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Stomatogenesis in cyrtophorid ciliates
European Journal of
Europ.]. Protisto!' 23, 165-184 (1987)
PROTISTOLOGY
Stomatogenesis in Cyrtophorid Ciliates II. ChilodonelJa cyprini (Moroff, 1902): The Kinetofragment as an Anlagen-Complex Angelika H. Hofmann Institut fUr Biologie III, Universitat Tiibingen
SUMMARY Non-dividers of Chilodonella cyprini possess two circum-oral kineties and one preoral kinety. These oral kineties are inverted kineties and consist of dikinetids. The cytostome is accompanied by a large microtubular organelle, the cyrtos. The latter consists of an outer ring of nematodesmata and an inner ring of cytopharyngeal microtubular lamellae. In stomatogenesis five somatic kineties are involved. Each of them produces one kinetofragment. These kinetofragments are anlagen-complexes for the oral kineties and the cyrtos. The somatic monokinetid is the elementary unit of stomatogenesis. In front of almost all somatic kinetosomes of a kinetofragment a new kinetosome is assembled, at an angle of 400 to the kinety axis. The somatic cilium disappears and the somatic mother kinetosome transforms into the non-ciliated kinetosome of the oral kinetosomal pair. The newly synthesized anterior daughter kinetosome bears the oral cilium and becomes the ciliated kinetosome of the oral kinetosomal pair. At the anterior end of each of these oral kineties anlagen there are two to four non-ciliated single somatic kinetosomes. Their triplets are covered with electron-dense materia!' Together with their subkinetal microtubules each of these non-ciliated single somatic kinetosomes forms one nematodesmata anlage. The postciliary microtubules of a part of the non-ciliated kinetosomes of the paired kinetosomes in the oral kineties anlagen and of the non-ciliated single kinetosomes increase in number and align to form one cytopharyngeallamella anlage. Subsequently all components of the anlagen-complexes separate . Seen from outside the cell the five oral kineties anlagen perform an anti-clockwise circular movement of approximately 1800 around the center of the new oral field of the posterior daughter cell, and rearrange to form the circum-oral kineties and the preoral kinety. In the nematodesmata anlagen the nonciliated kinetosome is resorbed. The electron-dense material covering the triplets transforms into the matrix plate of the nematodesma and the subkinetal microtubules increase in number. In the center of the new oral field the nematodesmata anlagen (subkinetal microtubules) and the cytopharyngeal tube lamella anlagen (postciliary microtubules) form the new cyrtos. The anlagen-complexes develop at the anterior ends of the somatic kineties of the posterior daughter cell; the Cyrtophorida show the telokinetal type of stomatogenesis.
Abbreviations A aki cks ct eta ctf ctl ctla cvp cy de df DK DKA
= anterior pole of the
cell = anterior end of the kinety = ciliated kinetosome = cytopharyngeal tube = cytopharyngeal tube anlage = cytopharyngeal tube fragment = cytopharyngeal tube lamella = cytopharyngeal tube lamella anlage = contractile vacuole pore = eytostome = "dent" = division furrow = dorsal kinety = dorsal kinety anlage
© 1988 by Gustav Fischer Verlag, Stuttgart
dks = daughter kinetosome dmt = distal microtubules dmta = distal microtubules anlage dtf = dense transverse fibril em = electron-dense material ex = extrusome fl = fine lamella of electron-dense material ICK = inner circum-oral kinety kd = kinetodesmal fibril = left side of the cell L mks = mother kinetosome mp = matrix plate mtu = membraneous tubules ncks = non-ciliated kinetosome ncr = non-ciliated region nd = nematodesma
nda oc OCK ofa OLK P pcmt PK ps R sc skmt sks st STK tmt
nematodesma anlage oral cilium outer circum-oral kinery oral field anlage outermost left kinety = posterior pole of the cell = postciliary microtubules = preoral kinety = parasomal sac = right side of the cell = somatic cilium = subkinetal microtubules = somatic kinetosome = stalk = stomatogenic kinety = transverse microtubules = = = = =
0932-4739/88/0023-0165$0.00/0
Fig. 1. Chilodonella cyprini - scanning electron micrograph s. - A) Ventral side of a non-dividing cell, the cytostome (cy) (x 2500).B) Dorsal side of a non-di viding cell, the dorsal kinety (DK) (x 2500). - C) The oral field of a non-d ividing cell; it consists of thre e oral kineties (preoral kinety (PK), out er circum-oral kinety (O CK), inner circum-oral kinety (IC K)) and the cyrtos (cytopharyngeal tube (et) and outer circle of nernatodesmata (nd)) . The oral cilia are shorterthan the somatic cilia and more densely packed (x 10 000). D) The oral field anlage of a dividing cell at stage 3 of stomatogenesis (late divider); synchronou s to the ingrowth of the division furrow (df) the oral field anlage (ofa) moves towards the posterior cell pole; in the center of the anlage the new cyrtos is visible (x 3600).
Stomatogenesis in Chilodonella cyprini . 167 Introduction The cyrtophorid ciliate Chilodonella cyprini is well known by ichthyoparasitologists as the organism that causes Chilodonella-disease [28] . The'parasite lives on the skin and gills of freshwater fishes and is often found in breeding ponds of carps and trouts. The morphology of C. cyprini has been examinedon the level of light microscopy by several authors [1, 19,20,24]. According to these studies the ciliate is dorso-ventrally flattened. With the exception of a dorsal kinety, the ciliature is restricted to the ventral side of the cell (Fig. 1 A, B). The ventral ciliature is dividedinto a left and a right ciliary field , separated by an unciliated middle field of the cortex. This character distinguishes the genus Chilodonella from the closely related genus Trithigmostoma, which shows a continuous somatic ciliature [13]. The cytostome of C. cyprini lies in the anterior half of the cell. It is accompanied by a large microtubular organelle (the cyrtos) and three oral kineties, which show the same arrangement as in the genus Trithigmostoma [12 ]. Using the light microscopic techniqueof protargol silver impregnation, Deroux [4,5] has suggested the identity between the stomatogenesis in the genus Chilodonella and the genus Trithigmostoma. This fact can only be proved by a study of the stomatogenesis on the electron microscopic level. Of special importance are the ultrastructure and orientation of the oral kineties in the non-dividing cell and the differentiation of the oral kineties anlagen in the morphogenetic field of an early divider. The ultrastructure of the cyrtos of the Cyrtophorida has been studied by different authors [26,29,30] but there are only a few data about the development of this microtubular organelle during stomatogenesis [22]. In the somatic and oral morphogenesis of the hypotrich ciliate Paraurostyla weissei [14-17] the complex patterns of the somatic and oral cortex (cirri, membranelles) have their origin in simple cortical patterns like monokinetids and dikinetids. The kinetosomes in the cortex of dividing cells are associated with fibrils that cannot be observedin the cortex of non-dividing cells. Hence the characters of stomatogenesis on the electron microscopic level prove to be very valuable for phylogenetic considerations. The Cyrtophorida show the telokinetal type of stomatogenesis, which is, according to Corliss [3], the most primitivetype of stomatogenesis in ciliates. The study of this stomatogenesis on the electron microscopic level should reveal some primitive characters of the ciliate cortex. Rapidly growing populations of C. cyprini are rich in dividing cells and therefore this ciliate is an ideal system for an electron microscopic study to clear some of the open questions about the stomatogenesis of the Cyrtophorida. Material and Methods ,
Cultivation of Cbilodonella cyprini was not possible. Without their host fish the organisms rapidly died. The populations of Chilodonella eyprini examined were isolated from the skin of a heavily infected schlei (Tinea tinea) . For purification the probe
was filtered over a fine gauze (width ofmesh: 50 11m) andwashed in Ean Volvic. The preparations forthelight andelectron microscopic studies were done as described previously [12]. All Figs. are oriented as seen from outside thecell. The triplets of thekinetosomes arenumerated according to de Puytorac [25].
Results
Light microscopy Non-divider A non-dividing cell of Chilodonella cyprini (Fig. 2 A) is 30-60 urn long and 20-40 urn wide. The posterior pole of the cell is marked by an indentation at its left side. The oral ciliature consists of two circum-oral kineties and one preoral kinety, which show a concentricarrangement. The inner circum-oral kinety is the shortest one and lies in front of the cytostome. The longer outer circum-oral kinety is situated in front of the inner circum-oral kinety and reaches the right side of the cytostorne. The right end of the preoral kinety liesin front of the left end of the outer circum-oral kinety. The left end of the preoral kinety is situated in the preoral suture. The left field of the somatic ciliature consists of 11-15 longitudinally arranged kineties. The anterior ends of these left somatic kineties (the two short innermost kineties excluded) face the preoral kinety or the anterior ends of the right somatic kineties. The short outermost left kinety does not extend into the .region of the division furrow. The right field of the somatic ciliature is composed of 10-13 kineties. The five to six innermost right kineties are postoral kineties and their anterior ends face the outer circum-oral kinety. The remaining right kineties bend left in front of the oral field and reach the preoral kinety or the anterior ends of the left somatic kineties. The anterior ends of both the right somatic kineties and the left somatic kineties are arranged to form the preoral suture. The posterior ends of the somatic kineties are arranged to form a postoral suture, close to the indentation of the posterior cell pole. In each field of the somatic ciliature opens the pore of one contractile vacuole.
Stage 1 of stomatogenesis (early divider) An early dividerof Chilodonella cyprini (Fig. 2 B,4 A)is 50-80 urn long and 25-45 urn wide. The morphogenetic field is situated in the left field of the somatic ciliature. The five innermost left kineties segment and develop one kinetofragment each, which are numbered 1 to 5 from right to left. Kinetofragment 1 is the anlage of the preoral kinety and kinetofragment 2 is the anlage of the inner circum-oral kinety. Together the kinetofragments 3, 4 and 5 are the anlage of the outer circum-oral kinety. In Chatton-Lwoff silverline preparations a non-ciliated region is visible in front of the anterior ends of the oral kineties anlagen. The innermost somatic kinety of the left ciliary field is the stomatogenickinery, Anterior to kinetofragment 1, the
168 . A. H. Hofmann
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Fig. 2. Chilodonella cyprini - schematic drawings based on Chatton-Lwoff silverline preparations. - A) Kinetome of the ventral side of a non-dividing cell. The three oral kineties: preoral kinety (PK), outer circum-oral kinety (OCK), inner circum-oral kinety (I CK). The orientation in the cell: anterior pole of the cell (A), posterior pole of the cell (P), right side of the cell (R), left side of the cell (L) .B) Kinetome of the ventral side of a cell at stage 1 of stomatogenesis (early divider); the morphogenetic field consists of five kinetofragments (1-5), segment I' and a non-ciliated region (ncr) in front of the anterior ends of the kinetofragments; the innermost kinety of the left field of the soma tic ciliature is the stoma togenic kinety (STK).
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Fig. 4. Stomatogenesis of binary division, schematic drawings based on Chatton-Lwoff silverline preparations. For numeration of the kinetofragments see Figs. 2 Band 5. - A) Stage 1 of stomatogenesis (early divider); the segmentation of five somatic kineties and the development of five kinetofragrnents, segment l ' and a non-ciliated region in front of the anterior ends of the kinetofragments. - B-D) Stage 2 of stomatogenesis (middle divider); the circular movement of the kinetofragments (and segment 1') around the center of the oral field anlage and their arrangement to the oral kineties of the posterior daughter cell. - E-F ) Srage 3 of stomatogenesis (late divider); the first movement of the oral field anlage towards the right somatic kineties, followed by a second movement towards the posterior pole of the cell; the allometric growth of the anterior ends of the right somatic kineties, synchronous to the ingrowth of the division furrow; the development of segment I' to the innermost somatic kinery of the left ciliary field, to maintain a constant number of left somatic kineties.
