I. Introduction A. DEFINITIONOF EXTRUSIVEORGANELLES Extrusive organelles are membrane-bounded structures of protists, usually located in the cortical cytoplasm of these cells. Although they have different type-specific structures and functions, they all exhibit one general characteristic: They are readily discharged when subjected to a wide range of stimuli (mechanical, electrical, and chemical). During the transition from the resting state to the ejected form, which in several cases takes place within milliseconds, the organelles undergo characteristic morphological changes. In the following disThis article is dedicated to Professors R. Hovasse, Fr. Kriiger, and K. E. WohlfarthBottermann.

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cussion the term “extrusome” is used as a general name for all kinds of extrusive organelles (according to Grell, 1973).

B. HISTORICALBACKGROUND As far as can be determined, the first observation of extruded extrusomes was made in Paramecium by Ellis in 1769; however, these organelles were first described in detail by Allman in 1855. Later investigations were made by Kolsch (1902), Maier (19O3),Schuberg (1905), Mitrophanow (19O5), and Khainsky (1911). In 1914 Tonniges reviewed the literature on extrusomes in an article on the nature of these organelles in the ciliate Frontonia (Tonniges, 1914). Following Saunders’ 1925 studies, Kriiger (1929, 1930, 1931a,b, 1934a,b) initiated his extensive work on extrusomes using the darkfield light microscope; the results are summarized in his 1936 review article. Development of the electron microscope provided new opportunities for studying extrusomes, and since its introduction our knowledge of these organelles has rapidly increased (for reviews, see Hausmann, 1972a; Hovasse, 1965a, 1969; Hovasse and Mignot, 1975). Today many different kinds of extrusomes are known. In some cases electron microscope studies have shown the macromolecular arrangement of filamentous elements making up special parts of several extrusomes. In addition, the extrusion mechanism of some of these organelles has been revealed by electron microscope visualization of different stages of expulsion interrupted in progress. Many remaining unanswered questions are also discussed in this report. 11. Methods

A. PREPARATION OF MATERIAL One of the easiest ways to prepare extrusomes for light microscope examination is to press the protists between a microscope slide and a cover glass. By this procedure the cells are destroyed, and ejected extrusomes become visible. However, a triggering of the extrusion by cover glass pressure is not always possible because of the small size of the cells (e.g., flagellates), or not desirable if special methods are required (e.g., negative staining or shadow casting for electron microscopy). Fumes of formaldehyde or similar solutions also produce extrusion (Kriiger, 1931a, 1934a, 1950). If an increased quantity of inhibited extrusomes is required, one can use a solution of 5% potassium ferrocyanide (Kriiger, 1930), which is added to the cells prior to their destruction or irritation.

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For special problems it is possible to induce extrusion of all the extrusomes and still keep the cells alive. Geranium juice is reported to be such a stimulant (Schuster et al., 1967), and the application of a moderate electric shock has also been used in some cases (Hausmann and Allen, 1976; Wohlfarth-Bottermann, 1953; Yusa, 1963, 1965).

B. LIGHT MICROSCOPY It is not necessary to employ special staining methods for light microscope examinations of extrusomes if one makes use of the properties of the phase-contrast microscope, the anoptral-contrast microscope, and the differential-interference-contrast microscope. Kriiger (1936) has reviewed the different light microscope staining methods for extrusomes. C. ELECTRONMICROSCOPY Basically all the conventional electron microscope techniques can be employed for the study of extrusomes, although it is frequently useful to change or modify the concentration of the fixative, the kind of buffer system, or the pH of the solution to prevent an inopportune extrusion. It is known that some buffers trigger the discharge of extru-

somes (Allen and Wolf, 1974), and different negative-staining procedures have been reported to influence the fine structure of these organelles (Hausmann, 1973a; Hausmann and Stockem, 1973). For other methods (freeze-fracture, shadow casting, scanning electron microscopy, and so on) see Allen and Hausmann (1976), Bachmann et al. (1972), Hausmann (1974), Jakus and Hall (1M6), Marczalek and Small (1969), Plattner et al. (1973b), and Small and Marczalek (1969).

111. Characterization and Distribution of Extrusomes Extrusomes are listed alphabetically in Table I. Their morphology in the resting and ejected state is described and information on their mode of ejection is provided in the table. In 1936, Chadefaud attempted to use extrusomes as a characteristic for taxonomic purposes. But the presence of these organelles does not seem to be correlated with other characteristics used to classify protists. Kriiier (1936) has pointed out, however, that extrusomes in ciliates are restricted mainly to holotrichs. The distribution of the various types of extrusomes in protists is summarized in Table 11.

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KLAUS HAUSMANN TABLE I CHARACTERIZATION OF EXTRUSOMES Name

Resting state

Discobolocyst (Fig. 46)

Spherical body with a diskshaped ring at one pole

Ejectisome (taeniobolocyst) (Fig. 44)

Spirally wound ribbon, usually bipartite

Haptocyst (missile-like body or phialocyst) (Fig. 42)

Bottlelike organelle with a complex internal structure composed of several components Compound organelle consisting of a central element enveloped by a ringlike jacket Saclike vesicle filled with an unordered material Polyhedral paracrystalline body

Kinetocyst (Fig. 29)

Muciferous body (Fig. 26) Mucocyst (Figs. 20 and 21)

Nematocyst (cnidocyst) (Fig. 48) Rhabdocyst (Fig. 43)

Spindle-shaped capsule with a coiled tube Stick-shaped organelle

Spindle trichocyst

Spindle-shaped or

Ejected state

Mode of ejection

Solid ring with the same dimensions as in the resting state furnished with a long tail Tubelike; smaller and longer than the resting state

Unknown (stretching?)

Partly everted; excretion of poisonous material

Sudden unrolling of the ribbon and formation of a tube by rerolling laterally Unknown

The central element lies in front of the open jacket

Unknown

Amorphous mucilage

Secretion of the material through a pellicular pore

Polyhedral paracrystalline body whose diameter and length are multiples of those of the resting state Capsule with an everted tube

Secretion lasting several seconds; unfolding of a preexisting network of filaments

Tubelike structure with the same length and plus or minus the same diameter as the resting form Thread-shaped

Eversion of the tube Telescopic expulsion

Sudden (lasting

(Continued)

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(Continued)

Name

Resting state

Ejected state

Mode of ejection

(acontobolocyst) (Figs. 6 and 7)

rhomboid paracrystalline body; sometimes furnished with a specially constructed tip

paracrystalline filament with plus or minus the same diameter as the resting state

less than a second) unfolding of a preexisting three-dimensional network of protein filaments

Toxicyst (including pexicyst and cyrtocyst) (Figs. 36 and 37)

Inverted tube inside a capsule

Polar filament (of cnidosporidians)”

4 coiled tube in-

side the spore

Capsule with an everted tube of the same width and length as in the resting state; excretion of poisonous material Everted tube in contact with the spore

Sudden eversion and/or telescopic expulsion of a tube

Evagination of the tube

This structure is not an extrusomesensu stricto, since it is not a membrane-bounded organelle (see Section &A). TABLE I1 EXAMPLESOF THE OCCURRENCE OF EXTRUSOMES IN THE DIFFERENT TAXONOMIC CATEGORIES OF PROTISTS‘ Systematic group

Type of extrusome

Flagellata

Discobolocyst Ejectisome Muciferous body Mucocyst Nematocyst Spindle trichocyst Toxicyst Kinetocyst Haptocyst Mucocyst Rhabdocyst Spindle trichocyst Toxicyst Polar filament

Rhizopoda Sporozoa Ciliata

Cnidosporidia I.”

Example

Ochromonas tuberculatus Chilomonas paramecium Euglena spirogyra Euglena splendens Pol ykrikos schwartzi Oryrrhis marina Colponema loxodes Acanthocystis aculeata Acineta tuberosa Tetrahymena pyrifomis Kentrophoros latum Paramecium caudatum Didinium nasutum Nosema bombycis

Some protists possess several different types of extrusomes; for example, Didinium

-..---.,”+.. ....,I

c-4

-.,..

tr

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IV. Fine Structure, Extrusion Mechanism, Function, and Origin of the Different Types of Extrusomes The choice of extrusomes and order in which they are discussed are based on their similarities to each other and on the extent to which they have been investigated. A. SPINDLETRICHOCYSTS 1. Resting Spindle Trichocysts of Ciliates

In ciliates, resting spindle trichocysts are located in the cortex of the cell (Fig. 1).In Paramecium there are about 6000 to 8000 trichocysts in each cell, and they are located at predictable positions within the pellicular system (Fig. 2) (Allen, 1971; Ehret and de Haller, 1963; Ehret and McArdle, 1974; Ehret and Powers, 1959; Grimstone, 1961; Hufnagel, 1969; Jacobson, 1931; Jurand and Selman, 1969; Khainsky, 1911; Klein, 1952; Pitelka, 1963, 1965; Plattner et al., 1973a; Schneider and Wohlfarth-Bottermann, 1964; Schuberg, 1905; Sedar and Porter, 1955; Stewart and Muir, 1963; Wichterman, 1953). In Paramecium the spindle-shaped or rhomboid trichocyst bodies (tb in Fig. 6a), furnished with a specially constructed tip (tt in Fig. 6a), regularly alternate with single or paired basal bodies (Fig. 2a and b). Their tips (tt in Fig. 2c) pass through the space between adjacent alveoli (a in Fig. 2c), where they closely underlie the plasma membrane. In freeze-fracture replicas the P face of the plasma membrane displays regular ringlike aggregates of membrane-intercalated particles located directly above the trichocyst tips (Figs. 3a and b; arrows in Fig. 4a). [In this article the freeze-etching nomenclature proposed by Branton et al. (1975) is used.] These aggregates consist of an outermost single or double ring, 300 nm in diameter (a-type granules), either a concentric middle ring or a diffuse zone of particles, 180 nm in diameter (b-type granules), and finally a central rosette, 80 nm in diameter (c-type granules) (Figs. 3a and b; arrows in Fig. 4a) (Janisch, 1972; Plattner et al., 1973a; Satir, 1974b; Satir and Satir, 1974). The trichocyst membrane is also connected to the alveolar membrane by particles (e-type granules) (e in Figs. 3b and c, and 4b). Plattner et al. (1973a) interpret these particle accumulations to be membrane-to-membrane attachment sites, providing the trichocyst tip with a solid anchorage in the plasma membrane and a connection with the alveoli. This explanation, which is supported by isolation experiments (Anderer and Hausmann, 1977), seems to be plausible, since the expulsion, which takes place within milliseconds (Pitelka, 1963),

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FIG. 1. Diagram (a) and electron micrograph (b) of the general organization of P . cuudutum. The resting spindle trichocysts (tr) are located in the cortex of the cell. ma, Macronucleus; mi, micronucleus; cv, contractile vacuole; fv, food vacuole; pe, peristome; ve, vestibulum. (b) x800. (a) After Grell, 1973.

must expose the orifice of the opened trichocyst membrane to extremely high forces which could tear the trichocyst membrane or pull it out of the cell if it were not firmly anchored to the surrounding pellicle (Allen and Hausmann, 1976). According to D. R. Pitelka (personal communication) the function of the particles in the plasma membrane-at least the c-type granulesmay be, in addition to providing attachment sites, to prevent inopportune fusion of the plasma membrane with the trichocyst membranean idea also proposed independently by Bardele (1976a) for the kinetocysts in heliozoan axopods (see Section IV,B,8).

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FIG.3. Diagram of the distribution of membrane-intercalated particles in the cortex of Paramecium after freeze-fracture (a and c), and thin sections (b). In the thin sections the particles are located inside the membranes rather than in between as shown. (b) is drawn in this way to show clearly the position of the particles. a, b, c, and e , Different aggregates of particles associated with the plasma membrane (pm), the trichocyst membrane (tm), and the alveolar membrane (am). al, Alveolus; d, depression; EF-tm, external face of the trichocyst membrane; p, particle; PF-pm, protoplasmic face of the plasma membrane; PF-tm, protoplasmic face of the trichocyst membrane; tb, trichocyst body; tc, tubular collar; tt, trichocyst tip. (c) From Allen and Hausmann, 1976.

