J. Promzool., 30(3). 1983, pp. 519-529 0 1983 by the Society of Protozoologists

Morphogenetic Pattern in Laurentiella acuminata (Ciliophora, Hypotrichida): Its Significance for the Comprehension of Ontogeny and

Phylogeny of Hypotrichous Ciliates JESUS MARTIN,* CONCEPCION FEDRIANI,** and JULIO PEREZ-SILVA**

*Departamento de Microbiologia, Facultad de Ciencias, Universidad de Cbrdoba, Esparla. and **Departamento de Microbiologia. Facultad de Biologi‘a, Universidad de Sevilla, Esparia

ABSTRACT. Morphogenetic events during division, physiological reorganization, and postraumatical regeneration, the last being induced both chemically and microsurgically, were studied by light microscopy on protargol-impregnated specimens of the hypotrichous ciliate, Laurentiella acuminata. Parakinetal stomatogenesis, from transverse cirrus- I during division and reorganization, changes during regeneration to a parakinetal one which characterizes more primitive members of Hypotrichida in the S. 0. Stichotrichina, but solely when the AZM is damaged. These morphogenetic events a) confirm the previous inclusion of L. acuminata among the Oxytrichidae on the basis of its morphological characters and indicate that it is a primitive species of this family related with the Stichotrichina through genera Pleurotricha and Paraurostyla; b) suggest a synthetic model that explains both the positioning and timing of cortical morphogenesis in the cell cycle. The key point of this model is the attribution to the AZM of a repressive capacity on the stomatogenic area, the last one being positioned according to the system of gradients of morphogenetic activity proposed by Jerka-Dziadosz to explain location of primordia in urostylids. This repression is manifested not as a gradient, as indicated by De Terra, but as a long-term repression limited to a certain distance. Simultaneous repression and stimulation occurring in a growing cortex with the AZM remaining constant in size could explain the critical ratio, buccal cortex/somatic cortex, at which stomatogenesis is triggered as indicated by De Terra.

OR more than 30 years (1 l), attention has been focused F upon the study of morphogenetic processes in ciliates. As this approach has proved to be a valuable taxonomic criterion, several attempts at revising Kahl’s classic taxonomy of different taxa of ciliates have been published. Reviews dealing with the Order Hypotrichida include that published by Faurk-Fremiet ( 1 2) , who divided the order into two suborders, Stichotnchina and Sporadotrichina, each containing four families. Both sub- orders have been accepted by De Puytorac et al. (47) and Corliss (6, 7 ) in their revisions of the major taxa of the Phylum Cil- iophora partly on the basis of their morphogenetical pattern. Levine et al. (42) also accept the Stichotrichina and Sporado- trichina as suborders in their recently revised classification of the protozoa. Another attempt which deserves special mention is that published by Borror (2 ) , more recently outlined in two papers ( 3 , 4), in which the genera of Hypotrichida are separated into six families, their morphogenetical patterns being part of the basis of this classification. Since the available morphoge- netical data are still fragmentary, however, gaps necessarily exist in such schemes and many genera are only tentatively placed. This is the case of the genus Laurentiella described by Dragesco et al. (9, 10). Finally, Hemberger (30) filled a great number of these gaps in the most extensive study realized at present on the taxonomic application of morphogenetic traits in hypo- trichs.

There is also a great interest in the study of mechanisms of control of the cortical production, positioning, timing, and pat- terning of new organelles which appear in the cortex during the morphogenesis of ciliates, because they are processes of cellular differentiation that occur in primitive eukaryotes.

Hypotrichous ciliates, due to their structural complexity, have been used preferentially by many authors interested in this sub- ject; Jerka-Dziadosz (33-4 l), Frankel (1 5 , 16) and Grimes (1 8- 27) are particularly noteworthy. On the other hand, others- e.g. Tartar (52). De Terra (53-58), Pelvat (46)-have preferred heterotrichous ciliates for the study of morphogenesis, mainly because of the ease of microsurgery on these ciliates. Aufder- heide et al. ( I ) have recently reviewed the state of knowledge of cortical morphogenesis in protozoa.

In this report, the different variants of the basic morphoge-

I The authors wish to thank Dr. Jerka-Dziadosz for criticism of the manuscript.

netic pattern that are produced in Laurentiella acuminata ( 1 3 ) after both physiological (division and reorganization) and ex- perimental (regeneration) stimulation are described. These re- sults are discussed with the aim of offering a model explaining satisfactorily both the perfect synchrony of cortical morpho- genetic processes with the cellular cycle and the evolution of the stomatogenic pattern among Hypotnchida. The taxonomic and phylogenetic position of the genus Laurentiella is also discussed.