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Fig. 3. Chatton-Lwoff silverline preparations of dividing cells. For numeration of the kinetofragments see Fig. 2B and 5 (x 2000). A) Stage 2 of stomatogenesis (middle divider); as a first sign of the circular movement the kinetofragmems 2-5 move closer together and kinetofragment 5 migrates in front of kinetofragment 4. - B) Stage 2 of stomatogenesis (middle divider); the five kinetofragments and segment l ' perform a circular movement around the center of the developing oral field anlage; the left somatic kineties split in an anterior and a posterior half. - C) Stage 3 of stomatogenesis (late divider); the oral field anlage moves towards the right field of the somatic ciliarure; synchronous to the beginning ingrowth of the division furrow, the right somatic kineties split in an anterior and a posterior half. - 0 ) Stage 3 of stomatogenesis (late divider); the oral field anlage moves towards the posterior cell pole, due to the allometric growth of the anter ior ends of the right somatic kineties, synchrono us to the ingrowth of the division furrow.
170 . A. H. Hofmann
stomatogenic kinety develops one segment 1', which is the primordium of the stomatogenic kinety of the posterior daughter cell. Parallel to the outermost right somatic kinety, and in the region of the division furrow, lies the anlage of the short dorsal kinety.
Stage 2 of stomatogenesis (middle divider) In the second stage of stomatogenesis the kinetofragments perform an anti-clockwise circular movement of approximately 1800 around the center of the developing oral field anlage. As they move, the kinetofragments follow two concentric paths and rearrange to form the new oral kineties (Fig. 3 A-B, Fig. 4 B-D, 5): as a first sign of the circular movement the kinetofragments 2-5 move closer together and kinetofragment 5 migrates in front of kinetofragment 4. On the outer circle, kinetofragments 4 and 3 follow kinetofragment 5 and they join to form the outer circum-oral kinety anlage. On the inner circle, kinetofragment 2 moves parallel to kinetofragment 3 and forms the inner circum-oral kinety anlage. Kinetofragment 1 starts last. Its anterior end bends to the left and follows the posterior ends of the kinetofragments 2-3. Finally the
anterior end of kinetofragment 1 reaches the position of the preoral kinety. Segment I' migrates towards the posterior pole of the cell and completes the circle of the moving anlagen. At this stage of stomatogenesis the left somatic kineties split into an anterior and a posterior half, except for the outermost left kinety. This short somatic kinety remains totally in the anterior daughter cell.
Stage 3 of stomatogenesis (late divider) The third stage of stomatogenesis is characterized by a first movement of the oral field anlage towards the right side of the cell, followed by a second movement of the oral field anlage towards the posterior pole of the cell (Fig. 3 C-D, 4E-F). The right somatic kineties split into an anterior and a posterior half. Synchronous to the beginning ingrowth of the division furrow, the anterior ends of the right somatic kineties of the posterior daughter cell increase in length. As the oral field anlage reaches the right ciliary field, the anterior ends of the future postoral kineties face the outer circum-oral kinety anlage. Synchronous to the further ingrowth of the division furrow, the anterior ends of the
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Fig. 5. The number of kinetofragments and their arrangement to the oral kineties (two circum-oral kineties and one preoral kinety) of the posterior daughter cell; schematic drawingbased on Chatton-Lwoff silverline preparations. The kinetofragments and segment I' are shown as arrows; the arrows mark the direction of the movement and (segment I' excluded) also the polarity of the kinetofragments; the circular movement performs with a strict order and sequence; kinetofragment 1 = preoral kinety anlage; kinetofragment 2 = inner circum-oral kinety anlage; kinetofragments 3,4 and 5 = outer circum-oral kinety anlage; segment I' = primordium of the stomatogenic kinety of the posterior daughter cell.
Stornatogenesis in Chilodonella cyprini . 171
Fig. 6. Tangential section of the somatic and oral cortex of a non-dividing cell, showing the som atic monokinetids and the oral dikinetids, and also the structural elements of the cyrtos. The oral dikinetids are arranged in a zigzag-pattern and with an inverted polarity relative to the somatic monokinetids (see Figs. 7, 8 for comparison). Each kinetosomal pair consists of a posterior non-ciliated kinetosome (ncks) bearing postc iliary microtubules (pcmt) and an anterior ciliated kinetosome (cks) bearing a dense transverse fibril
(dtf) (x 24000).
172 . A. H. Hofmann remaining right kineties bend to the left side of the cell. As they meet the anterior ends of the left somatic kineties, the preoral kinety anlage becomes situated in the preoral suture. Due to the allometric growth of the anterior ends of the right somatic kineties, the oral field anlage moves towards the posterior pole of the cell. Segment l' develops into the innermost kinety of the left ciliary field. It becomes the new stomatogenic kinety of the posterior daughter cell and compensates the loss of the short outermost left kinety. Electron microscopy
Non-divider The somatic cortex of the ventral side. The somatic kine ties are separated by steep cytoplasmic ridges, which are covered with flat pellicular alveoli. The somatic kineties consist of monokinetids. At the proximal end of each somatic kinetosome five types of associated fibrils arise, which show the "phyllopharyngid pattern" according to Lynn [23J (Figs. 6, 7, 8 A): a postciliary ribbon of three microtubules at triplet number 9, an anterolaterally oriented kinetodesmal fibril at triplets number 5 and 6, two transverse microtubules in radial position to triplet number 5, a dense transverse fibril at triplet number 3 and also six subkinetal microtubules underneath the kinetosome cylinder. One parasomal sac accompanies the soma-
see Fig .8
tic cilium, situated at its right side. The associated fibrils of the somatic kinetosome show the same arrangement and extension as described for Trithigmostoma steini [12J. The unciliated middle field of the somatic cortex and the unciliated left and right margins of the ventral side are crowded with mucocyst-like extrusomes. The extrusomes are small, membrane-bounded, pyriform bodies, approximately 160 nm in diameter and 240 nm in height (Figs. 10, 11). They are filled with electron-dense material, which does not show the net-like structure that was described for the mucocysts of Tetrahymena [11]. The extrusomes lie freely in the cytoplasm or are attached to the plasmamembrane. They are restricted to the ventral side of the cell.
The oral cortex of the ventral side: the cyrtos. The cytostome of Chilodonella cyprini is accompanied by a large microtubular organelle, the cyrtos, The cyrtos consists of the cytopharyngeal tube and an outer ring of nematodesmata (Figs. 1 C, 6, 7). The cytopharyngeal tube is composed of 34-37 radially arranged cytopharyngeal tube lamellae. Each lamella is a single-layered ribbon of 12-15 microtubules. The microtubules extend into the cytoplasm, at an angle of 90° to the cell surface. The distal end of each cytopharyngeal tube lamella is accompanied by a single-layered ribbon of three to four microtubules. These distal microtubules extend towards the nematodesmata, parallel to the cell surface.
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Fig. 7. The fine structure of the oral field, schematic drawingbasedon ultrathin sections. The three oral kinetics (preoral kinety (PK), outer circum-oral kinety (OCK), inner circum-oral kinety (ICK)) show an inverted polarity relative to the somatic kineties. Their anterior ends (aki) point to the posterior pole of the cell (P) or to its right side (R), their posterior ends (pki) point to the left side of the cell (L). The cyrtos consists of an cytopharyngeal tube (et) and an outer circle of nematodesmara (nd). The distal end of each cytopharyngeal tube lamella (etl) is accompanied by a ribbon of distal microtubules (dmt).
Stomatogenesis in Chilodonella cyprini . 173
An outer ring of 14-16 nematodesmata surrounds the cytopharyngeal tube. Each nematodesma is a rigid bundle of microtubules, which show a hexagonal arrangement. At their distal ends, the nematoclesmal microtubules are covered with a matrix plate of electron-dense material. Connected to the matrix plate is a stalk, which bears two "dents". The stalk and the "dents" consist of the same electron-dense material as the matrix-plate. The phagoplasm of the cyrtos contains a lot of membraneous tubules, which often show a hexagonal arrangement. They correspond to the "tubules complexes" described by Pyne and Tuffrau [26] for Chilodonella un-
situated on the outer side of the kinety, not facing the cytostome. The posterior kinetosome of the kinetosomal pair is non-ciliated and a ribbon of three to four postciliary microtubules arises at triplet number 9. The non-ciliated kinetosome is situated on the inner side of the kinety, facing the cytostome. It stands deeper in the cytoplasm than its ciliated partner. The associated fibrils of the oral kinetosomal pair show the same arrangement and extension, as described for Trithigmostoma steini [12]. At the proximal end of the kinetosome cylinder, electron-dense material connects the kinetosomes of each pair and the kinetosomal pairs of each oral kinety. Two parasomal sacs accompany the oral cilium. The first one is situated on the outer side of the kinety, posterior to the dense transverse fibril. The second one lies on the inner side of the kinety, between the non-ciliated kinetosomes.
cinata. The oral cortex of the ventral side: the oral kineties. Two circum-oral kineties and one preoral kinety are situated at the anterior and right margin of the oral field. The oral kineties consist of single rows of cilia. The oral cilia are shorter than the somatic cilia and more densely packed (Figs. 1 A, 1 C). The circum-oral kineties and the preoral kinety show the same ultrastructure. Their orientation is inverted, relative to the orientation of the somatic kineties. Their anterior ends point to the posterior pole of the cell or to its right side. Their posterior ends point to the left side of the cell (Figs. 6, 7). The oral kineties consist of dikinetids (Figs. 6, 7, 8 B). The kinetosomal pairs are arranged in a zigzag-pattern, with the axis of each pair and the kinety axis forming an included angle of 45°. The anterior kinetosome of the oral kinetosomal pair bears the oral cilium and a dense transverse fibril at triplet number 3. The ciliated kinetosome is
Stage 1 of stomatogenesis (early divider) The kinetofragment as an anlagen-complex. In ChattonLwoff silverline preparations the morphogenetic field of an early divider is characterized by the kinetofragments 1-5 (oral kineties anlagen), segment l ' and also a nonciliated region in front of the anterior ends of the kinetofragments (Fig. 2 B). In ultrathin sections of the morphogenetic field the kinetofragments extend into the non-ciliated region. They are anlagen-complexes for the dikinetids of the oral kineties and also for the nematodesmata, cytopharyngeal tube lamellae and distal microtubules of the cyrtos. The somatic monokinetid is revealed to be the elementary unit of stomatogenesis (Figs. 12, 15 A).
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Fig. 8. The fine structure of somatic kineties and oral kineties; schematic drawings based on ultrathin sections; for orientation see frame Fig. 7. - A) Somatic monokinetids. - B) Oral dikinetids; the kinetosomes and their associated fibrils show an inverted polarity relative to the somatic monokinetids (inverse orientation of the anterior ends (aki) and posterior ends of the kineties (pki)).
