An Aspect of Morphogenesis in the Ciliate Protozoa

lume 14

The Journal of Protozoology February, 1961 Number 1

ROTOZOOL. 14(1), 1-8 (1967).

An Aspect of Morphogenesis in the Ciliate Protozoa*

JOHN 0. CORLISS

Dt-parf mt-nt of Biological Scit-nus, University of Illinois at Chicago Circle, Chicago, Illinois 60680

;OPSIS. Stomatogenesis or new mouth formation sensu Zato rep- nts an explicit example of a major morphogenetic phenomenon ie life cycle of most ciliate Protozoa. Investigations of the process i a number of approaches may yield data of considerable value. .he present paper 3 such approaches are treated in some detail. udies of stomatogenesis can be of importance in providing addi-

information of significance on the structure and physiology I given organism. Five major types or categories of stomato- :sis in ciliates are recognized. These are defined and discussed. mearch related to such an intrinsic value of the phenomenon the k carried out is generally only descriptive, and the most impor-

technic employed is some method of silver impregnation. a comparative appromh is used, then attention may be focused

possible homolcgies in stomatogenesis as i t occurs in diverse .ies of ciliates. Such data are (or potentially are) of value not

in the practical taxonomy of the organisms involved but also onsideration of phylogenetic and evolutionary interrelationships

r i g the higher groups comprising the entire subphylum Ciliophora.

6SEPH Needham, the brilliant British biochemical em- . bryologist, long ago defined the term morphogenesis as le coming-into-being of characteristic and specific form living organisms,” and the word difierentiation as “in- p e in complexity and organization . . . increase in mor- )logical heterogeneity by the appearance of form and .ern.” I t seems to me that, today, studies of what might called “embryology at the protozoan level” fulfill the

.’Je of Needham’s definitions as adequately as investiga-

.)s of metazoan material. Indeed, I believe that many logists will agree that not only has the time long since ‘&ed to consider morphogenesis at the cellular (and even v-ellular) level but that the most fruitful “cells” may if be certain species of Protozoa. Electron microscopy r revolutionized the approaches possible to our under- nding of the cytoarchitecture of such minute forms as k b a e , sporozoa, flagellates, and ciliates ; and combining rilstructural technics with an array of biophysical and chemical methods ( e . g . , most recently electron micro- )pic autoradiography) has begun to yield invaluable data tb respect to integration of form and function. When licient studies have been carried out to permit compara-

%ddress of Past President, Society of Protozoologists, delivered C$llege Park, Maryland, 19 August 1966. Support of National c ce Foundation Grant CB-2800 is gratefully acknowledged.

Such application of comparative studies may well become a very fruitful approach to certain problems in ciliate phylogeny ; 2 examples are discussed briefly. Silver impregnation technics are again indispensable.

Perhaps stomatogenesis will prove most valuable in the hands of developmental biologists who are seeking to solve 2 of the most intriguing problems in cell biology today: the exact origin of new kinetosomes (or centrioles), and their precise morphogenetic role in the life cycle of a cell or unicellular organism. Since this third approach to the study of stomatogenesis is of necessity a dynamic one, the researcher must employ sophisticated experimental technics to obtain data a t the molecular and macromolecular levels of both organization and function. Some progress in this area has already been made, but the availability of “ideal” organisms has perhaps not been as widely realized as it should. Certain of the “higher” ciliates have a mode of stomatogenesis which would lend itself beautifully to fruitful investigation of problems concerning both replication or neoformation of kinetosomes and their possible role in fibrillogenesis in unicellular organisms.

tive analyses-and if the investigators have not neglected earlier information gained from painstaking light microscopic work and from physiological observations on whole as well as fractionated organisms-then we may be in a position to draw some general conclusions of synthetic value to the entire area of cell biology.

Dr. Trager (99) , in his past-presidential address of 4 years ago, presented a most heuristic review of differentiation in protozoa, emphasizing important morphogenetic events which occur in their life cycles and drawing on diverse groups for his examples. In an exciting way, he stressed the value of protozoa in studies of cellular differentiation in general-or, as he modestly stated in his conclud- ing remark, he hoped that he had “indicated the special opportunities provided by protozoa in this central problem of biology.” In addition to Trager’s(99) quite recent review, a number of other analyses, including several classi- cal ones of years ago as well as some very recent ones, are available which treat in a significant way some of the major facets of morphogenesis in protozoa( 1,2,6a,7,7a,1011 1,20- 23,25 - 2 7,30,3 1,34,34a,35a,39,43,47,53,56 - 58,6 1,64,66,67,69, 71-76,79a183,89,92-94,100,101, 105,106,110,111, 117,115.) Thus here I should like to limit my own topic to a single aspect of morphogenesis, stomatogenesis, as it occurs in a

1

1 2 MORPHOGENESIS IN CILIATES

single major group, the Ciliophora, within the phylum Pr+ tozoa. I wish particularly to call attention to a virtually unexplored but potentially very important experimental a p proach to stomatogenesis; but I must first discuss 2 other more conventional ways in which the phenomenon may be utilized in studies of ciliates, since an understanding of such descriptive approaches is an essential prerequisite to the third kind of investigation.