However, other workers have postulated that similarly arranged particles in the plasma membrane of Tetruhyrnena, calledfusion rosettes (Fig. 22g), are necessary for fusion of the plasma membrane with the extrusome membrane in this ciliate (Satir et al., 1972, 1973) and may be a general feature of other membrane fusions (Satir, 1975; Satir and Satir, 1974). As indicated by Beisson et al. (1976),the true function of the particles in the plasma membrane could be discovered by examining paraFIG. 2. Electron micrograph (a) and simplified diagrams (b and c) of the pellicular system of P. caudatum. a, Alveolus; ci, cilium; kf, kinetodesmal fiber; pmt, posterior microtubules; tb, trichocyst body; tmt, transverse microtubules; tt, trichocyst tip. (a) x 40,000. (a) From Hausmann and Allen, 1976.

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mecia with mutations that affect the trichocysts, such as those reported by Jurand and Saxena (1974), Ruiz et al. (1976), and Pollack (1974). Since the membrane junctions in Paramecium resemble to some extent metazoan gap junctions (for reviews, see McNutt and Weinstein, 1973; Staehelin, 1974), which are generally considered sites of selective permeability for intercellular communication or coupling, Plattner et al. (1975)have analyzed, using the technique of high-resolution freeze-fracture and tracer experiments (microperoxidase, lanthanum, and cytochrome c), the morphology and functional role of the ringlike aggregates of intercalated particles in the plasma membrane ofParamecium. They showed that in none of the techniques did low-molecular-weight electron microscope tracers penetrate the membranes. Therefore the assumption of Satir and Satir (1974), that extrusome discharge can be triggered by an osmotic shift via transmembranous canals, has not yet been confirmed experimentally. However, these results cannot rule out the possibility that the particles operate as active transmembranous carriers of ions, for example, Ca2+, which can then induce exocytosis. [Plattner (1974) induced trichocyst discharge artificially b y using ionophoretic Ca2+ injections.] However, special electron microscope methods for detecting Ca2+ have never shown corresponding electron-dense deposits in the region of the membrane-intercalated particles of type a, b, or c (Plattner and Fuchs, 1975).This is not surprising, since it is likely that exocytosis is triggered from inside rather than from outside the cell. However, Ca2+may play a more prominent role in the actual fusion of the trichocyst membrane with the plasma membrane than in triggering discharge, since it is known to be important in other exocytic processes (Allison and Davies, 1974; Douglas, 1974; Poste and Allison, 1973). Recent localizations of Ca2+-accumulatingsites in Paramecium (Fisher et al., 1976; Plattner and Fuchs, 1975) support this possibility. Except at the distal part of its tip, the trichocyst has a membrane that shows the normal characteristics of biomembranes following freeze-fracture preparation (Figs. 3c and 4b). The P face possesses randomly distributed 10-nm-diameter particles (p in Fig. 3c; PF-tm in FIG.4. (a) In freeze-fracturepreparations of the P face of the plasma membrane (PFpm) particle rings (arrows) between the cilia (arrowheads)indicate the position of the underlying trichocyst tips. (b) The P face of the trichocyst membrane (PF-tm) possesses randomly distributed particles, whereas the E face (EF-tm) shows corresponding depressions. Most of the distal part of the tip is free of particles (bracket) except at the extreme end of the tip where one observes a conglomeration of particles (e).(a and b) x 27,000. (a) From R. D. Allen; (b) from Allen and Hausmann, 1976.

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FIG.5. Schematic three-dimensional reconstruction of the detailed structure of part of the trichocyst tip and ensheathing structures (1 to IV), cut away to expose the different layers. tc, Tubular collar. From Bannister, 1972.

Fig. 4b) which are present in a frequency of about 800 particles per square micrometer. The E face shows a corresponding number of depressions (d in Fig. 3c; EF-tm in Fig. 4b). The membrane surrounding the distal third of the trichocyst tip is encased in a tubular collar (tc in Figs. 3b and c, and 5). This external specialization of the trichocyst membrane is complemented by a differentiation in the adjacent intramembranous region; both the P face and the E face are free of particles and depressions and are smooth (tc in Fig. 3c; bracket in Fig. 4b). The particles that were in this area during trichocyst development (Allen and Hausmann, 1976) are now concentrated within the trichocyst membrane at its distal tip (e in Figs. 3c and 4b). Ultrastructurally the undischarged trichocyst consists of several different components (Bannister, 1972; Hausmann et al., 1972b):

1. The crystalline matrix of the trichocyst tip (I in Fig. 5; Fig. 6b) and the body, which reveals a periodic, 7-nm striation (Fig. 6c and d). 2. A meshlike sheath surrounding the body of the organelle, which is best seen in isolated trichocysts (Anderer and Hausmann, 1977) (arrowheads in Fig. 6d).

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3. An inner sheath surrounding the tip, consisting of four helically arranged envelopes with a square net substructure (I1 in Fig. 5). 4. An outer sheath around the tip composed of tubular structures (I11 and IV in Fig. 5). 5. A membranous trichocyst sac which has an apical region surrounded by a cylinder of tubular structures joined to each other by dense material, that is, the tubular collar (tc in Figs. 3c and 5).

2 . Discharged Spindle Trichocysts of Ciliates The discharged spindle trichocysts of Paramecium caudatum, which measure 25-35 pm in length and 0.5-0.6 pm in width (Fig. 6e

FIG.6. Resting (a-c) and ejected trichocysts of P. caudaturn (d-h). The trichocyst is a bipartite structure consisting of a tip (tt) and a body (tb). In the electron microscope the tip (b)as well as the body (c and d) of the resting stage show a regular pattern. The ejected form is elongated by a factor of 8 (e).The shaft discloses electron-dense (d) and electron-light (1) cross striations. The striae are due to a regular arrangement of fila. x95,OOO. (c and d ) x80,OOO. (e) ments (points and asterisks) (h). (a and e) ~ 8 0 0(b) ~ 4 5 0 0 (f . and g ) x 110,000. (f) From Hausmann, 1971.

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and f), are characterized by transverse striations with a period of 55 nm (Fig. 6f and g). Electron-dense (d in Fig. 6g) and electron-lucent (1 in Fig. 6g) bands make up this periodic structure (Bannister, 1972; Beyersdorfer and Dragesco, 1952b; Ehret and McArdle, 1974; Hausmann, l971,1973b,d; Hausmann and Stockem, 1973; Hausmann et al., 1972a; Jakus, 1945; Jakus and Hall, 1946; Knoch and Konig, 1951; Kriiger and Wohlfarth-Bottermann, 1952; Nemetschek et al., 1953; Pease, 1947; Wohlfarth-Bottermann, 1950, 1953; Wohlfarth-Bottermann and Pfefferkorn, 1952). The same or a similar structure can be observed in spindle trichocysts of other species of ciliates (Beyers-

FIG. 7. The various fine-structural features of the discharged trichocyst shaft (a-d) can he explained by a three-dimensional model (a‘-d‘). Depending on the plane of view different patterns can be seen. Note the marks at the bottom of the model. (a-d) X 210,000. From Hausmann et al., 1972a.

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dorfer and Dragesco, 1952a; Bretschneider, 1950; Dragesco, 1968; Hausmann, 1973b; Kawakami and Yagiu, 1960; Kriiger et al., 1952; Luporini and Magagnini, 1970; Nilsson, 1969; Potts, 1955; Rouiller and Faur6-Fremiet, 1957). The alternation of electron-dense and electron-transparent striae is caused by a regular three-dimensional arrangement of thin filaments (Fig. 6h) constituting the structural elements of the trichocyst shaft at the macromolecular level (Bannister, 1972; Hausmann et al., 1972a). The filaments are connected in a very regular manner (Figs. 6h and 7). One of the striations is always the same (0in Fig. 6h), while the other alternates in its fine structure (asterisk in Fig. 6h). However, it can be shown by means of a three-dimensional model of the trichocyst shaft that these periods always have the same structure; the bands are just twisted against each other at an angle of 90" (Fig. 7) (Hausmann et al., 1972a). 3. Comparison of Resting and Ejected Spindle Trichocysts of Ciliates On comparing the morphological features of resting and ejected trichocysts of Paramecium the following facts emerge:

1. The resting trichocyst is 3-4 pm long; the ejected form is 25-35 pm long and is therefore about eight times longer than the resting trichocyst. 2. Both resting and ejected trichocysts have about 500 periodic cross-striations. 3. The cross-striations of the resting trichocyst have a periodicity of 7 nm (Fig. 8a'); that of the striations in the ejected trichocyst is 55 nm (Fig. 8b'). Besides an eightfold increase in the length of the trichocyst, the width of the periodicity increases by the same factor of 8. 4. The resting trichocyst has a homogeneous cross-striation (Figs. 6c and d, and 8a'), whereas the striae of the ejected form are divided into several subperiods (Fig. 6h, 7, and 8b').

4. Inhibited Spindle Trichocysts of Ciliates The question arises, what is the mechanism of trichocyst expulsion which takes only a few milliseconds to complete (Jahn and Bovee, 1967; Pitelka, 1963)? The extrusion process cannot be analyzed in the light microscope in oivo, because of the rapidity of the discharge. Even high-speed microcinematography was unable to show the details of expulsion (Miller et al., 1968; Pitelka, 1963). Nevertheless, Kriiger (1930) studied the discharge indirectly. He inhibited the ex-

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a'

b'

FIG. 8. Schematic comparison of resting (a) and ejected trichocysts of Paramec:ium

(b). After discharge the homogeneous cross-striationof the resting stageI (a') widens and becomes divided into subperiods (b'). From Hausmann et al., 1972a.

pulsion by using a solution of 5% potassium ferrocyanide. The various trichocyst forms resulting from this treatment can be used to reconstruct the possible course of the discharge (Fig. 9a-f) (Hausmann, 1971). However, the light microscope shows only elongation of the trichocyst and is not able to provide more detailed information. Electron micrographs of these specimens disclose that the essential process of discharge is a rearrangement (recrystallization) of filaments (Figs. 9g-i, 10, and 13a) from a highly ordered structure with a period of 7 nm (Fig. 6c and d; rt in Fig. 10) to another highly ordered structure with a period of 55 nm (Figs. 6f-h, and 7; dt in Fig. 10). Therefore trichocyst expulsion is due to the rapid stretching and unfolding of a network of filaments which are initially packed very close together and are preformed in the resting stage (rt in Fig. 10; Fig. 13a). Such a model has been proposed by Schmidt in 1939, postulating that

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an unfolding of filamentous molecules is the basic event in trichocyst discharge; his conclusions were based on polarizing microscope observations. What happens at the molecular level during the expulsion remains to be explained. There is almost no information regarding this problem, with the exception of observations that ATP inhibits the discharge (Anderer and Hausmann, 1977; Hoffmann-Berling, 1960, 1961), whereas bivalent ions and many different stimuli may trigger the expulsion. The reported cytochemical localization of ATPase activity in trichocysts during extrusion (Kawakami, 1971) needs to be verified.

5. Spindle Trichocysts of Microthoracidae The holotrichous ciliate Pseudomicrothorax dubius, family Mi-

crothoracidae, is a rather rare protozoan with an uncertain systematic position (Corliss, 1958; Thompson and Corliss, 1958). Like other members of this family, for example, Drepanomonas dentata (Hausmann, 1973d; Hausmann and Mignot, 1975; Prelle, 1968) and Leptopharynx costatus (Prelle and Aguesse, 1968), this ciliate possesses a rather different type of extrusome (tr in Fig. 11)(FaurBFremiet and Andre, 1967; Peck, 1971, 1974); its spindle trichocyst has a quadripartite tip structure (tr in Fig. 11; Fig. 12). The shaft of this trichocyst is structurally and cytochemically similar to the spindle trichocyst of Paramecium (Hausmann and Mignot, 1975); a periodically striped structure is seen in both the resting and elongated stages with periods of 12 and 50 nm, respectively (Fig. 12a and c). But the four apical tubes of the tip are the most striking feature of this extrusome (t in Fig. 12a and b). This tip is a dynamic structure (Figs. 12e-g, and 13b). During or following elongation of the shaft the tubes spread and at the same time excrete a very electron-dense material (arrows in Fig. 12g) which can be seen, even in the light microscope, as four drops at the very tip of the trichocyst (5in Fig. 13b)(Hausmann and Mignot, 1975; Pknard, 1922). The significance of these tubes has not yet been clarified. A problem, especially for the classification of ciliates, is the appearance of similar trichocysts in the scuticociliate Ctedoctema (P. Didier, personal communication). 6. Spindle Trichocysts of Flagellates Flagellates are known to possess extrusomes (for reviews, see Cachon et ul., 1974, 1975; Dodge, 1973; Hall, 1946). Many dinoflagellates have spindle trichocysts, for example, species of Amphidinium (Dodge and Crawford, 1968; Dragesco and Hollande, 1965), species of

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FIG.10. This partially inhibited spindle trichocyst ofPurumecium demonstrates the mode of ejection in which the resting body (rt) is transformed into the discharged shaft (dt) by a sudden expansion. tt, Trichocyst tip. x 25,000. From Hausmann, 1973d. FIG.9. Inhibited spindle trichocysts of Paramecium in light micrographs (a-f) and electron micrographs (g-i). The different stages found in the light microscope are represented in the electron microscope as a gradual disorganization of the network of the trichocyst filaments. (a-f) x 2100. (g) x 30,000. ( h and i) x 8000. From Hausmann et ul., 1972b.