MATERIALS AND METHODS All the observations reported below have been made on a

strain of Laurentiella acuminata, a hypotrichous ciliate isolated from a sample of water collected in the “Parque de Maria Luisa” (Sevilla). (Laurentiella acuminata is synonymous with Lauren- tia acuminala [ 13,59,60] and Onychodromus acuminatus [32].) It was cultivated in Pringsheim’s medium at 20 -t 1°C and fed daily with Colpidium sp., growing in the same medium with integral flour added and previously filtered, or with Chlorogo- nium sp. that had been concentrated by centrifugation. The last food organism was maintained in the same medium supple- mented with sodium acetate, meat extract, yeast extract, and bacteriological tryptone, all at 1o/oo (w/v).

We induced full reorganization of the ciliary apparatus by

-starvation for more than 24 h, after which cells ceased motion, sank, altered their shape, and began physiological reorgani- zation.

-treating starved cells (having no food in cytoplasm) with urea (3% [w/v] final concentration) for 30 sec, causing lysis of most of the AZM and subsequent synchronous regeneration.

-microsurgical removal of a cell fragment.

To see different stages of the following morphogenetic process (except for division and reorganization which are asynchronous processes), we fixed treated and untreated animals from the same culture every half hour and stained them with protargol (61). Dividing and reorganizing cells were observed in random sam- ples of growing or reorganizing cultures respectively.

RESULTS Morphology

Laurentiella acuminata (Fig. 1) is oval with a broad anterior end and a pointed posterior end (1 3). The AZM extends to at

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520 J. PROTOZOOL., VOL. 30, NO. 3, AUGUST 1983

Figs. 1-23. Figs. 1 4 .

Laurentiella acuminala. Protargol method. 1. Ventral view of an individual showing the pellicular and subpellicular organelles. AZM = adoral zone of membranelles; 1 (BC) =

buccal cirrus; (2-5) FC = frontal cim; I = inner paroral membrane; LMC = left marginal cirri; Ma = macronuclear node; 0 = outer paroral membrane; RMC = right marginal cim; TC = transverse cirri; VC = ventral cirri. 2. Oral primordium (OP) is just appearing at left and close to the first transverse cirrus (TCl). 3. Simultaneous emigration and proliferation of kinetosomes of the OP. 4. Club-shaped OP from which protrudes a line of kinetosomes which finally becomes fragmented: anterior and posterior tracts are indicated by small arrows.

least half the length of the body. The frontoventral cirri (FV) are arranged usually in six longitudinal rows which form during morphogenesis but become difficult to recognize in adult ciliates. Specimens with a higher number of FV rows of cirri are rela- tively frequent. Row I is represented only by a paroral or buccal cirrus 1 (BC) located anterior to the paroral apparatus. Row I1 consists of 4-6 cirri running parallel and very close to the outer (0) and inner (I) paroral membranes, which delimit the right border of the peristome. Row I11 usually is made up of seven

cirri; it runs parallel to row 11. These three rows are made of cirri larger than the remaining ones and begin in the same level at the anterior end of the animal. Row IV extends from about the middle of the frontal field to the transverse cirri (TC) and consists of 7-8 cirri. Row V, made up of 6-8 cirri, extends from about the level of the posterior end of the peristome to the transverse cirri. Row VI runs parallel to the right marginal cirri but, in most of the cases, a break in continuity is observed, so that the anterior tract comprises 3-5 cirri and the posterior one,

MARTIN ET AL.-MORPHOGENETIC PATTERN IN LAURENTIELW AC‘UMINATA 52 1

Figs. 5-8. 5 . The early stage of the organization of the OP (small arrow) into membranelles is simultaneous with the alignment of fronto- ventro-transverse streaks and the formation of the paroral primordium (PP) of the opisthe. Fragmentation of streak I1 can also be seen (large arrow). 6. A more advanced stage in the organization of the OP (small arrow). Continuity between proter and opisthe primordia is suggested (large arrow). Note that kinetosomes of the PP are more sparse than the FVT streaks. 7. Left and right marginal primordia (LGM’sP, RMG’sP) of both proter and opisthe are developing at right of old marginal rows. Small arrows indicate gaps in continuity of old marginal rows. Frontal cirri 1-3 and the inner paroral membrane (1) of the opisthe are still visible. 8. Patterning of the ventral ciliature is concluding and the tinal positioning of the ventral ciliary organelles is beginning. Note the different size of cirri of the frontal field (1-5), a short “surplus” ciliary streak (arrow). and the new dorsomarginal kineties (nDmK) now arising from the anterior edge of the new marginal rows.