STOMATOGENESIS

Stomatogenesis ( L‘new-mouth-formation,” taken in its broadest sense) represents an explicit and often dramatic example of a major morphogenetic phenomenon in the life cycle of most ciliates. Although it lends itself beautifully to study, the difficulties-in practice-of getting any particular species to cooperate with the investigator should not go unmentioned. Nothing could be more frustrating to the eager student of the phenomenon than a species which might divide but once every 11 months, and then only during a 1-hour period from 2 : 00 to 3 : 00 a.m. ( I don’t believe that any explicit reference to such a negative finding can be found in the literature, but it may be true!)

INTRINSIC VALUE

I t must be agreed that study of stomatogemsis in a given ciliate species is of considerable value purely from the point of view of providing additional data concerning the structure and physiology of the organism itself thruout its full life cycle or ontogeny. The ideal way to make such studies-restricting ourselves for the moment to ,the level of light microscopy and to essentially morphologic investigation-is to employ various methods of silver impregnation, treatments which reveal beautifully the positions of the all-important basal bodies or kinetosomes of the infraciliature and, in some instances, the external ciliature as well. The Chatton-Lwoff and protargol technics have proven themselves to be among the best( 14,16). The latter method, in the hands of the experts (e.g., 17,19,23,40,41,59-61,90, 90a,91,104,105,108,114) delicately stains the cilia as well as the kinetosomes; this is a matter of some importance in the most precise investigations.

Consideration of the relationships-topologic and morphogenetic-of the mouth or associated buccal organelles being produced anew to that (or those) of the parental organism has made possible a classification of the kinds of stomatogenesis which ciliates may have( 15). Note that all terminology-in my treatment of the subject below-comes from but one principal frame of reference: that is, the more or less gross relationship of the newly formed oral com- plexes to those existing in the parental or pre-dividing ciliate in the trophont stage of its life cycle. If there appears to be no direct relationship to the parental oral apparatus then one looks for other sources of the anlage(n), either unique permanent primordia, the kinetosomes of the regu- larly arranged somatic ciliary meridians, erratic non-ciliated (or barren, to use an apt term originally proposed, I believe, by P. C. Bradbury) basal bodies, or special kineto-

somes possibly neoformed from the cytoplasm at certi sites during appropriate times in the ontogeny of the cil ate. The scheme on types or categories of stomatogenes which I proposed 2-3 years ago(15,23) remains a tentati\ one, necessarily awaiting accumulation of much more know edge about many more species before refinement is possibl And there may be overlapping in certain of its categorie Nevertheless, i t will be useful to recall that classificatio briefly here: t

(1) When the anlage(n) of a set of buccal organelles fc the opisthe (posterior filial organism during homothetogeni fission) appear(s) to be directly and solely produced by (

very intimately associated topologically with the (kinetc somes of the) parental oral apparatus (which may or ma not be preserved intact for the proter), the process is clai sified as autonomous. In other words, the old “mouth” ap pears to be responsible for production of the new; hen6 in this sense, the process is autonomous. The hymenostome Paramecium and Frontonia may serve as examples here( 5 i 83,91,118).

(2) If stomatogenesis involves the parental organelle (i.e., their infraciliary bases) to only a partial extent i may be called semi-autonomous. In these cases special pri k mordial, “germinal,” or stomatogenous fields of non-orb and non-meridianal kinetosomes play a role which may b at least as striking as that of the parental buccal baa bodies. Clear-cut recent examples would include species i! the taxonomically enigmatic genera with the long “false‘ names: Pseudomicrothorax and Pseudocohnilembus (23,98) Small(90a), has very recently considered several othe genera in this group, Thigmotrichs and peritrichs, as wa as certain hymenostomes, may be assignable to this cat6 gory(61-63).

(3) When the kinetosomes of one or more of the regula: somatic meridians or kineties appear to be directly involvet in stomatogenesis, the phenomenon may be considered non autonomous or, better, somatic-meridional in nature. Tht parental oral apparatus appears to be involved in no wad? a t least with respect to the new mouth of the opisthe. Therf are 2 sub-categories here, depending on the complexity oi the organization of the oral ciliature. Examples from thi “lower” ciliates include the trichostome Colpoda ( 10 1 ) Among the “higher” forms may be mentioned the weir known Tetrahymena(9a,48,114) among holotrichs: a% Spirostomum and Condylostoma among spirotrichs ( 109 ). A number of genera conventionally assigned here I have rG moved to category 5, to be discussed below.