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FIG. 11. Localization of compound trichocysts (tr and arrows) in the pellicular system of the ciliate P . dubius. a, Alveolus; ci, cilium; ep, epiplasm; epr, epiplasmic ridge; mt, microtubule; ps, parasomal sac.

Blastodinium (Soyer, 1970b), Gonyaulax polyedra (Bouck and Sweeney, 1966; Sweeney and Bouck, 1966), species of Gymnodinium (Dragesco and Hollande, 1965; Hausmann, 1973d), Nematodinium armatum (Mornin and Francis, 1967),Noctiluca miliaris (Soyer, 1968, 1969, 1970a,b), Oodinium cyprinodontum (Lom and Lawler, 1973), Oxyrrhis marina (Dodge and Crawford, 1971; Dragesco, 195213; Dragesco and Hollande, 1965; Hausmann, 1973a,d), Peridinium westii (Messer and Ben Shaul, 1969, 1971),Prorocentrmm micans (Dragesco and Hollande, 1965; Sweeney and Bouck, 1966), Strippsiella sweeneyi (Sweeney and Bouck, 1966),Thecadinium kofoidi (Dragesco and Hollande, 1965),Warnovinia pulchra (Greuet, 1969),and Woloczynskia micra (Leadbeater and Dodge, 1966). In the resting stage the trichocyst tip of 0. marina assumes a tubular configuration (tt in Fig. 14a); the material of the shaft shows a striation of 8 nm (tb Fig. 14a). The elongated form is characterized by a cross-striation with a period of 68 nm, which is subdivided into 16- to 17-nm subperiods (Fig. 14d). The smallest structural components of

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the shaft are l-nm filaments. This striated pattern is a general characteristic of ejected trichocysts of dinoflagellates, for example, 0. marina (Fig. 14d) and Gymnodinium fuscum (Fig. 14e). The mechanism of discharge of potassium ferrocyanide-treated trichocysts is a sudden change from one paracrystalline state to another (Fig. 14b and c ) ,like the unfolding and elongating process of the Paramecium trichocyst (see Section IV,A,4). A less common and not yet understood spindle trichocyst is found in Gonyostomum semen (Hall, 1946; Mignot and Hovasse, 1974- 1975). Further investigation will be required before its structure and mechanism of extrusion can be determined. 7. Chemical Composition of Spindle Trichocysts Determination of the isoelectric point (IEP) of spindle trichocysts was made by Jakus (1945) and by Wohlfarth-Bottermann and Schwantes (1952). An IEP of pH 4.1 was found for both resting and ejected trichocysts. The chemical composition of spindle trichocysts has been studied by Pollack and Steers (1973) and by Steers et al. (1969). It was shown that trichocysts, isolated for biochemical experiments, are composed of proteins containing no detectable carbohydrate or nucleic acid moieties. When analyzed by acrylamide disk gel electrophoresis in gels containing sodium dodecyl sulfate, two components are detected. These two forms are estimated to have molecular weights of 17,000 and 36,000, respectively. These investigators suggest the name “trichynin” for this structural protein. The trichocysts of Paramecium have frequently been compared with collagen, because of some structural similarities (Beyersdorfer, 1951; Jakus, 1945; Nemetschek et al., 1953; Wohlfarth-Bottermann and Pfefferkom, 1952). However, amino acid analysis indicates that the Paramecium trichocyst is not collagenous (Steers et al., 1969). Moreover, it is significant that a similar noncollagenous amino acid profile has also been reported for the trichocyst protein of the flagellate Peridinium (Messer and Ben Shaul, 1971). Cytochemical experiments have also added to our understanding of the chemical composition of the Paramecium trichocyst. In contrast to the results from biochemical studies a fine staining for glycoprotein material was found in the periphery of the in situ trichocyst body, that is, the meshlike sheath (see Section IV,A,l) (Esthve, 1974). A similar staining is also found in the compound trichocysts of D . dentata (Hausmann and Mignot, 1975). Further studies should clarify the significance of this sheath.

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FIG.13. Comparison of the stretching mechanism of the Parumecium trichocyst (aJ4) and the stretching (b,l-4)and spreading process (b,5) of the Microthoracidae extrusome (b). From Hausmann and Mignot, 1975.

8 . Membrane Behavior of Trichocyst Vacuoles during and after Trichocyst Discharge in Ciliates For trichocyst discharge to occur the trichocyst membranes must fuse with the plasma membrane (tmand pm in Fig. 15a and e). The first visible step in the ejection process is the formation of an extremely narrow opening of less than 40 nm diameter (Hausmann and Allen, 1976). This opening is smaller than the diameter of the rosette of particles (c type) found on the P face of the adjacent plasma membrane (Figs. 3a and 4a). As ejection proceeds, this opening widens until it attains the diameter of the tubular collar (Fig. 15e) (Hausmann and Allen, 1976; Plattner et al., 1973a).The tubular collar may reinforce and protect the membrane at the distal tip of the trichocyst (Allen and Hausmann, 1976).As the trichocyst passes through the tubular collar, as through a nozzle, it is transformed into an elongated structure with a 55-nm periodicity (Fig. 15a and e). Immediately after expulsion the membrane of the vacuole is again separated from the plasma membrane and released back into the cytoplasm (Fig. 15b and e) (de Haller and ten Heggeler, 1969; Hausmann and Allen, 1976; Pitelka, 1965; Plattner, 1976). This process takes place very rapidly, in FIG. 12. Compound trichocyst of Microthoracidae in electron micrographs. The trichocyst consists of a body (tb) and four tubes (t). After ejection the elongated shaft (d) shows a periodic cross-striation (c). The tubes spread (e-g) and excrete an electron-opaque material (arrows). (a) ~22,000.(b) x 34,OOO. (c) ~54,000.(d) X3500. (e-g) x 9500. (a) From R. K. Peck; (d-g) from Hausmann and Mignot, 1975.

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FIG. 14. Spindle trichocysts of the dinoflagellates 0. marina (a-d) and G .fuscum

(e).(a) Resting stage. (b and c) During expulsion. (d and e) Ejected stage. tb, Trichocyst

body; tt, trichocyst tip. (a and b) x 35,000.(c) x 25,000.(d and e) x 200,000. (a-d) From Hausmann, 1973a.

less than a second. The membrane then vesiculates into small units (Fig. 15c-e), which can no longer be distinguished from vesicles of the same dimensions that normally exist within the cell's cytoplasm. The entire process is completed within 5-10 minutes. By this mechanism of trichocyst discharge the complex pellicular

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FIG.15. Membrane behavior of the trichocyst vacuole in Paramecium. During the discharge the trichocyst membrane and plasma membrane fuse (a). Afterward the trichocyst vacuole is pinched off (b) and broken down into small vesicles (c and d). (e) Schematic representation of this process. am, Alveolar membrane; pm, plasma membrane; tb, trichocyst body; tc, tubular collar; tm, trichocyst membrane; tt, trichocyst tip. (a-d) x 30,000. From Hausmann and Allen, 1976.

system of the ciliate (Allen, 1971) is not disturbed. A similar situation has been reported for another exocytic event known to occur in ciliates: The membranes of defecation vacuoles are not incorporated into the plasmalemma but are pinched off into the cytoplasm after exocytosis (Allen and Wolf, 1974).

9. Origin and Cycle of Spindle Trichocysts In those cells that have been investigated (Ehret and de Haller, 1963; Selman and Jurand, 1970; Yusa, 1963, 1965) the spindle trichocysts of ciliates originate from the endoplasmic reticulum. The,first stage seen is a membrane-limited endoplasmic vesicle enclosing a homogeneous mass (asterisk in Fig. 16a and b) which is itself a condensation of closely packed, linearly oriented fibrous elements (0in Fig. 16a and b; 1in Fig. 18).This paracrystalline core is the first definitive sign of the presumptive trichocyst. The form of the juvenile trichocysts, recognizable as a system of closely packed, multilayered, fibrous sheets, becomes increasingly evident (2 and 3 in Fig. 18).Matu-

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FIG. 16. Early stages of trichocyst development in P. cuudatum. A paracrystalline structure (0)lies embedded in an amorphous matrix (asterisk). (a and b) x 25,000.

ration of the trichocyst includes formation of the cap, elaboration of the characteristic periodic striation of the body, and development of the tubular collar (4 in Fig. 18). Development of the compound trichocysts in P. dubius shows a variation. Obviously the four arms are composed of dictyosomederived globules which consolidate to form ultimately the rodlike tubes of the trichocysts (1-5 in Fig. 17) (Hausmann, 1977a). The flagellate's spindle trichocysts are reported to originate from swollen vesicles which arise in the vicinity of the Golgi apparatus (Bouck and Sweeney, 1966; Leadbeater and Dodge, 1966). The succeeding steps in their development are quite similar to those for ciliate trichocysts. After trichocyst discharge (5 in Fig. 18) the trichocyst membrane remains inside the ciliate (6 in Fig. 18) and is transformed into small vesicles (7 in Fig. 18) which probably return to the endoplasmic reticulum (er in Fig. 18) or are engulfed by autolysosomes (Fig. 18).

FIG. 17. Development of' compound trichocysts. Dictyosoma-derived vesicles fuse with a vacuole (1). The contents of these vesicles are the material for the rodlike tubes of the mature trichocyst (5). 2-4, different developmental stages. From Hausmann,

1977a.

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FIG. 18. Scheme of the spindle trichocyst cycle in Paramecium. Vesicles with a paracrystalline core (l),presumably derived from the endoplasmic reticulum (er), mature to normal-shaped trichocysts (2-4) which then become fixed to the pellicle a t special sites (4). During expulsion the trichocyst membrane and plasmalemma fuse (5);the vacuole is then pinched off (6)and broken down into small vesicles (7) which may then be incorporated into the endoplasmic reticulum or into autophagosomes.

At present the manner in which young trichocysts find their way to their destinations in the complex architecture of the pellicle is not yet clear. Recently, however, Peck (1977a,b) reported that specific proteins in the epiplasm of P . dubius are localized at the trichocyst insertion sites. These proteins can be selectively dissolved and identified electrophoreticall y.

10. Function of Spindle Trichocysts Originally spindle trichocysts were thought to be organelles of defense. For example, Didinium, when attacking Paramecium, is sometimes forced back mechanically by a massive discharge of trichocysts (Mast, 1909).But Didinium, feeding only on Paramecium, can survive and multiply very well. However, some less specialized enemies are probably chased away by trichocyst discharge. A second possibility is that trichocysts are used by Paramecium to adhere to surfaces (Saunders, 1925). But this idea cannot explain the abundance of trichocysts in a single cell (6000 to 8000). Wohlfarth-Bottennann

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(1950,1953) has suggested an osmoregulatory function for the spindle trichocysts ofParumecium in which the trichocysts may operate as ion carriers. However, this concept was not confirmed when more sensitive experimental techniques became available, for example, electron probe x-ray analysis. The function proposed for dinoflagellate trichocysts by Ukeles and Sweeney (1969) seems to be of secondary rather than primary importance. Ejected trichocysts are said to block the mouth orifice of the bivalve mollusc larvae Crussostrea virginicu, which prevents them from taking in food. B.