3-6 cirri. Transverse cirri, usually 5 in number, are located just behind the posterior end of the frontoventral rows, their inser- tion track being J-shaped. One row of cirri is situated on each margin ofthe animal. The left marginal row starts on the middle region near the peristomial vertex.

Three caudal cirri are located in the posterior end of the body.

Dorsal cilia are short and inserted in typical cortical pits ar- ranged into a large number of dorsal kineties, which have a complicated distribution in vegetative forms.

Four macronuclear nodes arranged like the number 7 and a variable number of micronuclei situated near them compose the nuclear apparatus, whose DNA content, nuclear cycles, and

522 J. PROTOZOOL., VOL. 30, NO. 3, AUGUST 1983

cell cycle alterations were studied earlier in our laboratory (29, 59, 60).

Morphogenesis of Division A few kinetosomes appear at the left border of the first trans-

verse cirrus (TCI) (Fig. 2), as the reorganization bands of the macronucleus have already run about ‘11 of the length of each node. Kinetosomes increase in number forming a patch which extends anteriorly (Fig. 3) and eventually reaches the proximity of the peristomial vertex. The resulting oral primordium (OP) is club-shaped, rounded anteriorly, and pointed posteriorly. From its anterior end, a group of kinetosomes diverges and parallels the right peristomial margin (Fig. 4) and finally segregates as a line of kinetosomes, the anterior tract of which, after breaking, becomes the ciliary streak I1 of the proter (Figs. 4, 5). The posterior tract becomes the ciliary streak I1 of the opisthe (Fig. 5) and two more primordial streaks arise at both sides of its enlarged base; the left one becomes the paroral primordium (PP) of the opisthe; the right one becomes the ciliary streak 111 of the opisthe. Some parental c im disintegrate and rearrange their kinetosomes into the ciliary streaks appearing next, which usually are three for each tomite. Some of these streaks at the beginning are continuous with those corresponding to the other tomite (Figs. 5, 6). Sometimes more than six streaks develop. Frequently no transverse cirri or no ventral c im arise from these “surplus” primordia (Figs. 7, 8, lo), explaining known varia- tions and discordance between number of transverse cirri and number of cirral rows (1 3).

Development of cirral primordia is accomplished by growth, in both length and width, of thin streaks and posterior frag- mentation in cim, which progresses as an anteroposterior wave, transverse cim being the last and largest (except for the four frontal cirri which derive from the two anteriormost cirri of the rows I1 and 111) (Figs. 9-1 1). While this happens, the rest of the OP differentiates into membranelles. Alignment of membra- nelles starts at the anterior right edge of the OP and progresses diagonally (Figs. 5-8). The paroral primordium (PP) of the op- isthe develops as a long anarchic field of kinetosomes nearly parallel to the right border of the OP and remains connected with streaks I1 and I11 until the start of organization of the endoral membrane. The paroral membrane and the buccal cirrus arise by the arrangement of remaining kinetosomes of the PP (Figs. 5-9). Thus, the FVT system of the opisthe derives from usually six cirral primordia; three of these come from the OP and three (sometimes more) originate by proliferation and rear- rangement of kinetosomes from old ventral c im (Figs. 5-10).

The parental AZM and the endoral membrane are inherited by the proter without any appreciable change (Figs. 7, 8)

The paroral membrane undergoes a partial disorganization affecting its anterior tract; the kinetosomes of this tract lose temporarily their linear arrangement and, later on, rearrange and form again the paroral membrane and a new buccal cirrus (Fig. 10).

The FVT streak I1 of the proter comes from the anterior tract of the row of kinetosomes extending from the OP (Fig. 5); ki- netosomes ofthe remaining FVT streaks result, as in the opisthe, from disintegration of frontal cim. These primordia also are arranged in a variable number, mostly six, of parallel streaks (Figs. 7, 8, 10, 11).

New marginal cirri appear later than the FVT system, and are built from streaks always formed closely at the right of the old rows and from kinetosomes disaggregating as orderly lines of kinetosomes from successively disintegrating marginal cirri at two sites on each side, one anterior and the other posterior, to the future fission plane (figs. 7,8, 1 1). Anterior and posterior elongation of each primordium and subsequent fragmentation

forms new marginal cim. Old marginal c im and the infracilia- ture of disintegrated ones disappear after cytokinesis. The dorsal ciliature derives from nine kineties in two sets. One set of three kineties develops by proliferation of some kinetosomes of three parental dorsal ciliary rows: a deciliated region appears in old kineties near each short developing kinety (Fig. 13). A new kinety starts growing at the posterior end of the kinety closest to the right margin, developing in synchrony with the other two (t’ig. 14). When the last kinetosomes of these three kineties reach the posterior end of the future tomites, they become organized in small basal plates, which result in the three new caudal c im (Fig. 15). A second set of six kineties radiates from the anterior limit of each new developing right marginal row and then ex- tends to the posterior end (Fig. 12). All dorsal kineties appear composed at first of densely packed pairs of kinetosomes, only one being ciliated early.