174 . A. H. Hofmann
Fig. 9. Stage 1 of stomatogenesis (early divider); from somatic monokinetids to oral dikinetids; tangential section of the developing oral kineties anlagen 1-5. In front of those somatic kinetosomes (sks) of the kinetofragments which belong to the oral kineties anlagen a new anterior daughter kinetosome (dks) is synthesized, at an angle of 40° to the kinety axis. The somatic cilium (sc) disappears and the somatic mother kinetosome (mks) transforms into the non-ciliated kinetosome (ncks) of the oral kinetosomal pair. The anterior daughter kinetosome (dks) develops a new oral cilium (oc) and becomes the ciliated kinetosome (cks) of the oral kinetosomal pair. The oral dikinetids anlagen show the same polarity as the somatic monokinetids did before (x 30000).
Stomatogenesis in Chilodonella cyprini
175
Fig. 10. Stage 1 of stomatogenesis (early divider); from somatic monokinetids to nematodesmata anlagen (nda); tangential serial sections of the non-ciliated region in front of the anterior ends of the oral kineties anlagen 2-5; section B proceeds deeper in the cytoplasm than section A. At those somatic kinetosomes of the kinetofragments which belong not to the oral kineties anlagen the somatic cilium disappears and also the associated fibrils (subkinetal microtubules and in some cases postciliary microtubules excluded). Their triplets are covered with electron-dense material. Together with its subkinetal microtubules (skmt) each of these nonciliated single somatic kinetosomes forms one nematodesma anlage (arrow) (x 30000).
176 . A. H. Hofmann
Fig. 11. Stage 1 of stomatogenesis (early divider); from somatic postciliary microtubules (pemt) to cytopharyngeallamellae anlagen (etIa) and distal microtubules anlagen (dmta) ; tangential serial sections of the oral kineties anlagen 2 and 3; from A to C the sections proceed deeper in the cytoplasm (x 30000). - A) Two groups of microtubules associated with an oral cilium (arrow). - B) A loose ribbon of microtubules associated with an oral cilium (arrow). - C) The inner group of three to four postcilliary microtubules attached to the non-ciliated kinetosome of an oral kinetosomal pair (arrow).
Stornatogenesi s in Chilodonella cyprini . 177
In the posterior parts of the kinetofragments (visible in the light microscope after Chatton-Lwoff silver impregnation) , each somatic kinetosome develops an anterior daughter kinetosome, at an angle of 40° to the kinety axis. At the somatic mother kinetosome, the somatic cilium and also the transverse microtubules, dense trans verse fibril and kinetodesmal fibril disappear. The postciliary microtubules and the subkinetal micro tubules are still present. The somatic kinetosome transforms into the non-ciliated kinetosome of the oral kineto somal pair. The anterior daughter kinetosome develops a new oral cilium and a dense transverse fibril and becomes the ciliated kinetosome of the oral kinetosomal pair. Each oral cilium is accompanied by two parasomal sacs. The oral dikinetids anlagen show the same polarity as the somatic monokinetids did before. At stage 1 of stomatogenesis the oral kinety axis shows the same orientation as the somatic kinety axis (Fig. 9). In the anterior part of each kinetofragment (visible in the light microscope as a non-ciliated region after Chat ton-Lwoff silver impregnation), two to four somatic monokinetids transform into nematodesmata anlagen. The somatic cilium disappears and also the associated fibrils (subkinetal microtubules and in some cases postciliary microtubules excluded). The somatic kinetosome detaches from the epiplasm and the kinetosomal triplets are covered with electron-dense material. Together with its subkinetal microtubules each of these non-ciliated single somatic kinetosomes forms one nematodesma anlage (Fig. 10). The cytopharyngeal tube lamellae anlagen and the distal microtubules anlagen have their origin in the postciliary microtubules of a part of the somatic kinetosomes of the oral dikinetids anlagen and of the nematodesmata anlagen. The somatic postciliodesma disappears. The somatic postciliary microtubules increase in number from three up to five or six and form a loose microtubular ribbon. The ribbon divides into two groups of microtubules. In the oral dikinetids anlagen the inner group of three to four microtubules, facing the kinetosome, corresponds to the oral postciliary microtubules. In the nematodesmata anlagen the inner group of microtubules disappears. The remaining two to three microtubules of the outer group increase in number and rearrange to form one cytopharyngeal lamella anlage and one distal microtubules anlage (Fig. 11). In a non-dividing cell only a few extrusomes are situated in the ciliary fields of the ventral side. Synchronous to the development of the morphogenetic field, extrusomes accumulate beside the anterior ends of the anlagen -complexes (Figs. 10, 11).
anlagen of the cyrtos separate from the anlagen of the oral kineties. The separated anlagen perform the morphogenetic movement and rearrange into the new oral field of the posterior daughter cell. In each of the five kinetofragments the anlagen of the cytopharyngeal tube lamellae and of the distal microtubules detach from their mother kineto somes. They arrange to form five cytopharyngeal tube fragments , near the anterior ends of the oral kineties anlagen (Figs. 13 B, 15 B). In the following these five cytopharyngeal tube fragments migrate towards the center of the developing oral field anlage. As they move they join to close the circle of the cytopharyngeal tube anlage (Figs. 14, 15 C-E). At the anterior ends of the kinetofragments, the nematodesmata anlagen quit their places in the anlagencomplexes and move towards the center of the oral field anlage. Synchronous to this movement, the non-ciliated kinetosome of the nematodesma anlage disappears. The electron-dense material, which covers the kinetosomal triplets transforms into the matrix plate of the nematodesma. The subkinetal microtubules of the anlage increase in number and become the nematodesmal microtubules (Figs. 13 A, 15 B-D). Around the cytopharyngeal tube anlage the nematodesmata anlagen arrange to form the outer circle of the cyrtos . The free ends of the nematodesmal microtubules point into the cytoplasm , the matri x plates point towards the plasmamembrane (Figs. 14, 15 E-F). As a first sign of their circular movement the oral kineties anlagen 2-5 move closer together and oral kinety anlage 5 migrates in front of oral kinety anlage 4. As a result of this movement, a wide gap is formed between oral kinety anlage 1 and 2, to generate the space where the cyrtos anlage is assembled (Figs. 15 B-D). The oral kineties anlagen 1-5 perform their circular movement around the developing cyrto s anlage and rearrange as described (Fig. 5) to form two circum-oral kineties and one preoral kinety. Synchronous to the beginning movement of the oral kineties anlagen the subkinetal microtubul es, which still mark the somatic origin of the nonciliated kinetosomes, disappear and electron-dense material connects the kinetosomes of an oral kinety (Figs. 15 E-F). With the rearrangement of the oral kineties anlagen the assemblage of the oral field anlage of the posterior daughter cell is completed and the axis of the oral kineties shows an inverted orientation. At this stage of stomatogenesis the extrusomes of the morphogenetic field concentrate in the center of the developing oral field anlage (Figs. 13, 14).
Stage 3 of stomatogenesis (late divider) Stage 2 of stomatogenesis (middle divider) The disintegration of the anlagen-complexes and the assemblage of the oral field anlage. In Chatton-Lwoff silverline preparations the second stage of stomatogenesis is characterized by the circular movement of the kinetofragments and their rearrangement into the oral kineties of the new oral field. In ultrathin sections the morphogenetic field of a middle divider is characterized by the disintegration of the totally differentiated anlagen-complexes. The
The oral field anlage of a late divider (Fig. 1 D) undergoes a first movement towards the right side of the cell and a second movement towards the poster ior pole of the cell. Synchronous to this process, the cyrtos anlage becomes totally differentiated. The nematodesmal microtubules and also the microtubules of the cytopharyngeallamellae increase in length and in number. At the matrix plates of the nematodesmata anlagen the stalk and the "dents" develop. In the phagoplasm of the cyrtos anlage the mem-
178 . A. H. Hofmann
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, pki Fig. 12. The ultrastructure of the morphogenetic field of a dividing cell at stage 1 of stomatogenesis (early divider, see Fig. 2 B for comparison); schematic drawing based on serial sections. The kinetofragments 1-5 are anlagen-complexes for the dikinetids of the oral kineties and for the nematodesmata, cytopharyngeallamellae and distal microtubules of the cyrtos. The somatic monokinetid is the elementary unit of stomatogenesis: in the posterior parts of the kinetofragments (visible in the light microscope after ChattonLwoff silver impregnation) the somatic kinetosomes (sks) transform into the non-ciliated kinetosomes (neks) of the oral kinetosomal pairs and the anterior daughter kinetosomes become the ciliated kinetosomes (eks) of the oral kinetosomal pairs; in the anterior parts of the kinetofragments (visible in the light microscope as a non-ciliated region after Chatton-Lwoff silver impregnation) the somatic monokinetids transform into the nematodesmata anlagen (nda); the anlagen of the cytopharyngeallamellae (et/a) and of the distal microtubules (drnta) have their origin in the postciliary microtubules (pernt) of a part of the somatic kinetosomes of the oral dikinetids anlagen and nematodesmata anlagen.
Stomatogenesis in Chilodonella cyprini . 179
Fig. 13. Stage 2 of stornatogenesis (middle divider ); the disintegr ation of the anlagen-complexes; tangential serial sections of the or al kineries anlagen 1-3; section B proceeds deeper in the cytoplasm than section A (x 30000). - A) The nernat odesmata anlagen (nda) have quitted the anlagen-complex ; the non-ciliated kinetosome of the anlage is resorbed; the dense mat erial covering the triplets transforms into the matrix plate (mp) of the nematodesma; the subkinetal microtubules (skmt) increase in number and become the nernatodesmal microtubules. - B) The anlagen of the cytopharyngeal cube lamellae (etla) and of the distal microtubules (dmta) are det ached from their mother kinerosornes and are arranged to a cytoph ar yngeal tube fragment (etf) .
180 . A. H. Hofmann
Fig. 14. Stage 2 of stomatogenesis (middle divider); the assemblage of the new cyrtos and the new oral kineties of the posterior daughter cell; tangential section of the developing oral field anlage. The cytopharyngeal tube fragments have arranged to the cytopharyngeal tube anlage (eta), surrounded by an outer circle of nernatodesmata anlagen (nda) (x 30000).
Fig. 15. The development of the oral field anlage of the posterior daughter cell at stage 1 (A) and stage 2 (B-F) of stomatogenesis; ~ schematic drawings based on ultrathin sections; for the circular movement of the oral kineties anlagen, see Figs. 4 and 5 for comparison. - A) The totally differentiated morphogenetic field of an early divider: The kinetofragments 1-5 are anlagen-complexes for the dikinetids of the oral kineties and for the nematodesmata, cytopharyngeal lamellae and distal microtubules of the cyrtos; segment I' is the primordium of the stomatogenic kinety (innermost kinety of the left ciliary field) of the posterior daughter cell. B) The disintegration of the anlagen-complexes in the morphogenetic field of a middle divider: The anlagen of the cytopharyngeal tube lamellae and of the distal microtubules lose the contact to their mother kinetosomes and arrange to cytopharyngeal tube fragments (ctf); the nematodesmara anlagen (nda) quit their places at the anterior ends of the kinetofragments and move towards the center of the developing oral field; the oral kinety anlage 5 starts its circular movement; segment L' detaches from the stomatogenic kinety, - C) The cytopharyngeal tube fragments (ctf) quit their positions at the right side of the oral kineries anlagen and move
Stomatogenesis in Chilodonella eyprini
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Fig. 15 (continued from page 180) towards the center of the developing oral field; the somatic kinetosome of the nematodesma anlage (nda) disappears, its subkinetal microtubules increase in length and in number and become the nematodesmal microtubules, the electron-dense material of the anlage transforms into the matrix plate; the oral kinety anlage 4 follows the oral kinety anlage 5. - 0) The assemblage of the oralkineties and the eyrtos: The cytopharyngeal tube fragments close the circle of the cytopharyngeal tube anlage (eta); the oral kinety anlage 3 follows the oral kineties anlagen 4 and 5; segment l' increases the number of its somatic monokinetids and starts its movement towards the posterior cell pole. - E) The nematodesmata anlagen (nda) close the outer circle of the cyrtos; the free ends of the nematodesmal microtubules point into the cytoplasm, the matrix plates point towards the plasmamembrane; the oral kinery anlage 2 runs parallel to the oral kinety anlage 3. - F) The totallyassembled oralfield anlage of the posterior daughter cell with the new oral kineties and the new cyrtos.