MUCOCYSTS (INCLUDINGMUCIFEROUS BODIES, KINETOCYSTS, AND CLATHROCYSTS)

1. Resting Mucocysts of Ciliates In ciliates mucocysts are found in the resting stage, like spindle trichocysts, in predictable positions in the well-organized cortical system, for example, in Tetruhymena (mu in Fig. 19)(Allen, 1967; Elliott, 1973; Nilsson, 1976; Pitelka, 1961; Satir et al., 1973; Wunderlich and

FIG.19. Schematic representation of the regular position of the mucocysts (mu) in the pellicular system of T . pyriformis. For a description of the other details see the original article. From Allen, 1967.

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Speth, 1972).The appearance of these organelles is very similar in the different species of ciliates; they are membrane-limited, oval bodies (Fig. 20a) with paracrystalline contents (Fig. 20b-d), and they underlie the plasmalemma (Bohatier, 1970, 1972; Bohatier and Detcheva, 1973; Cheissin and Mosevich, 1962; Dragesco et al., 1965; Foissner and Simonsberger, 1975; Grain, 1968a,b, 1970; Grain and Golinska, 1969; Grasse and Mugard, 1961; Hausmann, 1972c; Kink, 1973; Kovaleva, 1974; de Puytorac, 1964b; de Puytorac and Grain, 1968; de Puytorac and Kattar, 1969; Rieder, 1971; Rodrigues d e Santa Rosa and Didier, 1975; Roque et al., 1965, 1967; Rosati Raffaelli, 1970; Tokuyasu and Scherbaum, 1965; Wessenberg and Antipa, 1968; Yagiu and Shigenaka, 1958a,b; Zebrun et ul., 1967). The organelles at times reveal an elaborate fine structure, particularly after the use of certain fixatives (Fig. 20d) (Hausmann, 1973c; Williams and Luft, 1968). Only rarely are other structures, besides the paracrystalline body, found in mucocysts, for example, tubules and bundles of filaments (Didier and Detcheva, 1974). In a few cases two or more morphologically discernable types of mucocysts have been described for the same cell (Grain, 1970; Kovaleva, 1974; Rodrigues de Santa Rosa, 1974; de Puytorac and Rodrigues de Santa Rosa, 1975). For example, Loxophyllum meleagris possesses normal mucocysts (Fig. 20a, c, and d), as well as conocysts (Hausmann, 1977b; Rodrigues de Santa Rosa, 1974).The significance of this variation has not been explained. The extrusive pigment granules (pigmentocysts)reported for Blepharisma (Inabaet d ,1958; Kennedy, 1965; Rao, 1963),Stentor (Tartar, 1961),Loxodes (Mashansky et al., 1963), and Truchelonema (Kovaleva, 1974; Kovaleva and Raikov, 1972; Raikov and Kovaleva, 1968) represent, in all likelihood, a special type of mucocyst. 2. Ejected Mucocysts of Ciliates Mucocysts have in general a specific form after ejection, as well as during the resting stage (Fig. 21a-c) (Bresslau, 1923; Hausmann, 1972c,d, 1973c,d; Hausmann and Bohatier, 1977; Hayashi, 1974; Kriiger, 1934a; Wohlfarth-Bottennann and Pfefferkorn, 1953a,b), and this is not a mucigenic, amorphous mass as previously reported (Satir et al., 1973). They are composed of filaments connected in a three-dimensional network (Fig. 21d'). This structure has been observed following negative staining (Fig. 21a and d-g), shadow casting (Fig. 21b), and thin-sectioning (Fig. 21c) (Hausmann and Stockem, 1973). In negatively stained specimens different network patterns can be visualized: (1)an unordered network (Fig. 21d and d'), (2) a pattern of

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rhombic structures (Fig. 21e and e’), (3) a lattice of hexagonal elements (Fig. 21f and f‘), and (4) a latticework composed of tetragonal units (Fig. 21g and g’). All these different patterns can be reduced to one three-dimensional structure (Fig. 21d’-g’), since the various patterns depend on the plane of view of the ejected mucocyst network (Hausmann, 1972d). 3 . Chemical Composition of Ciliate Mucocysts The chemical composition of mucocyst material was first studied by Alexander (1968). H e demonstrated that the mucocysts of Tetrahymenu are rich in acidic residues and contain two proteolipids and one protein. Moreover, the presence of acid mucopolysaccharides in mucocysts has been demonstrated b y cytochemical methods (Grain, 1968b). In a more recent article Hayashi (1974) described the influence of pH variations on discharged mucocysts, as well as the effect of different temperatures and changes in ionic strength. Hayashi also investigated the effect of some chemicals (EDTA, EGTA, ATP, GTP, calcium chloride, dithiothreitol, mercaptoethanol, colchicine, and vinblastine), and several enzymes (DNase, RNase, phosphodiesterase, lecithinase, lipase, lysozyme, pronase, a-chymotrypsin, and trypsin) on the secreted organelles. Hayashi’s work shows that isolated, discharged mucocysts consist mainly of proteins which can be solubilized under mild conditions (40°C for 30 minutes at pH 7.0).

4. Membrane Behavior during Mucocyst Discharge in Ciliates In order for the mucocyst contents to be discharged the mucocyst membrane (mum in Fig. 22a) must first fuse with .the plasmalemma (pm in Fig. 22a), an event that can be traced in thin sections (Fig. 22ac). During this process the material within the mucocyst’s vacuole expands and becomes less electron-opaque (Fig. 22c). ~

~

FIG.20. Thin sections of resting mucocysts of L. meleagris (a and d), T. pyriformis (b), and Phacodinium metchnikoffi (c). The paracrystalline bodies (mub) are membrane-bounded (mum) and lie below the plasma membrane (pm). Following the use of special fixatives more details of the periodic structure become visible (d).Conocysts of L. meleagris are present, in the resting stage (e-g) and in negatively stained preparations, as ejected organelles (h-k). This type of extrusome consists of an axial rod (+) enveloped by a conical jacket (e, f, and g). After ejection the jacket seems to be amorphous (h), whereas the rod exhibits a distinctive fine structure (i, k). (a) x50,OOO. (b) x77,OOO.(c) x74,000.(d) ~275,OOO.(e-g) x70,000.(h) x35,000.(iandk) ~53,OOo. (e-k) from Haus(b) From R. D. Allen; ( c ) from P. Didier; (d) from Hausmann, 1973~; mann, 197%.

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FIG. 21. Ejected mucocysts of L. meleagris show definite forms (a) which can b e shown following negative staining (a), shadow casting (b), and thin-sectioning (c). The various network patterns of filamentous structure (d-g) are explained by means of a three-dimensional model (d‘-g’), The form of the pattern depends on the plane in which the mucocyst is viewed [compare (d-g) with d’-g’l. (a) ~ 6 0 0 0 (b . and c) ~40,000.(d-g) x 75,000. (b-g’) From Hausmann, 1972d, 1973c.

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The freeze-fracture technique was employed by Satir et al. (1972, 1973) to reveal details of the process of fusion of the plasma membrane with the mucocyst membrane in Tetrahymena pyriformis. The undischarged mature mucocyst finds its way to a special site on the plasmalemma. A rosette of 15-nm-diameter particles forms within the plasma membrane (Fig. 22d; arrow in Fig. 22g) when the mucocyst reaches a critical distance from it. Adjacent to this site, within the mucocyst membrane, is an annulus of ll-nm particles, whose inner edge comes to encircle the rosette in the plasma membrane (Figs. 22e and 23a). During discharge the cytoplasm between the two matching membrane sites is squeezed out, and the membranes fuse (Fig. 22g). Steps in membrane reorganization can be reconstructed from changes in the appearance of the rosette in the fracture faces. First, a depression within the rosette, the fusion pocket, forms in the plasma membrane (Figs. 22h and 23b). The rosette particles spread at the lip of the pocket as the pocket deepens and enlarges from 60 to 200 nm (Figs. 22i and k,and 23c). The annulus particles of the mucocyst membrane then become visible at the open lip, indicating completed fusion of the P faces of the mucocyst and the plasma membrane (Fig. 22m and n). While the contents of the mucocyst are released, the edges of these faces join so that the unit membrane runs uninterruptedly from the plasma membrane around the lip and into the pocket of the mucocyst (Fig. 23d). What happens to the mucocyst membrane after the discharge of its contents? The hypothesis proposed by Satir (1974a,b, 1975) and Satir et al. (1976)postulates that in Tetrahymena at least the lipid portion of this membrane is incorporated into the plasma membrane. The protein portion may be excluded. Satir and Satir (1974) calculated that the membrane surface area of all the mucocysts of one cell is sufficient to produce the plasma membrane of a daughter individual. Thus it is concluded that mucocysts may be a source of new plasma membrane during cell growth and division. And yet, how the growth of other intimately associated pellicular membranes, for example, the alveoli, is accomplished was not discussed. Experimental work will be required before this hypothesis concerning the fate of mucocyst membranes in Tetrahymena can be confirmed. It is also possible that a similar mode of membrane uptake or reengulfment exists, as is evident for the trichocyst membranes in Paramecium (see Section IV,A,8). The recent micrographs of Nilsson (1976) support this alternative explanation, that is, a breakdown of the mucocyst membrane of Tetrahymena into small vesicles and their subsequent engulfment. In Litonotus duplostriatus, a ciliate whose plasma membrane has a well-developed

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surface coat, the mucocyst membranes do not have such a coat during the secretion process (Hausmann and Mocikat, 1976), and consequently they are not likely to become part of the plasma membrane.

5. Mechanism of Mucocyst Discharge The mechanism of mucocyst secretion is somewhat similar to the ejection of spindle trichocysts. One paracrystalline form gives rise to another paracrystalline form. In both mucocysts and spindle trichocysts threadlike structures are the subunits that make u p a three-dimensional network. The differences are that mucocyst discharge takes several seconds, while spindle trichocyst discharge lasts less than a second; that during discharge the mucocyst increases in all dimensions (length, width, and depth), while spindle trichocysts increase only in length; and that the chemical composition of the mucocyst seems to be less homogeneous than that of the spindle trichocyst. The mechanism of mucocyst secretion is, however, more similar to that of spindle trichocyst ejection than to any other type of extrusomal discharge. Mucocyst secretion results from the stretching and unfolding of a preexisting network of filaments, as does spindle trichocyst ejection. 6. The Origin of Mucocysts in Ciliates The origin of mucocysts has been studied by several investigators (Dragesco et al., 1965; Grain, 196813; Kovaleva and Raikov, 1972; Rieder, 1971; Tokuyasu and Scherbaum, 1965; Wessenberg and Antipa, 1968). Except for Grain (1968b), who proposed an elaborate cycle of mucocyst development in Balantidium, the various workers unaniFIG. 22. Mucocyst secretion seen in thin sections (a-c) and freeze-fracture preparations (d-n). The thin sections ofhrophyllvrn show the fusion (arrows) of the mucocyst membrane (mum) and plasma membrane (pm). The content is lost (c). Freeze-fracture preparations of mucocyst secretion in Tetrahymena have been interpreted in the following way. Prior to discharge (d) the resting mucocyst lies directly below a rosette of particles within the plasma membrane (arrowhead). In the vesicular membrane a matching site, the annulus, is present (e).The P faces of the plasma and mucocyst membranes fuse (f). Note the presence of the annulus at the joined region (arrowhead). In freeze-fracture micrographs the fusion process can be followed best on the P face of the plasma membrane. (g) Rosettes of particles are found before fusion. (h) A depression then appears within the rosette. (i) The depression widens and the particles of the rosette separate at the edge of the pocket. (k) Further separation of the rosette particles takes place, and the pocket deepens. (1 and m) The particles of the annulus become visible. (n) Different pocket depths can sometimes be seen within a single fracture. (a-c) x 60,000. (d) x 96,OOO. (e-n) x 72,000. (a-c) From Hausmann, 1973c; (d-f) from Satir and Satir, 1974; (g-n) from Satir et al., 1973.

FIG.23. Diagram of membrane fusion during mucocyst discharge. (a) A cross section of the plasma membrane (PM), which contains large rosette particles and an underlying mucocyst membrane (Mc) containing rows of smaller annulus particles just before fusion. Wavy lines indicate the fracture plane along which the membrane will split into A and B halves. (b) Fusion begins when a hinge is formed between the surface complex, corresponding to the rosette and the inner edge of the annulus. (c)After the rosette particles spread, evolution of the annulus takes place, and the fusion of the A faces (known now as the P faces) is completed. (d) Reorganization occurs around the fusion lip. The fracture line now passes through the expanding mucocyst content. From Satir et al., 1973.