All ciliary organelles- ventral, marginal, or dorsal - which do not participate in morphogenesis disappear after cytokinesis.

Morphogenesis of Reorganization and Regeneration Irrespective of the type of morphogenesis-divisional, reor-

ganizational, or regenerative-in L. acuminata, a common scheme of renewal of the ciliary structures is apparent. It is in broad lines similar to that of the opisthe, the main difference being the stomatogenic pattern exhibited in each case. The sto- matogenic pattern of physiologically reorganizing animals is in- distinguishable from dividing ones, except that there are no reorganization bands in the macronucleus when the new OP appears close to TC 1 (Fig. 16). Similarly, urea-induced regener- ants show no reorganization band when the new kinetosomes of the OP appear not only near TC 1 but, surprisingly, also near TC2 and a number of c im of the IV FV row (Figs. 17, 18); TC 1 disintegrates afterwards and old cilia are incorporated into the developing OP (Fig. 18). Long axonemes, however, cannot be seen in more advanced stages of the development. The major peculiarity of the stomatogenesis of these regenerants is that a new group of kinetosomes appear close at the left of each of four or five posterior c im of the meridional and centrally placed FV row IV which will become incorporated into the developing OP (Figs. 17, 18).

Microsurgically-induced regenerants may or may not retain their AZM. The topographic origin of kinetosomes of the OP is different in each case. In individuals whose AZM has been damaged, the OP has an origin identical to that already dem- onstrated for the urea-induced animals (Fig. 19). In individuals with an intact AZM but which have lost a posterior fragment, the origin of the OP is similar to its origin in reorganizing and dividing animals (Fig. 20). It is also noticeable that animals with a damaged AZM begin to regenerate earlier than those whose AZM remains intact (delayed I h), but we have not quantified this difference accurately.

After both physiological and traumatic stimulation, the de- velopment of the OP is similar to that described for dividing animals but the function of these new paramembranelles is to replace a reduced number of parental ones which resorb in situ at the posterior end of the AZM (Figs. 21, 22). A new paroral primordium, formed as in division, also fuses, as do new para- membranelles, with an anarchic field which comes from the reorganization of preexisting paroral membrane (reorganiza- tion) or a remaining fragment (regeneration) (Figs. 2 1, 22). The remaining old ciliature is also replaced by only one set of cirral and ciliary primordia, similar in number and developmental pattern to that set present in the proter or opisthe during di- vision.

It is also noteworthy that at the end of each cortical reorgan- ization or regeneration, as during division, some micronuclei

MARTIN ET AL.-MORPHOGENETIC PATTERN IN LA URENTIELLA ACUMINATA 523

Figs. 9-12. 9, 10. Patterning of paroral and fronto-ventro-transverse cilialure in the opisthe and proter respectively. Note formation of buccal cirrus ( 1 ) from the paroral membrane in both tomites. 10. Formation of c im from a “surplus” primordium. 11. Positioning of new structures is in progress when macronuclear nodes fuse. The position of Ev rows is the same as in the vegetative form; however, the rightmost F’VT row is still continuous. Note also that old structures are not yet resorbed. 12. Six dorsomarginal kineties (nDmK) arise close to new right marginal cirri (nRMC) in an opisthe. The kineties are composed of single rows of argentophilic ellipsoid granules. From each granule a single cilium arises laterally. The extent of the ellipsoid granule suggests the existence of two kinetosomes per ellipsoid, only one of them being ciliated.

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Figs. 13-17. 13-15. Successive stages in the development of the dorsal kineties. 13. Formation of three dorsal kineties from kinetosomes belonging to old dorsal kineties. Note the gaps originating at the same level as the new kineties. The old cortical pits are still visible. 14. A new dorsal kinety appears at the right of the posterior end of the rightmost new one (arrow). 15. Formation of caudal cirri at the posterior end of three of the four new dorsal kineties (arrows). Fragmentation of new dorsal kineties can also be observed. 16. Kinetosomes of the oral primordium appear close to TCI in an animal reorganizing after physiological stress. 17. Appearance of new kinetosomes of the nascent oral primordium close to transverse cirri I and 2 (TC1, TC2) in a urea-induced regenerant.