182 . A. H. Hofmann
braneous tubules appear. The extrusomes, which have been situated at the distal end of the cytopharyngeal tube, are now located at its proximal end.
Discussion The stomatogenesis of Trithigmostoma steini [12] and Chilodonella cyprini show the same basic principles. Both ciliates possess a stornatogenic kinety, which gives rise to the anlage of the preoral kinety and to one segment 1'. Segment I' is the primordium of the stomatogenic kinety of the posterior daughter cell and compensates the loss of the outermost left kinety, According to Deroux [5], this mechanism to maintain a constant number of left somatic kineties is the synapomorphic character for the order Cyrtophorida. In both species the oral kineties anlagen show the same arrangement in the morphogenetic field of an early divider and undergo the same rearrangement in stage 2 of stomatogenesis to form the circum-oral kineties and the preoral kinety, The outer circum-oral kinety consists of three oral kineties anlagen, which join "head to tail" during the morphogenetic movement. A similar process can be observed in the stomatogenesis of haptorid ciliates like Spathidium, Homalozoon and Bryophyllum [8]. A single circum-oral kinety surrounds the cytostome, which is situated at the anterior pole of the cell. The oral kineties anlagen develop at the anterior ends of the somatic kineties of the posterior daughter cell. They bend to the right and join "head to tail" to form the circum-oral kinety of these genera. The oral kineties of C. cyprini show the same ultrastructure as the oral kineties of T. steini. The orientation of the circum-oral kineties and of the preoral kinety is inverted, for the same reasons as discussed for T. steini [12]. The differentiation process of the oral kineties anlagen in the morphogenetic field of an early divider is almost identical. In both species the oral dikinetids originate in somatic monokinetids. The somatic kinetosome transforms into the non-ciliated kinetosome of the oral kinetosomal pair. A new daughter kinetosome is synthesized in front of the somat .rl--",~ 1,; ~'?tosome, at an angle of 40° to the kir.e r
kineties anlagen, described for T. steini and for C. cyprini, is a common character in the order Cyrtophorida and Chonotrichida. The results of this paper show, that the kinetofragments are anlagen-complexes for the dikinetids of the oral kineties and for the nematodesmata, cytopharyngeal lamellae and distal microtubules of the cyrtos. The nematodesmata anlagen are located at the anterior ends of the kinetofragments, visible in Chatton-Lwoff silverline preparations as a non-ciliated region. Hence the kinetofragments develop at the anterior ends of the somatic kineties of the posterior daughter cell and the Cyrtophorida show the telokinetal type of stomatogenesis. According to Deroux [5, 6] the Cyrtophorida undergo the same parakinetal type of stomatogenesis, as described for Nassulopsis [7]. In protargol silver impregnations the nematodesmata anlagen are visible as single kinetosornes, Deroux interpreted these single kinetosomes as the remainders of a somatic ciliature in front of the anterior ends of the kinetofragments, He suggested a homology between these single kinetosomes and the segments of somatic kineties that are situated between the hypostomial frange anlage and the region of the division furrow in a dividing cell of Nassulopsis. According to Deroux the kinetofragments of Trithigmostoma and Chilodonella correspond to the elements of the hypostomial frange anlage of Nassulopsis, which is interpreted as the most primitive member of the Hypostomata. This concept needs confirmation by an electron microscopic study of Nassulopsis and its stomatogenesis. The nematodesmata of the cyrtos originate in somatic monokinetids, At stage 1 of stomatogenesis, the nematodesrna anlage consists of a non-ciliated somatic kinetosome, its triplets covered with electron-dense material and a bundle of subkinetal microtubules. In C. cyprini the kinetosome of the anlage disappears at stage 2 of stornatogenesis, whereas in Brooklynella hostilis [22] the kinetosome of the anlage is still present at the distal end of the nematodesma of a non-dividing cell, associated with the electron-densematerial of the "dents". The subkinetal microtubules of the nematodesrna anlage transform into the nematodesmal microtubules. This fact proves the homology between nematodesmal microtubules and subkinetal microtubules, suggested by Raikov, GerassimovaMarvejeva and de Puytorac [27]. In Spathidium, Bryophyllum and Homalozoon [2,21] the circum-oral kinety also consists of dikinetids. The kinetosome of the oral kinetosomal pair facing the cytostome is non-ciliated and associated with nematodesmal microtubules and a microtubular ribbon, which is interpreted a~ .~'lnsverse microtubules. The kinetosome of the oral kinetosomal pair not facing the cytostome bears the oral cilium. The somatic kineties of haptorid ciliates consist of monokinetids [2]. The oral kineties anlagen differentiate at the anterior ends of the somatic kineties of the future posterior daughter cell. As shown by protargol silver impregnation, the oral kineties anlagen of Homalozoon consist of kinetosomal pairs and are associated with oral cilia and nematodesmal microtubules, before the rearrangement of the oral kineties anlagen to the circum-oral
Stomatogenesis in Chilodonella cyprini . 183
kinety starts [8]. The examination of the differentiation modus of the oral kineties anlagen on electron microscopic level would be of great interest. On condition that the oral dikinetid anlage of haptorid ciliates develops in the same way as described for T. steini and C. cyprini, the following predictions can be made: The somatic kinetosome transforms into the non-ciliated kinetosome of the oral kinetosomal pair and becomes associated with nematodesmal microtubules and a ribbon of postciliary microtubules. An anterior daughter kinetosome is synthesized, which develops into the ciliated kinetosome of the oral kinetosomal pair. In the oral field of a non-dividing cell the non-ciliated kinetosome faces the apically situated cytostome. To obtain this orientation of the oral dikinetid, the kinetosomal pair axis has to make a turn of 90° towards the left, that is, towards the cytostome, during the rearrangement of the oral kinetids anlagen to form the circum-oral kinety. This would mean that postciliary microtubules, instead of transverse microtubules, contribute to the oral apparatus of haptorid ciliates. According to ]erka-Dziadosz and Golinska [18] complex cortical patterns have their origin always in simple cortical patterns. The way of differentiation leads from monokinetids to dikinetids to polykinetids. In T. steini and C. cyprini the somatic monokinetid is the elementary unit of the telokinetal type of stomatogenesis. The somatic kinetosome and its associated fibrils are the origin of the complex structures of the oral field of these ciliates. The somatic monokinetid transforms into the oral dikinetid. The postciliary microtubules give rise to the cytopharyngeallamellae and to the distal microtubules of the cyrtos. The subkinetal (nematodes mal) microtubules become the nematodesmata of the cyrtos. According to the biogenetic law of Haeckel [10], the ontogenesis of an organism reflects the events of its phylogenesis. If this statement is true for the stomatogenesis of ciliates, then the monokinetid turns out to be more primitive than the dikinetid. A kinetosome associated with the basic set of fibrils typical for ciliates (kinetodesmal fibril, postciliary microtubules, transverse microtubules, nematodesmal microtubules) has the morphogenetic potency to develop all the complex patterns observed in the somatic and oral cortex of ciliates. The future study of stomatogenesis in ciliates on the electron microscopic level should reveal the evolutionary pathways of these organisms and also some basic modes of pattern formation in the cortex of these cells.
3
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Acknowledgements 18 I wish to thank Dr. C. F. Bardele for his support during my work and the many discussions we have had, and also H. Schoppmann for the technical assistance with scanning electron microscopy.
References
19
20 21
1 Andre E. (1912): Les chilodontes parasites des cyprinides. Rev. Siusse Zool., 20, 207-212. 2 Bohatier j., Iftode F., Didier P. and Fryd-Versavel G. (1978): Sur l'ultrastructure des genres Spathidium and Bryophyllum,
cilies Kinetophragmophora (de Puytorac et al., 1974). Protistologica, 14, 189-200. Corliss ]. O. (1973): Evolutionary trends in patterns of stomatogenesis in the ciliated protozoa. ]. Protozool., 20, 506. Deroux G. (1972): Identite de la stomatogenese entre les types ciliaires "cucullulus" et "uncinata" du g. Chilodonella. J. Protozool., 19, 64. Deroux G. (1976a): Le plan cortical des Cyrtophorida, unite d'expression et marges de variabilite, I. Le cas des Plesiotrichopidae, fam. nov., dans la nouvelle systematique. Protistologica, 12,469-481. Deroux G. (1976b): Le plan cortical des Cyrtophorida, unite d'expression et marges de variabilite, II. Cyrtophorida a thigmotactisme ventral generalise. Protistologica, 12, 483-500. Deroux G., Iftode F. and Fryd G. (1974): Le genre Nassulopsis et les cilies fondamentalement hypostomiens. C. R. Acad. Sci. Paris, 278, 2153-2156. Fryd-Versavel G., Iftode F. and Dragesco ]. (1975): Contribution a la connaissance de quelques cilies gymnostomes. II. Prostomiens, pleurostomiens: morphologie, stomatogenese. Protistologica, 11, 509-5~0. Grain]. and Batisse A. (1974): Etude ultrastructurale du cilie chonotriche Chilodochona quennerstedti Wallengren, 1895. I. Cortex et structures buccales. J. Protozool., 21, 95-111. Haeckel E. (1866): GenereIIe Morphologie der Organismen. II. Allgemeine Entwicklungsgeschichte der Organismen. G. Reimer, Berlin. Hausmann K. (1978): Extrusive organelles in protists. Int. Rev. Cytol., 52, 197-276. Hofmann A. H. and Bardele C. F. (1987): Stomatogenesis in cyrtophorid ciliates. I. Trithigmostoma steini (Blochmann, 1895): from somatic kineties to oral kineties. Europ. J. Protistol., 23, 2-17. Jankowski A. W. (1967): Systematics of the genus Chilodonella and a proposed new genus Trithigmostoma gen. n. Zool. zh., 46, 1247-1250 (in Russian). jerka-Dziadosz M. (1980): Ultrastructural study on development of the hypotrich ciliate Paraurostyla weissei. I. Formation and morphogenetic movements of ventral ciliary primordia. Pratistologica, 16, 571-589. ]erka-Dziadosz M. (1981a): Ultrastructural study on development of the hypotrich ciliate Paraurostyla weissei. II. Formation of the adoral zone of membranelles and its bearing on problems of ciliate morphogenesis. Pratistologica, 17, 67-81. jerka-Dziadosz M. (1981b): Ultrastructural study on development of the hypotrich ciliate Paraurostyla weissei. III. Formation of preoral membranelles and an essay on comparative morphogenesis. Pratistologica, 17, 83-97. ]erka-Dziadosz M. (1982): Ultrastructural study on development of the hypotrich ciliate Paraurostyla weissei. IV. Morphogenesis of dorsal bristles and caudal cirri. Protistologica, 18,237-251. ]erka-Dziadosz M. and Golinska K. (1977): Regulation of ciliary pattern in ciliates. J. Protozool., 24, 19-26. Kazubski S. L. and Migala K. (1974): Studies on the distinctness of Chilodonella cyprini (Moroff) and Ch. hexasticha (Kiernik) (Chlamydodontidae, Gymnostomatida), ciliate parasites of fishes. Acta Protozol., 13, 9-40. Krascheninnikow S. (1953): The silver-line system of Chilodonella cyprini (Moroff), J. Morph., 92, 79-114. Kuhlmann S., Patterson D. J. and Hausmann K. (1980): Untersuchungen zu Nahrungserwerb und Nahrungsaufnahme bei Homalozoon uermiculare, Stokes 1887. I. Nahrungserwerb und Feinstruktur der Oralregion. Protistologica, 16, 39-55.