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mously describe one developmental pathway. A small vesicle is postulated to arise from the endoplasmic reticulum (er in Fig. 24), whose contents are first granular and then crystalloid (1in Fig. 24). The vesicle grows to the normal size of a mucocyst (2 and 3 in Fig. 24) and then finds its way toward its final location in the pellicular system (4 in Fig. 24). After discharge (5in Fig. 24) the empty vacuole remains for a time fused with the plasmalemma (6 in Fig. 24). The fate of the mucocyst membrane is unknown, or at least controversial.

7. Mucocysts of Flagellates Different flagellates are known to have mucocysts (for reviews, see Cachon et al., 1974-1975; Hall, 1946; Hovasse, 1969). In some cases their fine structure is similar to that of the mucocysts of ciliates, but they are usually different. Very often their contents seem to be granular to amorphous, and sometimes honeycomblike or fibrillar. In the chloromonadine Chattonella subsala the mucocysts (mu in Fig. 25a) arise from the Golgi apparatus (Ga in Fig. 25a). After discharge, a large, hollow tube is formed by irregularly connected fila-

FIG. 24. Diagram of the development of mucocysts in Tetruhymenu. Originating from the endoplasmic reticulum (er), vesicles with a paracrystalline core (1)increase in size and assume the normal morphology of mucocysts (2,3, and 4). After discharge (5) the mucocyst membrane remains attached to the plasma membrane for some time (6). Further steps in the development of this membrane are unknown.

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FIG.25. The mucocysts of the chloromonadine C. subsala (mu) originate from the Golgi apparatus (Ga). After the discharge, filaments form a hollow tube (arrow). n, Nucleus. (a) x 20,000. (b) x 6000. From Mignot, 1976.

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FIG.26. Muciferous bodies of euglenoids. (a) Distigrnu proteus. (b) Colacium mucronuturn. ( c )Euglenu stellutu. From Mignot, 1966.

ments (Fig. 25b) (Mignot, 1976). A similar situation is found in the mucocysts of Euglena splendens (Hausmann and Mignot, 1977). In euglenoids, besides mucocysts, muciferous (mucigenic) bodies occur (for reviews, see Dodge, 1973; Leedale, 1967). These bodies, whose structure varies in different species (Fig. 26), are presumed to secrete mucilage continuously. The mucus is probably necessary for the movement of the cell. Special pores in the pellicle (Arnott and Walne, 1967; Mignot, 1965a, 1966, 1967a,b; Schwelitz et al., 1970; Sommer, 1965) allow the continuous secretion of mucus. Some correlation may exist between the presence of muciferous bodies and mucocysts and the palmella stage of flagellates (Hovasse and Mignot, 1975). Very little is known about the chemical nature of the mucocysts and muciferous bodies of flagellates. However, mucin isolated from cultures of Euglena gracilis var. bacillaris has been found to have a carbohydrate content of at least 82%. Chromatographic analysis of acid hydrolyzates of the mucin has shown the presence of glucose, galactose, mannose, xylose, fucose, rhamnose, and small amounts of unidentified slow-moving sugars (Barras and Stone, 1968). Several amino acids have also been detected in the acid hydrolyzates of the mucin, indicating the presence of a relatively small amount of protein in these preparations; whether this is part of the mucin structure is not yet known (Barras and Stone, 1968). A unique kind of extrusome, which may be a mucocyst, has been described in the euglenoid flagellate Entosiphon sulcatum (Mignot, 1963,1967b; Mignot and Hovasse, 1973). A similar type is reported in Zsonema nigricans (Schuster et al., 1968). In the resting stage these organelles are thick-walled tubes (Fig. 27a-c) surrounded by a mem-

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FIG. 27. Extrusomes of the euglenoid flagellate E . sulcatum. In the resting state tubelike structures (a and b) are surrounded by a membrane (m). The ejected stages (d and e) reveal a regular pattern of two kinds of filaments (fi and fi). (a) x 12,500. (b) x 25,000. (c and d) x 60,000. (e) x 75,000. (a, b) From Mignot, 1966; (c-e) from Mignot and Hovasse, 1973.

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brane (m in Fig. 27c), but after discharge the wall becomes thinner (Figs. 27d and 2%). Two sets of filaments make up the hollow tube formed at discharge (fi and f2 in Fig. 27e; Fig. 28b). These two kinds of filaments do not seem to have the same chemical composition, since they show different affinities for stains for polysaccharides; the fi filaments reveal a heavy precipitation, whereas the f2 filaments are devoid of stain. Both are unaffected by enzymic treatment (Mignot and Hovasse, 1973). No explanation has been pregented for these results. The discharge of this type of extrusome is characterized as a sudden unfolding and stretching of a preexisting pattern, similar to the ejection of spindle trichocysts in ciliates (see Section IV,A,4). Expansion occurs in length and width by factors of 3 and 1.5, respectively (Fig. 28a).

I -

FIG.28. Mode of ejection of the extrusome in E . sulcatum. The organelle increases in size, expanding in length by a factor of 3 and in width by a factor of 1.5 (a). During this process ordered filaments become visible (b). (b) After Hovasse and Mignot, 1975.

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8. Kinetocysts of Rhizopods Kinetocysts are particles in the axopods of centrohelidians (Fig. 29ac) (Bardele, 1969, 1971, 197213, 1975, 1976a; Hovasse, 1965c; Troyer and Hauser, 1977), which perform discontinuous, jerky, bidirectional movements within the axopods at a velocity of 1-5 pm per second. A

FIG.29. Kinetocysts of the pseudoheliozoan Clathrulina elegans-(a), and the centrohelidians Acanthocystis rnyriospina (b), and Heterophrys sp. (c). The organelle is bounded by a membrane (km) and consists of a central element enveloped by a ringlike jacket. Characteristically they have a position between the plasma membrane of the filopods (pm) and filopodial microtubules (mt).'(d) In Acanthocystis en'nnceoides the P face of the plasma membrane (PF-pm) displays numerous kinetocyst attachment domains (arrowheads). (a) x 30,000. (b) x 65,000. (d) x 60,000. (a, b) From Bardele, 1972b; (c) from Troyer and Hauser, 1978; (d) from Bardele, 1976a.

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kinetocyst is a complex polar organelle surrounded by a membrane (km in Fig. 29a and c). It contains an electron-opaque bipartite central element enclosed in a jacket of less electron-opaque material (Fig. 29c). The organelles are situated between bundles of microtubules (mt in Fig. 29a and b) and the plasma membrane (pm in Fig. 27b and c) (Bardele, 1972b; Troyer and Hauser, 1978). The dense globules, which give rise to the characteristic bumpy appearance of the filopods of the ameboid stage of Chrysamoeba radians (Hibberd, 1971), apparently represent a type of kinetocyst. The mucilage vesicles in the ameboflagellate Gyromitus limax (Swale and Belcher, 1975) may be a variation of the kinetocyst, too. In the presence of formaldehyde fumes, the centrohelidian kinetocyst is discharged, and negative staining reveals that the jacket has been expelled. The material of the jacket seems to function as a propellant for the central element which is observed lying in front of the burst jacket. Bardele (1976a) characterizes the kinetocyst as a special type of compound motile mucocyst which most likely engages in trapping food. In fact, centrohelidian axopods are sticky and have an immobilizing effect on certain other protozoa. Freeze-fracture studies of these extrusomes, initially performed by Davidson (1974), showed that the attachment site of kinetocysts to the plasmalemma bears an array of particles (Fig. 29d) (Bardele, 1976a; Davidson, 1976) somewhat similar to the rosette found at the mucocyst attachment site in Tetrahymena (see Section IV,B,4). However, the function of this particle array in centrohelidians, called an attachment domain (Bardele, 1976a), was interpreted quite differently from the proposed function of the fusion rosette of mucocysts. Although the attachment domain of the kinetocyst defines the site where the fusion of the organelle’s membrane with the plasma membrane will occur upon an adequate stimulus, the particular particle arrangement is interpreted as a membrane differentiation which may prevent the organelle from being discharged at the wrong time (Bardele, 1976a), rather than to prepare for its expulsion, as proposed for Tetrahymena (Satir et al., 1973). Since it has been claimed that membrane fusion requires areas of relatively high fluidity (Poste and Allison, 1973), a fusion-delaying function of particles seems to be obvious; the attachment domain probably represents a very stable, nonfluid membrane area. 9. Function of Mucocysts In investigating mucocyst function, Bresslau (1921a,b, 1923, 1924) discovered a previously unknown type of behavior in ciliates. Following treatment with several dyes (trypaflavin, neutral red, methylene

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blue, or cresyl blue), ciliates, in particular Colpidium colpodu, secreted a gelatinous capsule. At a later time the protozoan can leave the capsule (Fig. 30a, for Tetrahyrnena), especially after it has been returned to its normal culture medium. Moreover, Bresslau (1924, 1928) found that the capsules were composed of small, secreted, fused rods, Tektinstabchen. He felt that these rods, which in fact were discharged mucocysts, were somehow related to trichocysts, and he concluded that trichocysts are responsible for encystment, a conclusion which was at least partly correct. Later Schneider (1930) systematically examined 92 species of ciliated protozoans for Tektin. He showed the presence of this material in many ciliates which are able to encyst and are known today to have mucocysts . Light and electron microscope studies, performed in a manner similar to the experiments of Bresslau, have now been made with Tetruhymena (Tiedtke, 1976).The method used to trigger the secretion of capsules is to expose the cells to a 0.01% alcian blue solution which is reported to free cells of all mucocysts. Ciliates, at the time they leave the capsule (Fig. 30a), have almost no mucocysts (Fig. 30b). The capsule stains with the same dye that stains discharged mucocysts (Nilsson and Behnke, 1971) and is composed of filamentous material resembling similar material in ejected mucocysts (Fig. 30b and c). The filaments are connected in an irregular manner (Fig. 30d). This experimentally induced capsule is not a cyst wall, sensu stricto, but probably a precursor of the cyst wall. Generally mucocysts are thought to play an important role as organelles, providing the raw material for formation of the cyst wall (Cheissin and Mosevich, 1962; Dragesco et al., 1965; Kawakami and Yagiu, 1963a,b,c, 1964a,b,c; Grass6 and Mugard, 1961; Repak and Pfister, 1967; Rieder, 1971; Roque et ul., 1965; Zebrun et ul., 1967). In this context an observation of some special metazoan cells is interesting. Cortical granules with a paracrystalline fine structure similar to that of mucocysts (Afzelius, 1956) are present in sea urchin eggs. They are composed of polysaccharides as well as proteins (Monn6 and Harde, 1951; Monnk and Slautterback, 1950). These granules are FIG.30. Capsule shedding in Tetrahyrnena. After treatment with 0.01%alcian blue the ciliates form a capsule which they can leave at a later time (a). Then the cells are devoid of mucocysts (b). The capsule is built from discharged mucocysts (c). A higher magnification [compared with the rectangle in (c)] reveals the filamentous nature of the capsule [(d); see also (b)]. (a) x 1100. (b) ~33,000.(c) ~ 3 7 5 0 .(d) ~ 5 0 , 0 0 0 From . Tiedtke, 1976.

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ejected and become part of the fertilization membrane (Lallier, 1977; Schatten and Mazia, 1976; Vacquier, 1976), and in this way they resemble mucocysts which are discharged to become part of the cyst wall. In Didinium nasutum, a ciliate that readily encysts, just before cyst formation special extrusomes appear in the cytoplasm, the clathrocysts (cl in Fig. 31) (Holt and Chapman, 1971; Rieder, 1970). These organelles are most probably highly modified mucocysts. They supply

FIG.31. Clathrocysts (cl) during an early stage of encystment inD. nasutum. x 7900. From Holt and Chapman, 1971.