(4) In some of the allegedly simplest ciliates, in which a complex oral apparatus is absent and the apically locatch‘ new mouth of the opisthe appears to be formed without re; lationship to either the parental mouth or the kinetosomes of the somatic meridians, stomatogenesis may be ternid, de novo cjltoplasmic. Future work may reveal that no q ~ e - cies really belong in this category; it may well be a dubiou: grouping. For the moment, however, the rhabdophoriw

t Only a few examples of ciliate gcnera associated with each 19

these types are given here. More extensive lists may be found in Evans & Corliss(23).

MORPHOCENESIS IN CILIATES 3

gymnostome Didinium may be cited as a reasonable example. In many such gymnostomes the new nematodesmata of the cytopharynx of the opisthe are not yet known defi- hitely to be produced under specific influence of either regu- larly aligned or erratic somatic basal bodies. Thus such oral structures V Z Q ~ have a non-kinetosomal or cytoplasmic de novo origin.

( 5 ) Finally, when the anlage(n) of the buccal organelles of the opisthe appear(s) to arise without any relationship - d i r e c t or indirect-to the kinetosomes of either the parental oral apparatus or the somatic meridians, the process may also be termed de novo,t but with this important dif- ference from the preceding category: the origin of the new oral apparatus is definitely kinetosomal, not cytoplasmic. A field of kinetosomes is involved, but its origin is postu- lated to be distinct from parental buccal or regular-somatic

’infraciliature; nor is the field itself a permanently organized structure, as is found in category 2. The term de novo is here used in contrast to autonomous of category 1 or SO-

matic-meridional of category 3 . In all cases, with the important possible exception of category 4, the ultimate origin of the oral structure is, of course, kinetosomal in nature. Since it is the fully formed new “mouth” and associated structures which are implicated in the term stomatogenesis, rather than the kinetosomal anlage(n) for them, the present category, to avoid unnecessary confusion in terminology and to distinguish it from the preceding cytoplasmic cate- Tory 4, is perhaps best called de novo kinetosoma/. Exam- ples may be found among all orders of spirotrich ciliates: to list a few generic names-Diplodinium, Strombidium, Stentor, Euplotes. (Several works may be cited here-e.g., 7,8a,29,69,94,102,106,109,117-but recall that these investigators, with the possible exception of Noirot-Timothke,69, did not recognize this category of stomatogenesis.) As will be mentioned again later, there is not universal agreement concerning the appropriateness of my assignment of certain genera (such as Stentor and Euplotes) to this category: only further work will resolve such issues.

Consideration of these 5 categories of stomatogenesis raises the whole question of genetic continuity or cytotactic integrity( 2,67,92) with respect to the cortical organelles which we call kinetosomes in ciliates. This fascinating topic can be mentioned only briefly here. No problem exists for those cases in which the buccal or regular-somatic infraciliature of the parent is involved. Even category number 4, de novo cytoplasmic, may not present any great diffi- culty-for future studies will surely clarify the situation: either somatic basal bodies of one kind or another or no basal nematodesmata. Only with respect to category 5 , de novo

ehinetosomal, does the serious question arise: can basal bodies destined to be responsible for formation of complex buccal organelles be organized themselves completely inde- pendently of pre-existing, typical kinetosomes? I suggest that the answer to this is “yes.” I t is true that erratically

t As carefully pointed out in footnote 6 in Evans & Corliss(23, p. 364), usage of the term dr novo by workers such as myself differs en- tirely-and understandably-from that of investigators such as Ehret who are more interested in problems at the nzolectilar level of organization.

distributed, barren basal bodies, located considerably below the cell surface, could serve as the source of stomatogenous fields in those ciliates which I assign to category 5 ; but, to my knowledge, no ultrastructural evidence of this has yet been published. Pertinent data of any kind, however, are admittedly very fragmentary from work done on this problem to date. And the early light microscopic observations of Chatton and colleagues(8) on suctorians and the very recent beautiful electron microscopic work of Dippell (18a) on the gymnostome Didinium show beyond all doubt that erratic, non-cilia-bearing basal bodies do play a role in production of new ciliary structures during division of these particular ciliates. Note, however, that stomatogenesis was not involved in either of the elegant studies just cited.

In any case, my category 5 stands separate from the other 4 ; and it may appropriately be called de novo kinetosomal, whether barren basal bodies lurking deep in the cytoplasm are involved or not.