184 . A. H. Hofmann 22 Lorn]. and Corliss J. O. (1971): Morphogenesis and cortical ultrastructure of Brooklynella hostilis, a dysteriid ciliate ectoparasitic on marine fishes. J. Protozool., 18, 261-281. 23 Lynn D. H. (1981): The organization and evolution of microtubular organelles in ciliated protozoa. BioI. Rev., 56, 243-293. 24 Moroff T. (1902): Chilodon cyptini nov. spec. Zool. Anz., 26,5-8. 25 Puytorac P. de (1970): Definition of ciliate descriptive terms. ]. Protozool., 17, 358. 26 Pyne C. K. and Tuffrau M. (1970): Structure et ultrastructure de I'appareil cytopharyngien et des tubules complexes en relation avec celui-ci chez Ie cilie gymnostome Chilodonella uncinata Ehrenberg. J. Micros., 9, 503-516.
27 Raikov 1. B., Gerassimova-Matvejeva Z. P. and Putorac P. de (1975): Cytoplasmic fine structure of the marine psammobiotic ciliate Tracheloraphis dogieli Raikov. I. Somatic infraciliature and cortical organelles. Acta Protozool., 14, 17-42. 28 Schaperclaus W. (1934): Chilodon cyprini (Moroff) als Krankheitserreger bei Forellenbrut und seine fischpathologische Bedeutung im allgemeinen. Z. Parasitenk., 7, 447-464. 29 Soltynska M. (1971): Morphology and fine structure of Cbilodonella cucullulus (O.F.M.). Cortex and cytopharyngeal apparatus. Acta Protozool., 9, 49-82. 30 Tucker J. B. (1972): Microtubule arms and propulsion of food particles inside a large feeding organelle in the ciliate Phascolodon vorticella. ]. Cell Sci., 10, 883-903.
Key words: Cyrtophorida - Stomatogenesis - Oral kineties anlagen - Cyrtos anlage Angelika H. Hofmann, Institut f. Biologie III, Universitat Tiibingen, Auf der Morgenstelle 28, 7400 Tiibingen, FRG
Europ.]. Protisto!' 23, 165-184 (1987)
PROTISTOLOGY
Stomatogenesis in Cyrtophorid Ciliates II. ChilodonelJa cyprini (Moroff, 1902): The Kinetofragment as an Anlagen-Complex Angelika H. Hofmann Institut fUr Biologie III, Universitat Tiibingen
SUMMARY Non-dividers of Chilodonella cyprini possess two circum-oral kineties and one preoral kinety. These oral kineties are inverted kineties and consist of dikinetids. The cytostome is accompanied by a large microtubular organelle, the cyrtos. The latter consists of an outer ring of nematodesmata and an inner ring of cytopharyngeal microtubular lamellae. In stomatogenesis five somatic kineties are involved. Each of them produces one kinetofragment. These kinetofragments are anlagen-complexes for the oral kineties and the cyrtos. The somatic monokinetid is the elementary unit of stomatogenesis. In front of almost all somatic kinetosomes of a kinetofragment a new kinetosome is assembled, at an angle of 400 to the kinety axis. The somatic cilium disappears and the somatic mother kinetosome transforms into the non-ciliated kinetosome of the oral kinetosomal pair. The newly synthesized anterior daughter kinetosome bears the oral cilium and becomes the ciliated kinetosome of the oral kinetosomal pair. At the anterior end of each of these oral kineties anlagen there are two to four non-ciliated single somatic kinetosomes. Their triplets are covered with electron-dense materia!' Together with their subkinetal microtubules each of these non-ciliated single somatic kinetosomes forms one nematodesmata anlage. The postciliary microtubules of a part of the non-ciliated kinetosomes of the paired kinetosomes in the oral kineties anlagen and of the non-ciliated single kinetosomes increase in number and align to form one cytopharyngeallamella anlage. Subsequently all components of the anlagen-complexes separate . Seen from outside the cell the five oral kineties anlagen perform an anti-clockwise circular movement of approximately 1800 around the center of the new oral field of the posterior daughter cell, and rearrange to form the circum-oral kineties and the preoral kinety. In the nematodesmata anlagen the nonciliated kinetosome is resorbed. The electron-dense material covering the triplets transforms into the matrix plate of the nematodesma and the subkinetal microtubules increase in number. In the center of the new oral field the nematodesmata anlagen (subkinetal microtubules) and the cytopharyngeal tube lamella anlagen (postciliary microtubules) form the new cyrtos. The anlagen-complexes develop at the anterior ends of the somatic kineties of the posterior daughter cell; the Cyrtophorida show the telokinetal type of stomatogenesis.
Abbreviations A aki cks ct eta ctf ctl ctla cvp cy de df DK DKA
= anterior pole of the
cell = anterior end of the kinety = ciliated kinetosome = cytopharyngeal tube = cytopharyngeal tube anlage = cytopharyngeal tube fragment = cytopharyngeal tube lamella = cytopharyngeal tube lamella anlage = contractile vacuole pore = eytostome = "dent" = division furrow = dorsal kinety = dorsal kinety anlage
© 1988 by Gustav Fischer Verlag, Stuttgart
dks = daughter kinetosome dmt = distal microtubules dmta = distal microtubules anlage dtf = dense transverse fibril em = electron-dense material ex = extrusome fl = fine lamella of electron-dense material ICK = inner circum-oral kinety kd = kinetodesmal fibril = left side of the cell L mks = mother kinetosome mp = matrix plate mtu = membraneous tubules ncks = non-ciliated kinetosome ncr = non-ciliated region nd = nematodesma
nda oc OCK ofa OLK P pcmt PK ps R sc skmt sks st STK tmt
nematodesma anlage oral cilium outer circum-oral kinery oral field anlage outermost left kinety = posterior pole of the cell = postciliary microtubules = preoral kinety = parasomal sac = right side of the cell = somatic cilium = subkinetal microtubules = somatic kinetosome = stalk = stomatogenic kinety = transverse microtubules = = = = =
0932-4739/88/0023-0165$0.00/0
Fig. 1. Chilodonella cyprini - scanning electron micrograph s. - A) Ventral side of a non-dividing cell, the cytostome (cy) (x 2500).B) Dorsal side of a non-di viding cell, the dorsal kinety (DK) (x 2500). - C) The oral field of a non-d ividing cell; it consists of thre e oral kineties (preoral kinety (PK), out er circum-oral kinety (O CK), inner circum-oral kinety (IC K)) and the cyrtos (cytopharyngeal tube (et) and outer circle of nernatodesmata (nd)) . The oral cilia are shorterthan the somatic cilia and more densely packed (x 10 000). D) The oral field anlage of a dividing cell at stage 3 of stomatogenesis (late divider); synchronou s to the ingrowth of the division furrow (df) the oral field anlage (ofa) moves towards the posterior cell pole; in the center of the anlage the new cyrtos is visible (x 3600).
Stomatogenesis in Chilodonella cyprini . 167 Introduction The cyrtophorid ciliate Chilodonella cyprini is well known by ichthyoparasitologists as the organism that causes Chilodonella-disease [28] . The'parasite lives on the skin and gills of freshwater fishes and is often found in breeding ponds of carps and trouts. The morphology of C. cyprini has been examinedon the level of light microscopy by several authors [1, 19,20,24]. According to these studies the ciliate is dorso-ventrally flattened. With the exception of a dorsal kinety, the ciliature is restricted to the ventral side of the cell (Fig. 1 A, B). The ventral ciliature is dividedinto a left and a right ciliary field , separated by an unciliated middle field of the cortex. This character distinguishes the genus Chilodonella from the closely related genus Trithigmostoma, which shows a continuous somatic ciliature [13]. The cytostome of C. cyprini lies in the anterior half of the cell. It is accompanied by a large microtubular organelle (the cyrtos) and three oral kineties, which show the same arrangement as in the genus Trithigmostoma [12 ]. Using the light microscopic techniqueof protargol silver impregnation, Deroux [4,5] has suggested the identity between the stomatogenesis in the genus Chilodonella and the genus Trithigmostoma. This fact can only be proved by a study of the stomatogenesis on the electron microscopic level. Of special importance are the ultrastructure and orientation of the oral kineties in the non-dividing cell and the differentiation of the oral kineties anlagen in the morphogenetic field of an early divider. The ultrastructure of the cyrtos of the Cyrtophorida has been studied by different authors [26,29,30] but there are only a few data about the development of this microtubular organelle during stomatogenesis [22]. In the somatic and oral morphogenesis of the hypotrich ciliate Paraurostyla weissei [14-17] the complex patterns of the somatic and oral cortex (cirri, membranelles) have their origin in simple cortical patterns like monokinetids and dikinetids. The kinetosomes in the cortex of dividing cells are associated with fibrils that cannot be observedin the cortex of non-dividing cells. Hence the characters of stomatogenesis on the electron microscopic level prove to be very valuable for phylogenetic considerations. The Cyrtophorida show the telokinetal type of stomatogenesis, which is, according to Corliss [3], the most primitivetype of stomatogenesis in ciliates. The study of this stomatogenesis on the electron microscopic level should reveal some primitive characters of the ciliate cortex. Rapidly growing populations of C. cyprini are rich in dividing cells and therefore this ciliate is an ideal system for an electron microscopic study to clear some of the open questions about the stomatogenesis of the Cyrtophorida. Material and Methods ,
Cultivation of Cbilodonella cyprini was not possible. Without their host fish the organisms rapidly died. The populations of Chilodonella eyprini examined were isolated from the skin of a heavily infected schlei (Tinea tinea) . For purification the probe
was filtered over a fine gauze (width ofmesh: 50 11m) andwashed in Ean Volvic. The preparations forthelight andelectron microscopic studies were done as described previously [12]. All Figs. are oriented as seen from outside thecell. The triplets of thekinetosomes arenumerated according to de Puytorac [25].