MARTIN ET AL.-MORPHOGENETIC PATTERN IN LAURENTIELLA AC‘UMINATA 525

Figs. 18-23. 18. Incorporation with the growing oral primordium of cilia coming from the disintegration of TI (large arrow) and from new kinetosomes formed close to cirri of the equatorial tract of IV ventral row (small arrows) in a urea-induced regenerant. 19. Surgically induced regenerant with damaged AZM. Note the incorporation into the developing OP of “the novo” kinetosomes formed close to cirri of the IV FV row (arrows). 20. Surgically induced regenerant with undamaged AZM. Note that there is no participation of ventral cirri in the formation of the developing OP. 21, 22. Two successive stages in the process of fusion of the parental reorganizing paroral apparatus and the remaining parental niembranelles with respective primordia produced during reorganization (arrows). 23. Definitive positioning of new structures and resorption of old ones havc finished. A difference in size of new membranelles in respect to old ones is still noticeable. Renodulation of the macronucleus takes place, resulting in the characteristic multinodulated form. This reorganizing animal illustrates the last step of both reorganizing and regenerating animals.

526 J. PROTOZOOL., VOL. 30, NO. 3, AUGUST 1983

divide mitotically and the number of macronuclear nodes in- creases to eight usually (Fig. 23); however, two nuclear appa- ratuses are not formed.

We have frequently observed that during reorganization a little cytoplasmic fragment is lost as a bud. This fact is in ac- cordance with the observation of atypical forms that appear in starved cultures from which reorganizing animals are selected for fixation.

DISCUSSION Different authors offer different explanations for the control

of differentiation of ciliary organelles in ciliates (1 4-1 6, 2 1, 24, 26, 27, 33, 36, 41, 46, 51-53, 57, 58, 62) (for a recent review see 1). The variable stomatogenic pattern exhibited by Lauren- tiella acuminata offers an excellent opportunity for discussion and comparison of this ciliate with others, with the aim oftesting the applicability of the various hypotheses and the possibilities of complementing them.

Our most interesting finding is the change of stomatogenic pattern exhibited by regenerants losing, chemically or micro- surgically, an anterior fragment of their bodies, in comparison with dividers. The formation in AZM-damaged L. acuminata of new kinetosomes of the OP close to TC2 and to the ventral cirri of row IV is an anteroposterior amplification of the area of appearance of the OP which has become confirmed in other oxytrichs similarly damaged (5,45). This reinforces the previous assumption of the existence in all hypotrichs of an “organization area” (33, 4 I ) , or “stomatogenic area” (36), or “determinative region” (19-21). There appears to exist a median ventral area with similar properties to that which occurs in urostylids (1 ,36) in which unknown determinative factors are responsible for the formation of the OP, whose location is assumed to be controlled by a Cartesian system of gradients of morphogenetic activity. However, our data suggest that this area can be larger than is normally manifest. Positioning of this area after alteration of the topographic limits of the cell cannot be explained accurately, however, by the “positional information” hypothesis adopted by Jerka-Dziadosz & Frankel (1 5, 16, 36, 4 1). In fact, the ap- pearance of the OP close to TC1 and TC2 in urea-regenerants, the latter cirrus rarely participating during division or during reorganization, could be explained satisfactorily by the posi- tional information hypothesis as a consequence of the new in- formational value acquired by each point of the cortex remain- ing after reconstruction of the hypothesized anteroposterior gradient of morphogenetic activity subsequent to the loss of an anterior fragment. This autoregulation, however, cannot explain the participation of several ventral c im of row IV as “structural guides” (1 5 , 16) of new kinetosomes that are being incorporated into the developing OP, because it supposes an amplification of the natural stomatogenic area of the species towards the anterior part of the body. The more plausible explanation is that these ventral cirri are normally included in a quiescent part of the stomatogenic area, which only shows its total capacity when the anterior end of the ciliate is lost. We think that the partial or total loss of the AZM located on the anterior cytoplasmic frag- ment that is lost selectively during the treatment with urea is the reason for this anterior amplification of the stomatogenic area in regenerants that was described earlier. This conclusion is supported by the fact that microsurgical loss of a posterior fragment ofthe body does not lead to such anterior amplification although autoregulation of the anteroposterior gradient ought to lead to a forward shift of the stomatogenic area. An alternative way to explain this enhancement of the stomatogenic area in regenerants of L. acuminata is to assume a repressive effect of the AZM (or related subjacent microtubular cytoskeleton) on the stomatogenic capacity of this area. Examples of repression