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the largest portion of material for the multilayered cyst wall which

consists of polysaccharides, proteins, and lipids (Rieder, 1973). Normal mucocysts supply a smaller portion of cyst wall material. Other investigators have proposed different functions for mucocysts (Chapman-Andresen and Nilsson, 1968; Nilsson, 1972,1976). In Tetrahymena mucous material (mucocysts) has been shown, by using alcian blue, to have binding properties similar to those of the ameba mucous coat (Nilsson and Behnke, 1971). In amebas such mucous material is capable of concentrating solutes, proteins, and inorganic cations up to 10 times or more of that in the external medium. Alcian blue binds to the mucous coat of amebas and is taken up by endocytosis (Chapman-Andresen, 1972). In Tetrahymena, the dye-mucus complex also appears to be engulfed by food vacuoles, a finding interpreted to be an indication that the mucus derived from extruded mucocysts may be involved in the feeding process. Other cytological evidence suggests that not only alcian blue but also components of the growth medium are adsorbed by extruded mucocysts and in this way are ingested by food vacuoles (Nilsson, 1976).Thus, in Tetruhymena as in Amoeba,the mucous substances have been interpreted to be implicated in endocytic uptake of nutrients. However, the extruded mucocyst’s ability to adsorb and concentrate material may not be of much physiological significance to the ciliate, since normally the discharged mucocyst, unlike the glycocalyx of the ameba, is completely separated from the cell and probably quickly lost into the surrounding medium. The ingestion of stained and unstained mucocysts may be affected by the experimental conditions. However, mucocysts in several flagellates seem to be involved mechanically in food capture. For example, the dinoflagellate N . miliaris has a tentacle for grasping food. This tentacle is bordered with numerous mucocysts which make it sticky and allow it to hold the food particles (Soyer, 1969, 1970a,b).This function is similar to that of the kinetocysts in centrohelidian axopods (see Section IV,B,8). C. TOXICYSTS(INCLUDING CYRTOCYSTS, PEXICYSTS, AND HAPTOCYSTS)

1. Definition of Toxicysts, Cyrtocysts, Pexicysts, and Haptocysts Toxicyst was the original term for the kind of extrusome in ciliates that is extruded as a tubelike structure and which secretes poisonous material (Kriiger, 1931b, 1934a, 1936).

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Cyrtocysts, described in Didinium (Wessenberg and Antipa, 1968), are basically similar to prototype toxicysts but appear strongly curved. Pexicysts, also found in Didinium (Wessenberg and Antipa, 1968), resemble toxicysts but are said to be organelles for the fixation and fastening of prey. Haptocysts (Bardele and Grell,' 1967) (missile-like bodies; Rudzinska, 1965) (phialocysts; Batisse, 1967) are extrusomes which are nearly always found in suctorians. Probably the only exception to this is the ciliate Cyathodinium which has haptocysts within its endosprits (Paulin and Corliss, 1969). Unlike toxicysts, they have a bottle-shaped structure. They join the suctorian tentacle to the prey. 2. Distribution and Location of Toxicysts in Ciliates Numerous ciliates, which are mainly, but not exclusively, Gymnostomatidae, are reported to have toxicysts, for example, Actinobolina (Holt and Corliss, 1973; Holt et al., 1973; Kriiger, 1936; Moody, 1912; Wenrich, 1929),Acropisthium (Bohatier and Detcheva, 1973),Ancistrocoma (de Puytorac, 1969),Chaenea (Dragesco, 1952a, 1962; FaurBFremiet and Ganier, 1969), Coleps (Kruger, 1936),Didinium (Dragesco, 1952a, 1962; Krtiger, 1936; Rieder, 1968a,b, 1971; Schwartz, 1965; Wessenberg and Antipa, 1968,1970; Yagiu and Shigenaka, 1965),Dileptus (Dragesco, 1952a, 1962; Dragesco and MBtain, 1948; Dragesco et al., 1965; Dumont, 1961; Golinska, 1974; Golinska and Grain, 1969; Grain and Golinska, 1969; Hausmann and Bohatier, 1978; Hayes, 1938; Kink, 1973; Kriiger, 1936; Metzner, 1933; Miller, 1968; Studitsky, 1930; Visscher, 1923), Enchelys (Dragesco, 1962; Kriiger, 1936), Helicoprorodon (de Puytorac and Kattar, 1969), Hemiophrys (Kriiger, 1936), Holophrya (Kriiger, 1936), Homalozoon (Kriiger, 1936), Lacrymaria (Bohatier, 1970, 1972; Dragesco, 1962; Kriiger, 1936), Lagynophrya (Grain, 1970), Legendrea (Kahl, 1926; PBnard, 1914), Litonotus (Bohatier and Njine, 1973; Dragesco, 1952a, 1962; Kruger, 1936), Loxophyllum (Dragesco, 1952a; Fritzsche, 1911; Hausmann, 1972b; Hausmann and Hausmann, 1973; Hausmann and Wohlfarth-Bottermann, 1973; Kriiger, 1931b, 1936; Peschkowsky, 1931; de Puytorac and Rodrigues de Santa Rosa, 1975), Monodinium (Rodrigues de Santa Rosa and Didier, 1975), Prorodon (Dragesco, 1952a; Hausmann and Wohlfarth-Bottermann, 1973; Kriiger, 1934a, 1936; de Puytorac, 1964a; Wohlfarth-Bottermann and Pfefferkom, 1953a,b), Pseudoprorodon (Dragesco, 1952a; Kattar, 1972; Kriiger, 1936),Spathidium (Dragesco, 1952a; Kriiger, 1936; Moody, 1912),and Trachelophyllum (Kriiger, 1936). Toxicysts may be distributed randomly in the cortex of the cil-

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iate, as for example in Prorodon teres (Hausmann and WohlfarthBottermann, 1973), but more commonly they are found in quite specific parts of the cell or in special regions of the cortex. The proboscis is frequently well equipped with toxicysts, for example, in Didinium, Dileptus, Helicoprorodon, Lacrymaria, Lagynophrya, and Litonotus. Legendrea (Fig. 32a) and Actinobolina (Fig. 32b) have more unique locations for their toxicysts. These ciliates have retractable tentacles

-10

a --+

L-

I

i

\

-

C

FIG. 32. The tentacle-bearing ciliates Legendrea (a) and Actinobolina (b). Legendrea is drawn with retracted (1)and extended tentacles (2).A toxicyst is located at the tip of the tentacles ofActinobolina (to); the tentacle has an ordered fine structure (d).a, Alveolus; cv, contractile vacuole; mt, microtubules; n, nucleus; os, mouth; t, tentacle. (a) After PBnard, 1914; (b) after Wenrich, 1929.

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(1and 2 in Fig. 32a). Inside the end of each tentacle, one (Fig. 32b-d) to several toxicysts are situated (Fig. 32a). The protozoans float through the water with extended tentacles and catch prey organisms by using these toxicysts. The tentacles of Actinobolina smalii have an elaborate ultrastructure (Fig. 32d) (Holt and Corliss, 1973;'Holt et al., 1973). The extended tentacle averages 80-100 pm in length (Fig. 32c) and is limited by a unit membrane subtended by alveoli (a in Fig. 32d). Transverse sections through the tentacles display rings of microtubules (mt in Fig. 32d). In longitudinal view these microtubules are seen to penetrate into the endoplasm. Toward the tip the lumen of the rings is occupied by a toxicyst (to Fig. 32c and d). Loxophyllum meleagris shows another peculiarity regarding the position of toxicysts. This ciliate has protuberances (Fig. 33 a-c, arrows) filled with numerous toxicysts (Figs. 33d and e, and 34a) (Hausmann and Hausmann, 1973) of two kinds, a long, light type (tol in Fig. 34a) and a short, dark type (to, in Fig. 34a). It is possible that these two types are comparable to the two kinds of toxicysts inside the noselike proboscis of Didinium, the toxicysts sensu strict0 (to in Fig. 34b) and the pexicysts (p in Fig. 34b) (Wessen-

FIG.33. The protuberances in L. meleagris (arrows) disclose in thin sections numerous toxicysts (d and e). The dotted line in (d) and (e) indicates the plane of section through the protuberance. (a-c) x 125. (d and e) x 6500. From Hausmann and Hausmann, 1973.

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FIG. 34. Schematic comparison of the fine-structural features of the toxicyst protuberances ofLoxophyllurn (a) and the proboscis ofDidiniurn (b). f, Filaments; m, mucocyst; p, pexicyst; to, toxicyst. (b) From Wessenberg and Antipa, 1968.

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berg and Antipa, 1968; Yagiu and Shigenaka, 1965).Surprisingly, the toxicyst-containing warts of Loxophyllum have a fine structure quite similar to that of the proboscis of Didinium (compare Fig. 34a and b), except that the cytostome of Loxophyllum is situated directly opposite the warts, whereas it is the proboscis of Didinium itself that contains the cytostome.

FIG. 35. Schematics of the involvement of toxicysts in the prey-catching process of Didinium (a). The ciliate attacks a Purumeciurn (b) by firing pexicysts (p) and finally toxicysts (t). ci, Cilium; tr, spindle trichocysts. (a) From Wessenberg and Antipa, 1968; (b) after Mast, 1909; (c, d) from Wessenberg and Antipa, 1970.

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3. Function of Toxicysts in Ciliates In an excellent study the function of the toxicysts of Didinium were demonstrated on the electron microscope level (Wessenberg and Antipa, 1970) (Fig. 35).Didinium (Fig. 35a) is known to feed on Parumecium (Mast, 1909) (Fig. 3%). Feeding starts with the attachment of a Didinium to a Paramecium (Fig. 35b). A few milliseconds after contact is made between predator and prey the pexicysts (p in Fig. 35c) are discharged and become attached to the surface of the Puramecium. Just as the discharging toxicysts (t in Fig. 35c) begin to penetrate into the Paramecium, it in turn fires its spindle trichocysts (tr in Fig. 35c). A short while later the toxicysts have all been discharged, probably killing the prey (Fig. 35d). The feeding behavior of Didinium, which is easy to observe in the light microscope, convincingly demonstrates the function of the toxicysts: catching and killing prey. Similar observations, which can also b e demonstrated with microcinematography (Dragesco, 1960; Dragesco and MBtain, 1948; Grell, 1964), have been reported for many other ciliates (for reviews, see Dragesco, 1962; Dogiel, 1%5; Grell, 1973; Hall, 1953; Kudo, 1971; Pitelka, 1963; Sleigh, 1973; Westphal, 1974). Studies on the chemical nature of toxicysts have shown for seven species of ciliated protozoans that they are rich in acid phosphatase (FaurbFremiet, 1962, 1967). In this context the apparent similarities in structure, effect, and mode of function between the toxicysts of protozoa and the nematocysts of cnidaria should be mentioned (Brown, 1973). 4. Resting and Ejected Toxicysts in Ciliates Structurally the resting toxicysts are very complicated organelles which contain fully developed structures inside a capsule which can later be everted (Fig. 36). For example, tubes (arrowheads in Fig. 36b-e) lying within the capsule (c in Fig. 36b, c, and e) of both kinds of toxicysts in Loxophyllurn (all arrows in Fig. 36a) are filled with an ordered material which is probably the poison (0in Fig. 36c and e). Furthermore, an amorphous substance is detectable in the vicinity of the capsule wall (asterisk in Fig. 36b-e). During ejection the shorter type of toxicyst, found in Loxophyllum, everts a tube which is the same length as the capsule (Fig. 37a). Therefore in the resting state one tube with the same length as the capsule itself is housed within the capsule (arrowheads in Fig. 36b and c). The long type ejects a tube whose length is twice that of the

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FIG.36. Resting toxicysts of Loxophyllurn. (a) Between the two types of toxicysts (different arrows) mucocysts (mu) are seen. Higher magnifications of cross sections (b and d) and longitudinal views of negatively stained material (c and e) reveal the different internal structures (0, arrowheads, arrows, asterisks). c, Capsule. (a) x52,OOO. (b-e) x 120,000. From Hausmann and Wohlfarth-Bottermann, 1973.

capsule (Fig. 37b). So, in a cross section of the resting stage of this toxicyst, the one tube is detectable twice (arrowheads in Fig. 36d; arrowheads and arrows in Fig. 36e). The Prorodon toxicyst is even more complex. In its resting form the lumen of its tube, which is twice as long as the capsule, contains another tubule which is the length of the capsule (Fig. 39d).

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FIG.37. Ejected toxicysts ofloxophyllurn.(a)The shorter type secretes what appears to he a sticky material from a tube ( t ) .(h)The longer kind ejects a tube (t) which is twice the length of the capsule (c). (a) x 11,500. (h) x 5500. From Hausmann and WohlfarthBottermann, 1973.