Results
Light microscopy Non-divider A non-dividing cell of Chilodonella cyprini (Fig. 2 A) is 30-60 urn long and 20-40 urn wide. The posterior pole of the cell is marked by an indentation at its left side. The oral ciliature consists of two circum-oral kineties and one preoral kinety, which show a concentricarrangement. The inner circum-oral kinety is the shortest one and lies in front of the cytostome. The longer outer circum-oral kinety is situated in front of the inner circum-oral kinety and reaches the right side of the cytostorne. The right end of the preoral kinety liesin front of the left end of the outer circum-oral kinety. The left end of the preoral kinety is situated in the preoral suture. The left field of the somatic ciliature consists of 11-15 longitudinally arranged kineties. The anterior ends of these left somatic kineties (the two short innermost kineties excluded) face the preoral kinety or the anterior ends of the right somatic kineties. The short outermost left kinety does not extend into the .region of the division furrow. The right field of the somatic ciliature is composed of 10-13 kineties. The five to six innermost right kineties are postoral kineties and their anterior ends face the outer circum-oral kinety. The remaining right kineties bend left in front of the oral field and reach the preoral kinety or the anterior ends of the left somatic kineties. The anterior ends of both the right somatic kineties and the left somatic kineties are arranged to form the preoral suture. The posterior ends of the somatic kineties are arranged to form a postoral suture, close to the indentation of the posterior cell pole. In each field of the somatic ciliature opens the pore of one contractile vacuole.
Stage 1 of stomatogenesis (early divider) An early dividerof Chilodonella cyprini (Fig. 2 B,4 A)is 50-80 urn long and 25-45 urn wide. The morphogenetic field is situated in the left field of the somatic ciliature. The five innermost left kineties segment and develop one kinetofragment each, which are numbered 1 to 5 from right to left. Kinetofragment 1 is the anlage of the preoral kinety and kinetofragment 2 is the anlage of the inner circum-oral kinety. Together the kinetofragments 3, 4 and 5 are the anlage of the outer circum-oral kinety. In Chatton-Lwoff silverline preparations a non-ciliated region is visible in front of the anterior ends of the oral kineties anlagen. The innermost somatic kinety of the left ciliary field is the stomatogenickinery, Anterior to kinetofragment 1, the
168 . A. H. Hofmann
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Fig. 2. Chilodonella cyprini - schematic drawings based on Chatton-Lwoff silverline preparations. - A) Kinetome of the ventral side of a non-dividing cell. The three oral kineties: preoral kinety (PK), outer circum-oral kinety (OCK), inner circum-oral kinety (I CK). The orientation in the cell: anterior pole of the cell (A), posterior pole of the cell (P), right side of the cell (R), left side of the cell (L) .B) Kinetome of the ventral side of a cell at stage 1 of stomatogenesis (early divider); the morphogenetic field consists of five kinetofragments (1-5), segment I' and a non-ciliated region (ncr) in front of the anterior ends of the kinetofragments; the innermost kinety of the left field of the soma tic ciliature is the stoma togenic kinety (STK).
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Fig. 4. Stomatogenesis of binary division, schematic drawings based on Chatton-Lwoff silverline preparations. For numeration of the kinetofragments see Figs. 2 Band 5. - A) Stage 1 of stomatogenesis (early divider); the segmentation of five somatic kineties and the development of five kinetofragrnents, segment l ' and a non-ciliated region in front of the anterior ends of the kinetofragments. - B-D) Stage 2 of stomatogenesis (middle divider); the circular movement of the kinetofragments (and segment 1') around the center of the oral field anlage and their arrangement to the oral kineties of the posterior daughter cell. - E-F ) Srage 3 of stomatogenesis (late divider); the first movement of the oral field anlage towards the right somatic kineties, followed by a second movement towards the posterior pole of the cell; the allometric growth of the anterior ends of the right somatic kineties, synchronous to the ingrowth of the division furrow; the development of segment I' to the innermost somatic kinery of the left ciliary field, to maintain a constant number of left somatic kineties.
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Fig. 3. Chatton-Lwoff silverline preparations of dividing cells. For numeration of the kinetofragments see Fig. 2B and 5 (x 2000). A) Stage 2 of stomatogenesis (middle divider); as a first sign of the circular movement the kinetofragmems 2-5 move closer together and kinetofragment 5 migrates in front of kinetofragment 4. - B) Stage 2 of stomatogenesis (middle divider); the five kinetofragments and segment l ' perform a circular movement around the center of the developing oral field anlage; the left somatic kineties split in an anterior and a posterior half. - C) Stage 3 of stomatogenesis (late divider); the oral field anlage moves towards the right field of the somatic ciliarure; synchronous to the beginning ingrowth of the division furrow, the right somatic kineties split in an anterior and a posterior half. - 0 ) Stage 3 of stomatogenesis (late divider); the oral field anlage moves towards the posterior cell pole, due to the allometric growth of the anter ior ends of the right somatic kineties, synchrono us to the ingrowth of the division furrow.
170 . A. H. Hofmann
stomatogenic kinety develops one segment 1', which is the primordium of the stomatogenic kinety of the posterior daughter cell. Parallel to the outermost right somatic kinety, and in the region of the division furrow, lies the anlage of the short dorsal kinety.
Stage 2 of stomatogenesis (middle divider) In the second stage of stomatogenesis the kinetofragments perform an anti-clockwise circular movement of approximately 1800 around the center of the developing oral field anlage. As they move, the kinetofragments follow two concentric paths and rearrange to form the new oral kineties (Fig. 3 A-B, Fig. 4 B-D, 5): as a first sign of the circular movement the kinetofragments 2-5 move closer together and kinetofragment 5 migrates in front of kinetofragment 4. On the outer circle, kinetofragments 4 and 3 follow kinetofragment 5 and they join to form the outer circum-oral kinety anlage. On the inner circle, kinetofragment 2 moves parallel to kinetofragment 3 and forms the inner circum-oral kinety anlage. Kinetofragment 1 starts last. Its anterior end bends to the left and follows the posterior ends of the kinetofragments 2-3. Finally the
anterior end of kinetofragment 1 reaches the position of the preoral kinety. Segment I' migrates towards the posterior pole of the cell and completes the circle of the moving anlagen. At this stage of stomatogenesis the left somatic kineties split into an anterior and a posterior half, except for the outermost left kinety. This short somatic kinety remains totally in the anterior daughter cell.
Stage 3 of stomatogenesis (late divider) The third stage of stomatogenesis is characterized by a first movement of the oral field anlage towards the right side of the cell, followed by a second movement of the oral field anlage towards the posterior pole of the cell (Fig. 3 C-D, 4E-F). The right somatic kineties split into an anterior and a posterior half. Synchronous to the beginning ingrowth of the division furrow, the anterior ends of the right somatic kineties of the posterior daughter cell increase in length. As the oral field anlage reaches the right ciliary field, the anterior ends of the future postoral kineties face the outer circum-oral kinety anlage. Synchronous to the further ingrowth of the division furrow, the anterior ends of the
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Fig. 5. The number of kinetofragments and their arrangement to the oral kineties (two circum-oral kineties and one preoral kinety) of the posterior daughter cell; schematic drawingbased on Chatton-Lwoff silverline preparations. The kinetofragments and segment I' are shown as arrows; the arrows mark the direction of the movement and (segment I' excluded) also the polarity of the kinetofragments; the circular movement performs with a strict order and sequence; kinetofragment 1 = preoral kinety anlage; kinetofragment 2 = inner circum-oral kinety anlage; kinetofragments 3,4 and 5 = outer circum-oral kinety anlage; segment I' = primordium of the stomatogenic kinety of the posterior daughter cell.
Stornatogenesis in Chilodonella cyprini . 171
Fig. 6. Tangential section of the somatic and oral cortex of a non-dividing cell, showing the som atic monokinetids and the oral dikinetids, and also the structural elements of the cyrtos. The oral dikinetids are arranged in a zigzag-pattern and with an inverted polarity relative to the somatic monokinetids (see Figs. 7, 8 for comparison). Each kinetosomal pair consists of a posterior non-ciliated kinetosome (ncks) bearing postc iliary microtubules (pcmt) and an anterior ciliated kinetosome (cks) bearing a dense transverse fibril
(dtf) (x 24000).
172 . A. H. Hofmann remaining right kineties bend to the left side of the cell. As they meet the anterior ends of the left somatic kineties, the preoral kinety anlage becomes situated in the preoral suture. Due to the allometric growth of the anterior ends of the right somatic kineties, the oral field anlage moves towards the posterior pole of the cell. Segment l' develops into the innermost kinety of the left ciliary field. It becomes the new stomatogenic kinety of the posterior daughter cell and compensates the loss of the short outermost left kinety. Electron microscopy
Non-divider The somatic cortex of the ventral side. The somatic kine ties are separated by steep cytoplasmic ridges, which are covered with flat pellicular alveoli. The somatic kineties consist of monokinetids. At the proximal end of each somatic kinetosome five types of associated fibrils arise, which show the "phyllopharyngid pattern" according to Lynn [23J (Figs. 6, 7, 8 A): a postciliary ribbon of three microtubules at triplet number 9, an anterolaterally oriented kinetodesmal fibril at triplets number 5 and 6, two transverse microtubules in radial position to triplet number 5, a dense transverse fibril at triplet number 3 and also six subkinetal microtubules underneath the kinetosome cylinder. One parasomal sac accompanies the soma-
see Fig .8
tic cilium, situated at its right side. The associated fibrils of the somatic kinetosome show the same arrangement and extension as described for Trithigmostoma steini [12J. The unciliated middle field of the somatic cortex and the unciliated left and right margins of the ventral side are crowded with mucocyst-like extrusomes. The extrusomes are small, membrane-bounded, pyriform bodies, approximately 160 nm in diameter and 240 nm in height (Figs. 10, 11). They are filled with electron-dense material, which does not show the net-like structure that was described for the mucocysts of Tetrahymena [11]. The extrusomes lie freely in the cytoplasm or are attached to the plasmamembrane. They are restricted to the ventral side of the cell.
The oral cortex of the ventral side: the cyrtos. The cytostome of Chilodonella cyprini is accompanied by a large microtubular organelle, the cyrtos, The cyrtos consists of the cytopharyngeal tube and an outer ring of nematodesmata (Figs. 1 C, 6, 7). The cytopharyngeal tube is composed of 34-37 radially arranged cytopharyngeal tube lamellae. Each lamella is a single-layered ribbon of 12-15 microtubules. The microtubules extend into the cytoplasm, at an angle of 90° to the cell surface. The distal end of each cytopharyngeal tube lamella is accompanied by a single-layered ribbon of three to four microtubules. These distal microtubules extend towards the nematodesmata, parallel to the cell surface.
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Fig. 7. The fine structure of the oral field, schematic drawingbasedon ultrathin sections. The three oral kinetics (preoral kinety (PK), outer circum-oral kinety (OCK), inner circum-oral kinety (ICK)) show an inverted polarity relative to the somatic kineties. Their anterior ends (aki) point to the posterior pole of the cell (P) or to its right side (R), their posterior ends (pki) point to the left side of the cell (L). The cyrtos consists of an cytopharyngeal tube (et) and an outer circle of nematodesmara (nd). The distal end of each cytopharyngeal tube lamella (etl) is accompanied by a ribbon of distal microtubules (dmt).