by the AZM on stomatogenesis are not rare. Tartar & Uhlig (52), for instance, have found that in Stentor the possibilities of development of an OP in an artificially implanted stripe-con- trast zone (stomatogenic area in Stentor) increase with the dis- tance of the site of implantation from the natural one. Aufder- heide et al. ( I ) , in a manner somewhat presaging this discussion, said “. . . The same mechanism could maintain spatially graded relation of developmental dominance and subordination within a single cell or cell region, so that, the area with potential to produce primordia of ciliary organelles can be much larger than the area that actually produces the primordium in the intact cell.” The microsurgical exchange of AZM between stentors belonging to strains differing in AZM size also alters the timing of stomatogenesis in such a way that a repressive effect on sto- matogenesis has been ascribed to the AZM by De Terra (57). Finally, Hyvert et al. (3 1) have shown that implanting a small fragment of AZM in the cytoplasm of regenerating stentors de- lays stomatogenesis as compared to appropriate controls.

The delay that we have found in microsurgically damaged AZM regenerants compared to undamaged-AZM regenerants of L. acuminata (similar delays were found by Jerka-Dziadosz in promers compared to corresponding opimers in Urostyla grandis [33] and Pseudourostyla cristata [34]) can also be ac- counted for by an inhibiting action of the old oral membranelles existing in promers, reorganizants, and dividers-all three mor- phogenetic variations showing, in accordance, an identical sto- matogenic pattern.

From the above data, it is not unreasonable to assume the coexistence in the growing cortex of L. acuminata of two coun- terpoised activities responsible for the unusual stomatogenic behavior of this species; one of them is the morphogenetic ac- tivation of the cortex containing the determinative region whose stomatogenic area is positioned centrally “B la Jerka-Diadosz,” the other is a repressive effect of the AZM or related infracilia- ture on this capacity. This repression should be exerted at a limited distance but not as a gradient, as indicated by its all- or-none response to stimulation. On the basis of both antago- nistic activities, moreover, an explanation for other cytologic features of L. acuminata common to hypotrichs and other spi- rotrichs can be offered.

According to De Terra (53, 55-58), since the cortex grows during the cell cycle, a cyclic triggering of ciliary reorganization must be expected at a critical cell size, proportional to AZM size. In our opinion, this happens because the posterior end of the stomatogenic area escapes from the repressive influence of the AZM. In agreement, during division and reorganization, this derepressed posterior end appears to include the TC1 as the sole structural guide for new kinetosomes of the OP, but during posttraumatic regeneration, TC2 and ventral meridians act as indicators of the extension and derepression, respectively, of the stomatogenic area.

Thus, on the basis of the foregoing considerations, occurrence of reorganization can be explained as a cortical morphogenetic process, starting as in a normal predividing cell when critical size relative to AZM size is attained. The macronuclear reor- ganization bands are lacking, presumably because of the scarcity of DNA-precursors after starvation (the method of induction). The increase in number of the macronuclear nodes, also ob- served in Gastrostyla and Histriculus ( 5 , 4 9 , simultaneous mi- cronuclear mitosis (requiring few DNA-precursors), and the loss of a little cytoplasmic fragment-all taking place at the end of the reorganization (the first two also during regeneration) and at the same stage of cortical development as during macronu- clear division-reinforce our suggestion that the physiological reorganization is an abortive division. (For more detailed dis- cussion on the nuclear cycle and its alterations in L. acuminata,

MARTIN ET AL. - MORPHOGENETIC PATTERN IN LA URENTIELLA ACUMINATA 527

see 59,60.) In our opinion and concurring with that of De Terra (54) the occurrence of macronuclear re-nodulation and micronu- clear mitosis during regeneration indicate cortical control of nuclear events (46, 60).

The extreme synchrony of the urea-induced regeneration and the delay in the start of morphogenesis in microsurgicaily in- duced regenerants (i.e. those in which the AZM is not damaged as compared to those with a damaged AZM) are two features which agree well with the above postulated model. They are explained by the absence or persistence respectively of an intact repressive AZM with the capacity of “stabilize” the organization area; however, the occurrence of delayed regeneration on AZM- undamaged L. acuminata indicates that the alteration of the somatic cortex is also a stimulating factor of regeneration. In this paper we do not present data indicating the nature of either determinative or repressive factors assumed to exist in the cortex of the hypotrichous ciliates, but we would point out that the subpellicular palisade-like layer of microtubules already dem- onstrated in other hypotrichs (1 7,48, and Torres, personal com- munication) offer ultrastructural support for the transmission of both positional and repressive information by allosteric-co- operative means proposed in the gradionation hypothesis of Roth & Pilhaja (49). Finally, the extension of such a model to Sporadotrichina in general is possible in view of recent results obtained in our laboratory, showing that the above described change of stomatogenic pattern is a rule in Gastrostyla steinii (9, Histriculus muscorum (43 , and in species of the Oxytri- chidae.