5. Mode of Ejection of Toxicysts in Ciliates Two types of ejection mechanisms are found in toxicysts:

1.Telescopic discharge of a tube, as reported for the pexicysts ofDidinium (Fig. 3 9 4 (Rieder, 1971; Wessenberg and Antipa, 1970). 2. Discharge of a tubule via evagination, as shown for the toxicysts of Loxophyllum (Figs. 38a-c, and 39b and c) (Hausmann and Wohlfarth-Bottennann, 1973).

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The ejection of the toxicysts ofProrodon and Dileptus (long type) is a combination of both mechanisms (Fig. 39d) (Hausmann and Bohatier, 1978; Hausmann and Wohlfarth-Botterman, 1973; Wohlfarth-Bottermann and Pfefferkorn, 1953a,b). During or near the end of toxicyst expulsion a sticky (Fig. 37a) and/or poisonous material is secreted by the tubules (Figs. 38d and e, and 39) (Hausmann and Wohlfarth-Bottermann, 1973). Kriiger (1934a, 1936), using the darkfield microscope, postulated different categories of toxicysts, basing his scheme on the ratio of the length of the different parts of the ejected extrusomes: capsule (l),tubule (2), and Fadenendstuck (end tube) (3). Three classes were described:

1. Capsule/tubule ratio 1: 1 (Fig. 39a and b). Examples are the pexicysts of Didinium and the short toxicysts of Loxophyllum and Di1ep tus. 2. Capsule/tubule ratio 1:2 (Fig. 39c). Examples are the long toxicysts of Loxophyllum (it has been adequately demonstrated that the seemingly bipartite everted tube is indeed one structure: Hausmann and Wohlfarth-Bottermann, 1973). 3. Capsule/tubule/end tube ratio 1:2 : 1 (Fig. 39d). Examples are the toxicysts of Prorodon and Dileptus (long type). Any deviations from these ratios, if they exist, have not been reported. The motive forces for the expulsion of the tubes are still unknown. One can speculate that the material in the vicinity of the capsule wall (asterisk in Fig. 36b-e) can serve such a function. However, since the chemical nature of this material is unknown, assigning a function to it is premature at this time. 6. Origin of Toxicysts All reports on the origin of toxicysts show that they are initially synthesized by the endoplasmic reticulum (Bohatier and Detcheva, 1973; Dragesco et al., 1965; Rieder, 1968a, 1971; Wessenberg and Antipa, 1968). After a process of differentiation and maturation (Fig. 40)

FIG.38. Tube ejection (t) occurs essentially through evagination [arrows and arrowheads in (a-c)]. After the discharge, filamentous and/or amorphous material is secreted (d and e). The toxicyst-containing warts of Lorophyllurn have a brushlike appearance after toxicyst discharge [arrows in (f and g)] (c) x 50,000. (d and e) x 35,000. (f) x 300. (9) x 1500.(c-e) From Hausmann and Wohlfarth-Bottermann,1973.

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FIG.40. Different stages in the development of toxicysts in Litonotus (a and b) and Loxophyllum (c and d). (a, b, and c) x 18,000. (d) x 16,000.

they migrate to their predestined positions in the cortex of the protozoan. A recent study based on cytochemical methods reports D N A in the toxicysts of the ciliate Homalozoon vermiculare (Gautier and Fakan, 1974). If this is true, it would disagree with the principle of compartmentalization of the cell (Schnepf, 1966a,b), which states that active D N A can be located only in the nucleocytoplasmic matrix (Schnepf, FIG. 39. Schematic summary of the four ways in which toxicysts are known to discharge. (a) Telescopic discharge of a tube (t) which is the same length as the capsule (c) (example: Didinium, pexicyst). (b) Evagination of a tubule the same length as the capsule (examples: Loxophyllum and Dileptus, short toxicysts). (c) Evagination of a tube twice as long as the capsule (example: Loxophyllum, long toxicyst). (d) Evagination of a tube twice as long as the capsule combined with a telescopic discharge of a tubule which is the same length as the capsule (examples: Prorodon, Dileptus, long toxicyst).

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1977) and cannot be enclosed in vesicles, particularly those that will be excreted from the cell. 7. Structure and Function of Toxicysts in Flagellates Only one flagellate is known to possess toxicysts, the phagotrophic Colponema loxodes (Mignot and Brugerolle, 1975; Mignot and Hovasse, 1974-1975). The resting toxicysts are spindlelike structures which lie in an oval vacuole below the plasma membrane (arrows in Fig. 41a) and are composed o f a capsule (c in Fig. 41b) surrounding a

FIG.41. The toxicysts of the phagotrophic flagellate C . lorodes lie below the plasma membrane [arrows in (a)]. The main structures of the toxicysts are a tube (t) and a capsule (c). During food capture the toxicysts are discharged and a tube is evaginated [arrow in (c)]. p, Prey. (a) x 14,000. (b) ~80,000. (c) x 27,000. (a) From J.-P. Mignot; (b, c) from Mignot and Brugerolle, 1975.

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tube (t in Fig. 41b). This tube can be discharged, as is seen in a feeding flagellate (arrow in Fig. 41c). Although connections between toxicysts and prey have never been seen (p in Fig. 41c), the involvement of these toxicysts in food catching is obvious.

8. Structure and Function of Haptocysts in Suctorians The knob of many suctorian tentacles is equipped with a highly complex organelle, the haptocyst (missile-like body or phialocyst) (ha in Fig. 42a, b, and d) (Bardele, 1970, 1972a, 1974; Bardele and Grell, 1967; Batisse, 1965,1966,1967,1972; Curry and Butler, 1976; Hauser, 1970; Hauser and van Eys, 1976; Hitchen and Butler, 1974; Jurand and Bomford, 1965; Lom and Kozloff, 1967; Rudzinska, 1965, 1970, 1973; Spoon et al., 1976; Tucker and Mackie, 1975).The knob is covered by the plasma membrane (pm in Fig. 42d). The protoplasmic fracture face of this membrane (PF-pm in Fig. 42c) contains randomly distributed intramembranous particles with an average density of 2300/pm2. Highly ordered membrane domains can be seen where the haptocysts are attached to the plasma membrane (arrowhead in Fig. 42c). A rosette of 12-nm-diameter particles with a large particle in the center is found above every haptocyst. The 60-nm-diameter rosette is surrounded b y an annular area, 40 nm wide and free of particles. According to Bardele (1976b) the rosette and annulus can be regarded as a multifunctional membrane differentiation having both receptor and effector functions, that is, recognition of prey, as well as involvement in rearrangement of the membrane components to allow an orderly discharge of the haptocysts. Bardele (1976b) stresses that the general order and apparent rigidity of particle arrangements found at the attachment sites of various extrusomes (e.g., spindle trichocysts, mucocysts, kinetocysts, and haptocysts) may be important in preventing their untimely discharge (see Section IV,B,8). Haptocysts are discharged on contact with the prey and, by puncturing the pellicle of the prey, give rise to a firm connection between the two cells (arrows in Fig. 42e). As compared with that of undischarged haptocysts (Fig. 42d), the altered structure of discharged organelles (Fig. 42e) indicates that part of their contents has been injected into the prey. The complex structure of the haptocyst, with its several parts, suggests the presence of several enzymes which may be responsible for puncturing the prey’s pellicle, abolishing ciliary motion, and producing local solubilization of the prey’s cytoplasm (Bardele, 1969, 1974; Batisse, 1967; Evans, 1953; Rudzinska, 1973). Moreover, a very important function of the haptocysts is their role in the “fusion” of the

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plasma membranes of predator and prey (Bardele, 1974). The discharge of haptocysts may occur within milliseconds. At this point other organelles, characteristically found in the suctorian’s knob, should also be mentioned-the solenocysts (Hitchen and Butler, 1973) or osmiophilic granules (Bardele and Grell, 1967; Hauser, 1970). It has been suggested that these particles, containing electron-opaque, ordered material (Bardele and Grell, 1967; Batisse, 1968) may represent membrane precursors used to form the vacuole around the newly ingested food. Whether or not these organelles fit into the category of extrusomes is not clear. However, Hitchen and Butler (1973) interpret their electron micrographs as showing a blebbing activity of the solenocysts at the tip of the feeding tentacle of Choanophrya. After discharge the haptocysts must be replaced. Large areas showing numerous forming haptocysts were found in the cytoplasma of the cell (Fig. 42f) (Bardele, 1970). Mature haptocysts move upward through the tentacle, where they find their way to certain regularly distributed anchoring sites in the membrane of the knob, probably in a manner similar to the way other types of extrusomes find their anchoring sites.

D. RHABDOCYSTS In lower marine ciliates, for example, in Trachelonema sulcata and Tracheloraphis dogieli, a special extrusome is found, the rhabdocyst (Raikov, 1971-1972, 1974; Raikov et al., 1975).The internal structure

of a rhabdocyst consists of an apex (a in Fig. 43), a tube (t in Fig. 43), a dark band (db in Fig. 43), and a vesicular basal part (bp in Fig. 43). The organelle is covered by a membrane (m in Fig. 43). The mechanism of ejection resembles to some extent that of toxicysts (Fig. 39a), which involves the telescopic discharge of a tube (2 and 3 in Fig. 43). The process is thought to be initiated by a swelling of the basal vesicle. The pressure thus produced can be transferred to the dark band at the end of the tube which is ejected like an arrow being expelled from a blowpipe (I. B. Raikov, personal communicaFIG.42. Haptocysts ofAcineta (a, d, e, and f ) and Discophrja (b and c). In the knob of the tentacle, haptocysts (ha) underlie the plasma membrane (pm). In freeze-fracture preparations the P face of the plasma membrane (PF-pm) shows rings of particles over each haptocyst (arrowhead). A tentacular necklace [arrows in (c)] composed of tightly packed particles indicates the end of the alveoli. The haptocysts make contact between the prey and the tentacle [arrows in (e)]. Generative areas of haptocysts exist within the cell (f). (a) x 17,500. (b) x 15,000. (c and f) ~ 4 5 , 0 0 0 .(d) ~ 2 6 , 0 0 0 (e) . ~ 2 4 , 0 0 0 .(a, e) From Bardele and Grell, 1967; (c) from C. F. Bardele; (d, f ) from Bardele, 1970.

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L_ _ _ _ _ _ _ _ _ _ _ J

FIG.43. Schematic interpretation of the mode of extrusion of rhabdocysts. A tube (t) with a specially constructed apex (a) and a dark band (db) near the proximal end (1) is ejected (2) by swelling of the proximal part (bp) of the extrusome (3).The organelle is covered by a membrane (m).

tion). The morphology of the tube changes little during the extrusion; it becomes only slightly longer and thinner (3 in Fig. 43). The function of the rhabdocyst is entirely unknown.

E.