Stomatogenesis in Chilodonella cyprini . 173
An outer ring of 14-16 nematodesmata surrounds the cytopharyngeal tube. Each nematodesma is a rigid bundle of microtubules, which show a hexagonal arrangement. At their distal ends, the nematoclesmal microtubules are covered with a matrix plate of electron-dense material. Connected to the matrix plate is a stalk, which bears two "dents". The stalk and the "dents" consist of the same electron-dense material as the matrix-plate. The phagoplasm of the cyrtos contains a lot of membraneous tubules, which often show a hexagonal arrangement. They correspond to the "tubules complexes" described by Pyne and Tuffrau [26] for Chilodonella un-
situated on the outer side of the kinety, not facing the cytostome. The posterior kinetosome of the kinetosomal pair is non-ciliated and a ribbon of three to four postciliary microtubules arises at triplet number 9. The non-ciliated kinetosome is situated on the inner side of the kinety, facing the cytostome. It stands deeper in the cytoplasm than its ciliated partner. The associated fibrils of the oral kinetosomal pair show the same arrangement and extension, as described for Trithigmostoma steini [12]. At the proximal end of the kinetosome cylinder, electron-dense material connects the kinetosomes of each pair and the kinetosomal pairs of each oral kinety. Two parasomal sacs accompany the oral cilium. The first one is situated on the outer side of the kinety, posterior to the dense transverse fibril. The second one lies on the inner side of the kinety, between the non-ciliated kinetosomes.
cinata. The oral cortex of the ventral side: the oral kineties. Two circum-oral kineties and one preoral kinety are situated at the anterior and right margin of the oral field. The oral kineties consist of single rows of cilia. The oral cilia are shorter than the somatic cilia and more densely packed (Figs. 1 A, 1 C). The circum-oral kineties and the preoral kinety show the same ultrastructure. Their orientation is inverted, relative to the orientation of the somatic kineties. Their anterior ends point to the posterior pole of the cell or to its right side. Their posterior ends point to the left side of the cell (Figs. 6, 7). The oral kineties consist of dikinetids (Figs. 6, 7, 8 B). The kinetosomal pairs are arranged in a zigzag-pattern, with the axis of each pair and the kinety axis forming an included angle of 45°. The anterior kinetosome of the oral kinetosomal pair bears the oral cilium and a dense transverse fibril at triplet number 3. The ciliated kinetosome is
Stage 1 of stomatogenesis (early divider) The kinetofragment as an anlagen-complex. In ChattonLwoff silverline preparations the morphogenetic field of an early divider is characterized by the kinetofragments 1-5 (oral kineties anlagen), segment l ' and also a nonciliated region in front of the anterior ends of the kinetofragments (Fig. 2 B). In ultrathin sections of the morphogenetic field the kinetofragments extend into the non-ciliated region. They are anlagen-complexes for the dikinetids of the oral kineties and also for the nematodesmata, cytopharyngeal tube lamellae and distal microtubules of the cyrtos. The somatic monokinetid is revealed to be the elementary unit of stomatogenesis (Figs. 12, 15 A).
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Fig. 8. The fine structure of somatic kineties and oral kineties; schematic drawings based on ultrathin sections; for orientation see frame Fig. 7. - A) Somatic monokinetids. - B) Oral dikinetids; the kinetosomes and their associated fibrils show an inverted polarity relative to the somatic monokinetids (inverse orientation of the anterior ends (aki) and posterior ends of the kineties (pki)).
174 . A. H. Hofmann
Fig. 9. Stage 1 of stomatogenesis (early divider); from somatic monokinetids to oral dikinetids; tangential section of the developing oral kineties anlagen 1-5. In front of those somatic kinetosomes (sks) of the kinetofragments which belong to the oral kineties anlagen a new anterior daughter kinetosome (dks) is synthesized, at an angle of 40° to the kinety axis. The somatic cilium (sc) disappears and the somatic mother kinetosome (mks) transforms into the non-ciliated kinetosome (ncks) of the oral kinetosomal pair. The anterior daughter kinetosome (dks) develops a new oral cilium (oc) and becomes the ciliated kinetosome (cks) of the oral kinetosomal pair. The oral dikinetids anlagen show the same polarity as the somatic monokinetids did before (x 30000).
Stomatogenesis in Chilodonella cyprini
175
Fig. 10. Stage 1 of stomatogenesis (early divider); from somatic monokinetids to nematodesmata anlagen (nda); tangential serial sections of the non-ciliated region in front of the anterior ends of the oral kineties anlagen 2-5; section B proceeds deeper in the cytoplasm than section A. At those somatic kinetosomes of the kinetofragments which belong not to the oral kineties anlagen the somatic cilium disappears and also the associated fibrils (subkinetal microtubules and in some cases postciliary microtubules excluded). Their triplets are covered with electron-dense material. Together with its subkinetal microtubules (skmt) each of these nonciliated single somatic kinetosomes forms one nematodesma anlage (arrow) (x 30000).
176 . A. H. Hofmann
Fig. 11. Stage 1 of stomatogenesis (early divider); from somatic postciliary microtubules (pemt) to cytopharyngeallamellae anlagen (etIa) and distal microtubules anlagen (dmta) ; tangential serial sections of the oral kineties anlagen 2 and 3; from A to C the sections proceed deeper in the cytoplasm (x 30000). - A) Two groups of microtubules associated with an oral cilium (arrow). - B) A loose ribbon of microtubules associated with an oral cilium (arrow). - C) The inner group of three to four postcilliary microtubules attached to the non-ciliated kinetosome of an oral kinetosomal pair (arrow).
Stornatogenesi s in Chilodonella cyprini . 177
In the posterior parts of the kinetofragments (visible in the light microscope after Chatton-Lwoff silver impregnation) , each somatic kinetosome develops an anterior daughter kinetosome, at an angle of 40° to the kinety axis. At the somatic mother kinetosome, the somatic cilium and also the transverse microtubules, dense trans verse fibril and kinetodesmal fibril disappear. The postciliary microtubules and the subkinetal micro tubules are still present. The somatic kinetosome transforms into the non-ciliated kinetosome of the oral kineto somal pair. The anterior daughter kinetosome develops a new oral cilium and a dense transverse fibril and becomes the ciliated kinetosome of the oral kinetosomal pair. Each oral cilium is accompanied by two parasomal sacs. The oral dikinetids anlagen show the same polarity as the somatic monokinetids did before. At stage 1 of stomatogenesis the oral kinety axis shows the same orientation as the somatic kinety axis (Fig. 9). In the anterior part of each kinetofragment (visible in the light microscope as a non-ciliated region after Chat ton-Lwoff silver impregnation), two to four somatic monokinetids transform into nematodesmata anlagen. The somatic cilium disappears and also the associated fibrils (subkinetal microtubules and in some cases postciliary microtubules excluded). The somatic kinetosome detaches from the epiplasm and the kinetosomal triplets are covered with electron-dense material. Together with its subkinetal microtubules each of these non-ciliated single somatic kinetosomes forms one nematodesma anlage (Fig. 10). The cytopharyngeal tube lamellae anlagen and the distal microtubules anlagen have their origin in the postciliary microtubules of a part of the somatic kinetosomes of the oral dikinetids anlagen and of the nematodesmata anlagen. The somatic postciliodesma disappears. The somatic postciliary microtubules increase in number from three up to five or six and form a loose microtubular ribbon. The ribbon divides into two groups of microtubules. In the oral dikinetids anlagen the inner group of three to four microtubules, facing the kinetosome, corresponds to the oral postciliary microtubules. In the nematodesmata anlagen the inner group of microtubules disappears. The remaining two to three microtubules of the outer group increase in number and rearrange to form one cytopharyngeal lamella anlage and one distal microtubules anlage (Fig. 11). In a non-dividing cell only a few extrusomes are situated in the ciliary fields of the ventral side. Synchronous to the development of the morphogenetic field, extrusomes accumulate beside the anterior ends of the anlagen -complexes (Figs. 10, 11).
anlagen of the cyrtos separate from the anlagen of the oral kineties. The separated anlagen perform the morphogenetic movement and rearrange into the new oral field of the posterior daughter cell. In each of the five kinetofragments the anlagen of the cytopharyngeal tube lamellae and of the distal microtubules detach from their mother kineto somes. They arrange to form five cytopharyngeal tube fragments , near the anterior ends of the oral kineties anlagen (Figs. 13 B, 15 B). In the following these five cytopharyngeal tube fragments migrate towards the center of the developing oral field anlage. As they move they join to close the circle of the cytopharyngeal tube anlage (Figs. 14, 15 C-E). At the anterior ends of the kinetofragments, the nematodesmata anlagen quit their places in the anlagencomplexes and move towards the center of the oral field anlage. Synchronous to this movement, the non-ciliated kinetosome of the nematodesma anlage disappears. The electron-dense material, which covers the kinetosomal triplets transforms into the matrix plate of the nematodesma. The subkinetal microtubules of the anlage increase in number and become the nematodesmal microtubules (Figs. 13 A, 15 B-D). Around the cytopharyngeal tube anlage the nematodesmata anlagen arrange to form the outer circle of the cyrtos . The free ends of the nematodesmal microtubules point into the cytoplasm , the matri x plates point towards the plasmamembrane (Figs. 14, 15 E-F). As a first sign of their circular movement the oral kineties anlagen 2-5 move closer together and oral kinety anlage 5 migrates in front of oral kinety anlage 4. As a result of this movement, a wide gap is formed between oral kinety anlage 1 and 2, to generate the space where the cyrtos anlage is assembled (Figs. 15 B-D). The oral kineties anlagen 1-5 perform their circular movement around the developing cyrto s anlage and rearrange as described (Fig. 5) to form two circum-oral kineties and one preoral kinety. Synchronous to the beginning movement of the oral kineties anlagen the subkinetal microtubul es, which still mark the somatic origin of the nonciliated kinetosomes, disappear and electron-dense material connects the kinetosomes of an oral kinety (Figs. 15 E-F). With the rearrangement of the oral kineties anlagen the assemblage of the oral field anlage of the posterior daughter cell is completed and the axis of the oral kineties shows an inverted orientation. At this stage of stomatogenesis the extrusomes of the morphogenetic field concentrate in the center of the developing oral field anlage (Figs. 13, 14).
Stage 3 of stomatogenesis (late divider) Stage 2 of stomatogenesis (middle divider) The disintegration of the anlagen-complexes and the assemblage of the oral field anlage. In Chatton-Lwoff silverline preparations the second stage of stomatogenesis is characterized by the circular movement of the kinetofragments and their rearrangement into the oral kineties of the new oral field. In ultrathin sections the morphogenetic field of a middle divider is characterized by the disintegration of the totally differentiated anlagen-complexes. The
The oral field anlage of a late divider (Fig. 1 D) undergoes a first movement towards the right side of the cell and a second movement towards the poster ior pole of the cell. Synchronous to this process, the cyrtos anlage becomes totally differentiated. The nematodesmal microtubules and also the microtubules of the cytopharyngeallamellae increase in length and in number. At the matrix plates of the nematodesmata anlagen the stalk and the "dents" develop. In the phagoplasm of the cyrtos anlage the mem-
178 . A. H. Hofmann
ps
cks
, pki Fig. 12. The ultrastructure of the morphogenetic field of a dividing cell at stage 1 of stomatogenesis (early divider, see Fig. 2 B for comparison); schematic drawing based on serial sections. The kinetofragments 1-5 are anlagen-complexes for the dikinetids of the oral kineties and for the nematodesmata, cytopharyngeallamellae and distal microtubules of the cyrtos. The somatic monokinetid is the elementary unit of stomatogenesis: in the posterior parts of the kinetofragments (visible in the light microscope after ChattonLwoff silver impregnation) the somatic kinetosomes (sks) transform into the non-ciliated kinetosomes (neks) of the oral kinetosomal pairs and the anterior daughter kinetosomes become the ciliated kinetosomes (eks) of the oral kinetosomal pairs; in the anterior parts of the kinetofragments (visible in the light microscope as a non-ciliated region after Chatton-Lwoff silver impregnation) the somatic monokinetids transform into the nematodesmata anlagen (nda); the anlagen of the cytopharyngeallamellae (et/a) and of the distal microtubules (drnta) have their origin in the postciliary microtubules (pernt) of a part of the somatic kinetosomes of the oral dikinetids anlagen and nematodesmata anlagen.