The major innovation of the model discussed above, is the incorporation of the repressive character of the AZM on sto- matogenesis as a new cortical factor that modifies the spatial expression of the stomatogenic area postulated by Jerka-Dzia- dosz and explains the chronological coordination of morpho- genesis with the cell cycle. The explanation for the timing of morphogenesis in the life cycle is the cyclic derepression of the posterior end of the stomatogenic area due to cellular growth.

A second topic of discussion on the results offered by L. acuminata in the present study is the taxonomic position and phylogenetic significance of this species.

The morphogenetic pattern displayed by L. acuminata rein- forces its inclusion among the Oxytrichidae, a position which had earlier been justified on the basis of its morphology (2, 13) and discards the original proposition to include it among Ho- lostichidae (9, 10) which was recently accepted by Tuffrau (63). In fact, the development of FVT system of cirri from about six cirral primordia (five bearing transverse cirri), the appearance of the OP adjacent to TCl , and the reformation of dorsal cil- iature from two sets of kineties of topologically different origins are all morphogenetic features shared by oxytrichous ciliates (see 44 for revision) (28, 50).

Laurentiella acuminata shares with its congeners, L. mac- rostoma (9) and L. monilata (lo), and with Onychodromus grandis and Gastrostyla spp. ( 5 , 45, and personal observation), a tendency to maintain in its vegetative form dense lines of middle-sized FV cirri which reflect the primordial streaks from which they arise. Such a trend characterizes stichotrichs (1 2), a group whose more evolved forms, species of the genus Parau- rostyla, recently have been proposed to belong to the Oxytri- chidae (4, 63) because of the great similarity between the mor- phogenetic patterns of the two groups. Therefore, we can conceive of the genus Laurentiella as being well placed among Oxytri- chidae as proposed by Borror (2) but as a primitive stem genus phylogenetically connecting directly a part of Stichotrichina and Sporadotrichina (44). This is not in agreement with FaurC-Fre- miet’s original idea; he considered both suborders as divergent evolutionary lines ( 1 2). Present data are also an experimental

support for Corliss’s intuitive assumption (7) that the paraki- netal stomatogenesis characterizing “evolved” forms of hypo- trichs (Sporadotrichina) is derived from the parakinetal one characterizing more primitive hypotrichs (Stichotrichina). In effect this stomatogenic pattern “reappears” in Oxytrichidae only when the AZM become reduced during induction of re- generation, so that the ratio AZM size/body size or buccal cor- tex/somatic cortex artifically reaches values closer to those nat- urally existing among stichotrichs in predivision stages. Thus we consider that the stomatogenic patterns existing among Spo- radotrichina are more evolved and derived directly from that shown by Stichotrichina as a consequence of a drastic increase of the proportion of AZM size/body size in Sporadotrichina. This conclusion is supported by the presence of a stomatogenic pattern identical to that of AZM-damaged regenerants of L. acuminata, an oxytrich with a natural reduction ofAZM relative to the body size, which appears to be a secondary tendency among Oxytrichidae (44).

The development of dorsal ciliature also reinforces this sug- gestion. The dorsal development of L. acuminata agrees well with that described for other closely related oxytrichs such as Onychodromus grandis (personal observations) and Gastrostyla steinii ( 5 , 45) and more evolved ones-Stylonychia mytilus (2), Stylonychia grandis (30), S. pustulata, Oxytricha sp. (22, 30), Histriculus muscorum, H. similis ( 5 , 45)-and with that of sti- chotrichs such as Paraurostyla weissei (4 1) and P. hymenophora (25 ) . There is, however, a surprising similarity both in number and initial development between the six kineties radiating in L. acuminata from the anterior limit of the new right marginal primordia, for which we propose the name dorsomarginal ki- neties, and the five or seven marginal primordia which arise in Urostyla grandis or in Pseudourostyla cristata respectively (35) from within rows or from a common anarchic field appearing at the right of old marginal rows. This conclusion is strongly confirmed by the recent finding ofa set of five primordial streaks on the right margin of Pleurotricha tihanyensis (30), only three of which become marginal rows. The other two presumably will become dorsomarginal kineties. As recently shown in Stylo- nychia mytilus ( 2 1, 26), dorsomarginal kineties appear inevit- ably at the right of both left and right old marginal rows when they are experimentally implanted in the dorsal face. It seems highly probable that the coexistence of dorsomarginal kineties with right marginal rows is due to a common evolutionary or- igin. That no similar kineties develop from left marginal rows may be explained by the ventral surface having morphogenetic properties different from the dorsal surface (1, 14).