EJECTISOMES

(TAENIOBOLOCYSTS)

1. Structure of Resting and Discharged Ejectisomes Ejectisomes are types of extrusomes which occur exclusively in flagellates (for reviews, see Dodge, 1974; Hall, 1946; Hovasse, 1965a, 1969; Hovasse and Mignot, 1975). They are present in most Cryptophyceae (Anderson, 1962; Dodge, 1969; Dragesco, 1951; Hovasse 1965b; Joyon, 1963; Kriiger, 1934b; Mignot, 196513; Mignot et al., 1968,1970; Schuster, 1968,1970; Wehrmeyer, 1970) and in a few Prasinophyceae (Manton, 1969; Norris and Pearson, 1975). Undischarged ejectisomes are situated mainly toward the anterior end of the cell adjacent to the gullet (Fig. 44a and b). In some organisms small ejectisomes are also located around the periphery of the

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FIG. 44. Ejectisomes. In Chilomonas paramecium the ejectisomes are situated adjacent to the gullet (T).The resting ejectisomes are bipartite structures (c) composed of a tape coiled into tight spirals (d). After ejection the extrusome is a needlelike structure with a sharply bent tip [arrows in (e and f)]. The mechanism of discharge is an unrolling of the tape [arrows in (g)]. AM, Amphosome; B, basal body; C, cortical area; CV,contractile vacuole; F, flagellum; N, nucleus; NCL, nucleolus; P, inpocketing of plasma membrane; PB, parabasal body; PM, paramylum bodies; R, rhizoplast; T, trichocysts (ejectisomes); V, vestibulum. (b) X37,OOO. (c) ~ 2 5 , 0 0 0 (d . and f ) x40,OOO. (e) x 6500; (9) X 50,000. (a) From Anderson, 1962; (b-d) from Hovasse et al., 1967; (g) from Mignot et, al., 1970.

cell, adjacent to grooves in the periplast (Dodge, 1969; Mignot, 1965b). The internal structure of ejectisomes consists of two parts: a large cylinder composed of a tapelike structure coiled into a tight spiral (of about 50 turns), which surrounds a narrow, cone-shaped canal

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(Fig. 44c and d), and a second part of similarly coiled material (arrow in Fig. 44c), which is connected to the first structure (Fig. 45a). These extrusomes have a proteinaceous nature (Mignot and Hovasse, 1973). The discharged ejectisome, first investigated by Anderson (1962), is a hollow tube (Figs. 44e and 45c-e) with a sharply bent tip (arrows in Fig. 44e and f; Fig. 45b and e), with the exception that in Prasinophyceae ejectisomes do not have a bipartite structure but are single tubes after discharge. 2. Mode of Function and Regeneration of Ejectisomes The possible mechanisms by which ejectisomes form a tubular structure after discharge have been reviewed by Hovasse et al. (1967). The generally accepted theory of the expulsion of ejectisome is shown in Fig. 45. The coiled extrusome is unrolled (arrow in Fig. 44a; Fig. 45b), and a hollow tube is formed as the tape rolls up laterally (Fig. 45c and d ) (Mignot et al., 1970; Hovasse, 1965a; Hovasse and Mignot, 1975; Hovasse et al., 1967). The development of ejectisomes has been studied by several inves-

FIG. 45. Schematic reconstruction of the discharge of ejectisomes. The resting stage is a coiled ribbon (a) which unrolls during ejection (b) and forms a hollow tube by rolling up laterally ( c and d). This results in a needlelike structure (e).(a, c-e) From Hov a s e et d.,1967; (b) after Hovasse and Mignot, 1975.

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tigators (Anderson, 1962; Schuster, 1970; Wehrmeyer, 1970). It has been shown that vesicles derived from the Golgi apparatus contain an extrusomal tape of only few turns. During further maturation the number of turns increases, and a bipartite structure is then formed. 3. Comparison of Ejectisomes with the R Bodies of the Kappa Particles of Paramecium Ejectisomes are curious and almost unique structures. The closest comparable structures known seem to be the R bodies of the kappa particles (symbiotic bacteria) found in Paramecium aurelia (Hovasse, 1965a; Hovasse et al., 1967; Preer et al., Soldo, 1974).The R body is a ribbon which in its normal compact form is wound into a tight roll consisting of several turns, just like an ejectisome. On heating to 60°C or treating with sodium dodecyl sulfate or phosphotungstate, the rolled ribbon of a stock-1039 R body suddenly and irreversibly unrolls into a long tape. The ribbon of a stock-51 R body unrolls only in response to lowering the pH below 6. In this case the process is reversible and, when the pH is raised above 7, rerolling occurs (Preer et al., 1966). Preer et al. (1974) stated that the difficulty in imagining how convergent evolution could have produced such bizarre and similar structures in such widely different organisms is matched only by the problem of imagining how an apparently bacterial structure can be phylogenetically related to an algal structure.

F. DISCOBOLOCYSTS Discobolocysts are restricted to flagellates, especially those of the order Chrysophyceae. The species known to have this type of extrusome are Chromulina georgesiana (Bourrelly, 1957),Cyclonexis annularis (Hovasse, 1948, 1949), Ochromonas crenata (Conrad, 1926; Klebs, 1893; Kalina, 1964), Ochromonas hovassei (Bourrelly, 1957), and Ochromonas tuberculatus (Hibberd, 1970). In the resting stage the organelles in 0. tuberculatus are almost spherical (Fig. 46b; 4 in Fig. 47). The part containing a disk with approximately 20 radial canals and a central hole (d in Fig. 46b; Fig. 46c and d) protrudes above the general level of the cell surface (di in Fig. 46a). The extrusome, bounded by a unit membrane (arrows in Fig. 46b) is filled with a fibrous, reticulate material (asterisk in Fig. 4%) in the space not occupied by the apical disk. After ejection the disk of the discobolocyst seems to be unaltered in fine structure (d in Fig. 46e), whereas the rest of the organelle is transformed into a long tail (t in Fig. 46e; 5 in Fig. 47). The tail consists of an unordered, fibrous material.

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FIG.46. Discobolocysts of the Chrysophycea Ochromonas tuberculatus. The organelles protrude above the general level of the cell surface (di). The organelles, consisting of a disk (d) and an amorphous portion (asterisk),are enveloped by a membrane (arrows). After discharge the extrusomes show a long tail (t) composed of unordered filaments. ch, Chloroplast; cv, contractile vacuole; Ga, Golgi apparatus; lv, leukosine vesicle; nu, nucleus. (a) x 7500. (b) x 20,000. (c and d ) x 10,000. (e) x 8000. From Hibberd, 1970.

The ontogeny of discobolocysts starts with balloon-like vesicles derived from the Golgi apparatus (1in Fig. 47). These vesicles, filled with reticulate contents (2 in Fig. 47), increase in size to 250-500 nm in diameter (3 in Fig. 47) and then migrate between the plastids (3 in Fig. 47) to the surface of the cell where they develop into typical discobolocysts (4 in Fig. 47). When development is complete, the fully

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FIG.47. Cycle of discobolocysts. Vesicles (l),originating from the Golgi apparatus (Ca) and filled with a reticulate material ( 2 ) ,increase in size to become large vacuoles (3). These migrate between the chloroplasts (ch) to the plasma membrane, where they develop into their typical form (4), ready to be discharged (5).

formed organelles presumably move to positions at the periphery of the cell (4 in Fig. 47), since typical discoboloysts are always seen in the cytoplasm between the plastid and the cell membrane. The function of these organelles is unknown. G. NEMATOCYSTS(CNIDOCYSTS) Nematocysts, extrusomes found in dinoflagellates such as PoZykrikos (Chatton and Grass6, 1929; Faur6-Fremiet, 1913; Hovasse, 1963) and Nematodinium (Hovasse, 1951b; Mornin and Francis, 1967), are basically small capsules filled with a coiled tube (n in Fig. 48a; 1 in Fig. 48b). This tube is furnished at the tip with a stylet and can be discharged by a process of evagination (Fig. 48b) (Chatton, 1914). The organelle may have a defensive function. It was first suggested that these extrusomes are self-duplicating organelles and should consequently have a fairly complicated developmental cycle (Chatton and Hovasse, 1944; Hovasse, 1951a,b). However, recent electron microscope observations do not support this idea (Greuet, 1971,1972; Greuet and Hovasse, 1977).They are formed by a normal developmental process which is complicated only because of the morphological complexity of the mature cnidocyst. OF CNIDOSPORIDIANS H. POLARFILAMENT The polar filament of cnidosporidians is not an extrusome by definition (see Section 1,A). It is mentioned in this article because of its extrusive properties.

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I

'

a

FIG.48. Polykrikos schwartzi with nematocysts (cnidocysts) (n).The coiled tube inside a capsule (1)is ejected by evagination (2 and 3). K, Nucleus; p, parabasal body; t, trichocyst. (a) After Chatton and GrassB, 1929.

Myxosporidians and microsporidians are parasites with complicated life cycles. They infect their host as small ameboid organisms which emerge from ingested spores and multiply within the host. Eventually they differentiate to form characteristic spores which contain ameboid sporoplasm. These spores are provided with a tubelike filament, the polar filament, which can be extruded from the spore by a process of sudden evagination. The extruded tube retains morphological contact

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with the spore, making it possible for the sporoplasm to infect new cells. In microsporidians the motive force for the extrusion of the tube is located in two special organelles, the polaroplast and the posterior vacuole. These parts of the spore are thought to be capable of swelling, which generates a sudden pressure and causes evagination of the tube (for review, see VQvra, 1977). I. VAFUA Other extrusomes have also been reported in protozoa, but they cannot be classified, since they have seldom been seen or have been poorly described, for example, in Discotricha papillifera (Tuffrau, 1954) and Urostyla cristata (Fig. 49) (Jerka-Dziadosz, 1964, 1965, 1967, 1970; Suganuma, 1973; Weinke, 1972). However, there are some very well-known protozoan organelles which may be extruded but for which proof of extrusion is still lacking, for example, in Blepharisrna (Giese, 1973), Petalotricha (Laval, 1971, 1972), and Strombidium (FaurBFremiet and Ganier, 1970). Further detailed studies will be necessary to fill this gap.

V. Conclusions This article has attempted to show the structural and functional diversity of extrusomes in protozoa. Some have a wide distribution in flagellates as well as in ciliates (spindle trichocysts, mucocysts, and toxicysts); others are restricted to small systematic groups, for example, the discobolocysts of the flagellate order Chrysophyceae or the cnidocysts of a few species of dinoflagellates. Surprisingly, some enigmatic similarity exists between extrusomes and organelles of systematically widely differing organisms such as ejectisomes of algae and

FIG.49. This unique extrusome of the ciliate Urostyla cristata cannot be placed in one of the categories of extrusomes, since not enough is known about its method of expulsion. x 26,000. From Suganuma, 1973.

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the R bodies of the bacterial symbionts of Paramecium or the toxicysts of ciliates and the nematocysts of cnidarians. The physiological function of extrusomes is evident only in a few cases, for example, toxicysts are involved in food capture and mucocysts probably in encystment; the significance of most extrusomes is still obscure. Even the function of the well-known spindle trichocyst in Paramecium has not been clarified. Therefore further studies on extrusomes should focus on investigating the physiology of these organelles rather than on cataloging further morphological variations. ACKNOWLEDGMENTS

Without the help of my wife, Dr. E. Hausmann, this article would not have been written. I thank Dr. Richard D. Allen, University of Hawaii at Honolulu, for his help in translating the article and for his critical review of the manuscript. Collaborations and discussions with the following scientists are appreciated (the names of those who kindly provided illustrations are followed by an asterisk): R. D. Allen*, R. Anderer, G. Antipa, C. F. Bardele*, J. Bohatier, J. 0. Corliss, G. Deichgraber, P. Didier*, K. G. Grell, G. de Haller, M. Hauser*, J. Hibberd*, H. Hoffmann-Berling, P. Holt*, R. Hovasse, N. Hiilsmann, Fr. Kriiger, J. Lom, J. -P. Mignot*, D. J. Patterson, R. K. Peck*, D. Pitelka, P. de Puytorac, J. Raikov, B. Satir*, E. Schnepf, Y. Suganuma*, A. Tiedtke*, D. Troyer*, J. Vavra [I gratefully acknowledge Dr. VQvra’skindness in allowing me to see the unpublished manuscript for his article, “The Fine Structure of Microsporidia,” in The Microsporidia (J. V5vra and V. Sprague, Eds.) to be published by Plenum Press, New York], H. Wessenberg*, and K. E. Wohlfarth-Bottermann. I am indebted for technical assistance to P. Batta, B. Koeppen, D. Laupp, K. -L. Medved, A. Riiskens, M. Sauernheimer, and M. Ueno. Financial support was given in part by the Deutsche Forschungsgemeinschaft, BonnBad Godesberg, West Germany. REFERENCES Afzelius, B. A. (1956). Erp. Cell Res. 10,257. Alexander, J. B. (1968). E x p . Cell Res. 49,425. Allen, R. D. (1967).J . Protozool. 14,553. Allen, R. D. (1971).J . Cell Biol. 49, 1. Allen, R. D., and Hausmann, K. (1976).J.Ultrustruct. Res. 54,224. Allen, R. D., and Wolf, R. W. (1974).J.Cell Sci. 14,611. Allison, A. C., and Davies, P. (1974).In “Transport at the Cellular Level” (M. A. Sleigh and D. H. Jennings, eds.), p. 419. Cambridge Univ. Press, London and New York. Allman, G. J. (1855). Q. J. Microsc. Sci. [N.S.] 3, 177. Anderer, R., and Hausmann, K. (1977),J . Ultrastruct. Res. 60,21. Anderson, E. (1962).J . Protozool. 9,380. Arnott, H. J., and Walne, P. L. (1967).Protoplasma 64,330. Bachmann, L., Schmitt, L., and Plattner, H. (1972).Proc. Eur. Congr. Electron Microsc., 244.

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