Stomatogenesis in Chilodonella cyprini . 179
Fig. 13. Stage 2 of stornatogenesis (middle divider ); the disintegr ation of the anlagen-complexes; tangential serial sections of the or al kineries anlagen 1-3; section B proceeds deeper in the cytoplasm than section A (x 30000). - A) The nernat odesmata anlagen (nda) have quitted the anlagen-complex ; the non-ciliated kinetosome of the anlage is resorbed; the dense mat erial covering the triplets transforms into the matrix plate (mp) of the nematodesma; the subkinetal microtubules (skmt) increase in number and become the nernatodesmal microtubules. - B) The anlagen of the cytopharyngeal cube lamellae (etla) and of the distal microtubules (dmta) are det ached from their mother kinerosornes and are arranged to a cytoph ar yngeal tube fragment (etf) .
180 . A. H. Hofmann
Fig. 14. Stage 2 of stomatogenesis (middle divider); the assemblage of the new cyrtos and the new oral kineties of the posterior daughter cell; tangential section of the developing oral field anlage. The cytopharyngeal tube fragments have arranged to the cytopharyngeal tube anlage (eta), surrounded by an outer circle of nernatodesmata anlagen (nda) (x 30000).
Fig. 15. The development of the oral field anlage of the posterior daughter cell at stage 1 (A) and stage 2 (B-F) of stomatogenesis; ~ schematic drawings based on ultrathin sections; for the circular movement of the oral kineties anlagen, see Figs. 4 and 5 for comparison. - A) The totally differentiated morphogenetic field of an early divider: The kinetofragments 1-5 are anlagen-complexes for the dikinetids of the oral kineties and for the nematodesmata, cytopharyngeal lamellae and distal microtubules of the cyrtos; segment I' is the primordium of the stomatogenic kinety (innermost kinety of the left ciliary field) of the posterior daughter cell. B) The disintegration of the anlagen-complexes in the morphogenetic field of a middle divider: The anlagen of the cytopharyngeal tube lamellae and of the distal microtubules lose the contact to their mother kinetosomes and arrange to cytopharyngeal tube fragments (ctf); the nematodesmara anlagen (nda) quit their places at the anterior ends of the kinetofragments and move towards the center of the developing oral field; the oral kinety anlage 5 starts its circular movement; segment L' detaches from the stomatogenic kinety, - C) The cytopharyngeal tube fragments (ctf) quit their positions at the right side of the oral kineries anlagen and move
Stomatogenesis in Chilodonella eyprini
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Fig. 15 (continued from page 180) towards the center of the developing oral field; the somatic kinetosome of the nematodesma anlage (nda) disappears, its subkinetal microtubules increase in length and in number and become the nematodesmal microtubules, the electron-dense material of the anlage transforms into the matrix plate; the oral kinety anlage 4 follows the oral kinety anlage 5. - 0) The assemblage of the oralkineties and the eyrtos: The cytopharyngeal tube fragments close the circle of the cytopharyngeal tube anlage (eta); the oral kinety anlage 3 follows the oral kineties anlagen 4 and 5; segment l' increases the number of its somatic monokinetids and starts its movement towards the posterior cell pole. - E) The nematodesmata anlagen (nda) close the outer circle of the cyrtos; the free ends of the nematodesmal microtubules point into the cytoplasm, the matrix plates point towards the plasmamembrane; the oral kinery anlage 2 runs parallel to the oral kinety anlage 3. - F) The totallyassembled oralfield anlage of the posterior daughter cell with the new oral kineties and the new cyrtos.
182 . A. H. Hofmann
braneous tubules appear. The extrusomes, which have been situated at the distal end of the cytopharyngeal tube, are now located at its proximal end.
Discussion The stomatogenesis of Trithigmostoma steini [12] and Chilodonella cyprini show the same basic principles. Both ciliates possess a stornatogenic kinety, which gives rise to the anlage of the preoral kinety and to one segment 1'. Segment I' is the primordium of the stomatogenic kinety of the posterior daughter cell and compensates the loss of the outermost left kinety, According to Deroux [5], this mechanism to maintain a constant number of left somatic kineties is the synapomorphic character for the order Cyrtophorida. In both species the oral kineties anlagen show the same arrangement in the morphogenetic field of an early divider and undergo the same rearrangement in stage 2 of stomatogenesis to form the circum-oral kineties and the preoral kinety, The outer circum-oral kinety consists of three oral kineties anlagen, which join "head to tail" during the morphogenetic movement. A similar process can be observed in the stomatogenesis of haptorid ciliates like Spathidium, Homalozoon and Bryophyllum [8]. A single circum-oral kinety surrounds the cytostome, which is situated at the anterior pole of the cell. The oral kineties anlagen develop at the anterior ends of the somatic kineties of the posterior daughter cell. They bend to the right and join "head to tail" to form the circum-oral kinety of these genera. The oral kineties of C. cyprini show the same ultrastructure as the oral kineties of T. steini. The orientation of the circum-oral kineties and of the preoral kinety is inverted, for the same reasons as discussed for T. steini [12]. The differentiation process of the oral kineties anlagen in the morphogenetic field of an early divider is almost identical. In both species the oral dikinetids originate in somatic monokinetids. The somatic kinetosome transforms into the non-ciliated kinetosome of the oral kinetosomal pair. A new daughter kinetosome is synthesized in front of the somat .rl--",~ 1,; ~'?tosome, at an angle of 40° to the kir.e r
kineties anlagen, described for T. steini and for C. cyprini, is a common character in the order Cyrtophorida and Chonotrichida. The results of this paper show, that the kinetofragments are anlagen-complexes for the dikinetids of the oral kineties and for the nematodesmata, cytopharyngeal lamellae and distal microtubules of the cyrtos. The nematodesmata anlagen are located at the anterior ends of the kinetofragments, visible in Chatton-Lwoff silverline preparations as a non-ciliated region. Hence the kinetofragments develop at the anterior ends of the somatic kineties of the posterior daughter cell and the Cyrtophorida show the telokinetal type of stomatogenesis. According to Deroux [5, 6] the Cyrtophorida undergo the same parakinetal type of stomatogenesis, as described for Nassulopsis [7]. In protargol silver impregnations the nematodesmata anlagen are visible as single kinetosornes, Deroux interpreted these single kinetosomes as the remainders of a somatic ciliature in front of the anterior ends of the kinetofragments, He suggested a homology between these single kinetosomes and the segments of somatic kineties that are situated between the hypostomial frange anlage and the region of the division furrow in a dividing cell of Nassulopsis. According to Deroux the kinetofragments of Trithigmostoma and Chilodonella correspond to the elements of the hypostomial frange anlage of Nassulopsis, which is interpreted as the most primitive member of the Hypostomata. This concept needs confirmation by an electron microscopic study of Nassulopsis and its stomatogenesis. The nematodesmata of the cyrtos originate in somatic monokinetids, At stage 1 of stomatogenesis, the nematodesrna anlage consists of a non-ciliated somatic kinetosome, its triplets covered with electron-dense material and a bundle of subkinetal microtubules. In C. cyprini the kinetosome of the anlage disappears at stage 2 of stornatogenesis, whereas in Brooklynella hostilis [22] the kinetosome of the anlage is still present at the distal end of the nematodesma of a non-dividing cell, associated with the electron-densematerial of the "dents". The subkinetal microtubules of the nematodesrna anlage transform into the nematodesmal microtubules. This fact proves the homology between nematodesmal microtubules and subkinetal microtubules, suggested by Raikov, GerassimovaMarvejeva and de Puytorac [27]. In Spathidium, Bryophyllum and Homalozoon [2,21] the circum-oral kinety also consists of dikinetids. The kinetosome of the oral kinetosomal pair facing the cytostome is non-ciliated and associated with nematodesmal microtubules and a microtubular ribbon, which is interpreted a~ .~'lnsverse microtubules. The kinetosome of the oral kinetosomal pair not facing the cytostome bears the oral cilium. The somatic kineties of haptorid ciliates consist of monokinetids [2]. The oral kineties anlagen differentiate at the anterior ends of the somatic kineties of the future posterior daughter cell. As shown by protargol silver impregnation, the oral kineties anlagen of Homalozoon consist of kinetosomal pairs and are associated with oral cilia and nematodesmal microtubules, before the rearrangement of the oral kineties anlagen to the circum-oral
Stomatogenesis in Chilodonella cyprini . 183
kinety starts [8]. The examination of the differentiation modus of the oral kineties anlagen on electron microscopic level would be of great interest. On condition that the oral dikinetid anlage of haptorid ciliates develops in the same way as described for T. steini and C. cyprini, the following predictions can be made: The somatic kinetosome transforms into the non-ciliated kinetosome of the oral kinetosomal pair and becomes associated with nematodesmal microtubules and a ribbon of postciliary microtubules. An anterior daughter kinetosome is synthesized, which develops into the ciliated kinetosome of the oral kinetosomal pair. In the oral field of a non-dividing cell the non-ciliated kinetosome faces the apically situated cytostome. To obtain this orientation of the oral dikinetid, the kinetosomal pair axis has to make a turn of 90° towards the left, that is, towards the cytostome, during the rearrangement of the oral kinetids anlagen to form the circum-oral kinety. This would mean that postciliary microtubules, instead of transverse microtubules, contribute to the oral apparatus of haptorid ciliates. According to ]erka-Dziadosz and Golinska [18] complex cortical patterns have their origin always in simple cortical patterns. The way of differentiation leads from monokinetids to dikinetids to polykinetids. In T. steini and C. cyprini the somatic monokinetid is the elementary unit of the telokinetal type of stomatogenesis. The somatic kinetosome and its associated fibrils are the origin of the complex structures of the oral field of these ciliates. The somatic monokinetid transforms into the oral dikinetid. The postciliary microtubules give rise to the cytopharyngeallamellae and to the distal microtubules of the cyrtos. The subkinetal (nematodes mal) microtubules become the nematodesmata of the cyrtos. According to the biogenetic law of Haeckel [10], the ontogenesis of an organism reflects the events of its phylogenesis. If this statement is true for the stomatogenesis of ciliates, then the monokinetid turns out to be more primitive than the dikinetid. A kinetosome associated with the basic set of fibrils typical for ciliates (kinetodesmal fibril, postciliary microtubules, transverse microtubules, nematodesmal microtubules) has the morphogenetic potency to develop all the complex patterns observed in the somatic and oral cortex of ciliates. The future study of stomatogenesis in ciliates on the electron microscopic level should reveal the evolutionary pathways of these organisms and also some basic modes of pattern formation in the cortex of these cells.
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Acknowledgements 18 I wish to thank Dr. C. F. Bardele for his support during my work and the many discussions we have had, and also H. Schoppmann for the technical assistance with scanning electron microscopy.
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20 21
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Key words: Cyrtophorida - Stomatogenesis - Oral kineties anlagen - Cyrtos anlage Angelika H. Hofmann, Institut f. Biologie III, Universitat Tiibingen, Auf der Morgenstelle 28, 7400 Tiibingen, FRG