The evolution of the dorsal ciliature of hypotrichous ciliates, a topic which currently receives special attention in our labo- ratory, is a new morphogenetic criterion offered to taxonomists to resolve both taxonomic and/or phylogenetic problems in the Order (43). The relatively high number of dorsomarginal ki- neties of L. acuminata further supports the assumption that L. acurninata is transitional between FaurC-Fremiet’s suborders which, in our opinion, can no longer be considered as divergent evolutionary lines (for more current reviews on this subject see 44, 64, 65).

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Received 7 I V 82; accepted 23 V 83

J ProrozooL, 30(3), 1983, pp. 529-535 0 1983 by the Society of Protozoologsts

Morphology and Morphogenesis of Fuscheria terricola n. sp. and Spathidium muscorum (Ciliophora: Kinetofragminophora)

HELMUT BERGER, WILHELM FOISSNER, and HANS ADAM Institut , f i r Zoologie der Universitat Salzburg, Akademiestrasse 26, A-5020 Salzburg, Austria

ABSTRACT. The morphology and morphogenesis of the kinetofragminophoran soil ciliates, Fuscheria terricola n. sp. and Spathidiurn rnuscorum Dragesco & Dragesco-Kerneis, 1979, are described. Stained specimens (protargol) are characterized biometrically. The new species differs from the other species of the genus in its body size, body shape, number of kineties, length of extrusomes, and habitat. Both species have telokinetal stomatogenesis, which commences with a proliferation of kinetosomes at those kineties which bear the brosse. Fuscheria terricola does not have a complex perioral ciliature; indeed, it might be that this species has only monokinetids. Thus only a proliferation of kinetosomes and the separation of the kineties takes place in the prospective division furrow. In contrast, S. inuseorurn differentiates short dikinetid kinetofragments in the region of the division furrow, which are arranged to form the perioral kinety of the opisthe in the intermediate and late stages of the stomatogenesis. The right part of the penoral kinety develops first. This and other studies show that telokinetal stomatogenesis proceeds very differently depending on the differentiation of the oral ciliature; however, detailed studies on the morphogenesis of kinetofragminophoran ciliates are still too few in number for subtypes to be defined.

EW detailed studies about the morphogenesis of free-living F gymnostome ciliates are available (7, 14), perhaps because they are so difficult to culture. The stomatogenesis of the ki- netofragminophoran ciliates is telokinetal (5). The published descriptions of stomatogenesis reveal that it proceeds in very different modes, indicating that this voluminous and probably artificial group is very heterogeneous. Telokinetal stomatogen- esis obviously requires classification into subtypes ( S ) , but this should not be done until more data are available. Toward this end, we have investigated the morphology and morphogenesis of two ‘‘lower’’ ciliates.

MATERIALS AND METHODS Two populations of Fuscheria terricola were investigated.

Population 1 occurred in the soil of a bottomland near Grafen- worth, Lower Austria. Population 2 was isolated from soil in the Schlossalm, Bad Hofgastein, Salzburg. Both populations were cultured by the method of Foissner (10). For revealing the in- fraciliature the protargol silver staining method according to Tuffrau ( 1 9) as modified by Foissner ( 1 3) was used. The silver-

’ The authors wish to express their thanks to the Austrian MaB-6 programme of the Austrian Academy of Science and the Bundes- ministerium fur Gesundheit und Umweltschutz for financial support.

line system was studied in specimens impregnated by the Chat- ton-Lwoff silver method (3).

Spathidiuin muscorum occurred in soil in the Schlossalm. Identification was made according to the descriptions of Dra- gesco & Dragesco Kerneis (6) and Foissner (1 2). As a culture medium, Eau de Volvic was used, with yeast and a species of the “Tetrahymena pyriformis” complex added as the food sup- ply. The protargol method was used to reveal the infraciliature (13, 19).

All statistical procedures follow methods described in Sokal & Rohlf ( 1 8).

RESULTS Fuscheria terricola n. sp.

(Figs. 1-1 7, Table I) Diagnosis. In vivo ca. 80-100 pm in length and ca. 27 p m in

width (n = Body cylindrical to slightly bottle-shaped. Fif- teen somatic kineties on the average. Many 5-7-bm-long extru- somes.

Type location. Moderately frequent in the soil ofa bottomland near Grafenworth, Lower Austria.

Protargol impregnation generally causes a shrinkage of about 20- 30%.