VALUE IN PHYLOGENY

The preceding discussion has stressed the intrinsic value of stomatogenesis; this aspect has, in practice, been mainly treated by descriptive studies concerned primarily with static stages in the process, involving use of only light microscopic methods and generally restricted to single species of ciliates. (Physiological work, such as recent investigations by Frankel, Stone, Williams, and Buhse, cited later in the present paper, represent important exceptions.) Now I should like to consider-again, rather briefly-a second aspect, viz., the extended value of stomatogenesis when coinparative studies are made and when attention is focussed on possible homologies in various stages of the phenomenon as i t occurs in diverse species. The technics are the same as those mentioned in the preceding section, but the use of the data obtained is vastly expanded. The comparative approach presents the taxonomist with an important additional tool in his practical attempt to determine the most reasonable classification of his organism. From a more theoretical but equally important point of view, such an approach will provide information usable in considering phylogenetic and evolutionary interrelationships among the higher taxa within the subphylum Ciliophora( 10-13,28,35a).

Stomatogenesis is a dynamic process; a t the same time there is, happily, growing evidence that it is a conservative phenomenon. For example, there is much more diversity in patterns of the definitive buccal apparatus, as is abundantly clear from the works of Thompson and others( 12,13,24,95- 97), than there is in patterns of stomatogenesis( 15,23,35, 49,50,90a). This strongly suggests that basic kinds of new-mouth formation may have arisen long ago in the evolution of ciliates and that thus interrelationships among various major taxonomic groups may be revealed thru recognition of their types of stomatogenesis. On the other hand, a word of caution is in order: the old spectres of parallel evolution and of independent origin of similar morphogenetic processes must not be ignored. And we cannot always be absolutely certain that the structures

4 MORPHOGENESIS IN CILIATES

involved are homologous, either. For example, the term L‘membranelle” sensu Zato is now often tacitly considered to include variations ranging from isolated tufts or fields of non-coordinated buccal ciliature of some hymenostomes and the “syncilia” of ophryoscolecids through the true mem- branelles sensu stricto of Tetrahymena and Euplotes to the complex peniculi of Paramecium and the unique p l y - kineties of thigmotrichs and peritrichs(63). In spite of potential pitfalls, however, several examples of fruitful application of comparative studies of stomatogenesis in ontogeny to problems of phylogeny have emerged; and I am optimistic concerning the future. Two arbitrarily chosen instances in which such investigations should prove of great value may be cited:

(1) In the plastic and presumably pivotal holotrich order Hymenostomatida( 10,11,13) there exists considerable un- certainty concerning the exact boundaries of subordinal groups; indeed, even the number of suborders is under dispute among the specialists on species of hymenostomes. Intensive-and much more extensive than to date-studies on the ktomatogenous processes in various “representative” forms will certainly aid in determining affinities and in establishing taxonomic borders. In the reshuffling of pres- ently recognized suborders, generally set up with little aid from studies of stomatogenesis, since such studies were not available at the time, a t least 1 new group may be indicated. This group would contain a considerable number of superficially diverse species having the semi-autonomous mode of stomatogenesis exemplified by persistence of a primordial field of kinetosomes, seemingly unrelated to either the parental oral apparatus or the regular somatic kineties, which functions “in collusion” with kinetosomes from the undulating membrane in production of a new mouth for the opisthe. At least some of the genera discussed by Small(90a) will fall into this group; for example, Pseudocohnilembus, Anophrys, Philaster, Cohnilembus.

( 2 ) A second example involves a still higher level in the taxonomic hierarchy of ciliates, the degree of closeness of phylogenetic affinities among the orders of hymenwtomes, thigmotrichs, and peritrichs. When stomatogenesis is studied, or restudied with greater precision and attention to details, in certain arhynchodine thigmotrichs and both sessiline and mobiline peritrichs, I predict that patterns of stomatogenesis will be found which will bear a startling resemblance to some of those well established for a number of hymenostome species, especially modes belonging to the categories referred to earlier as autonomous and semi- autonomous. Such data would not necessarily be interpreted as supplying evidence for a direct phylogenetic line, hymenostomes-giving-rise-to-thigmotrichs-giving-rise-to-peritrichs; but they would strongly suggest origin for all 3 groups from a common ancestral form or forms. FaurC- Fremiet, Lom, and others( 7a,35,39,40,59,61,63,70,79,83,84) have already laid solid groundwork for investigations of such potentially far-reaching significance in their work on the comparative structure of mouth-parts in species of the 3 orders, investigations which have recently involved electron microscopy to great advantage(e.g., 35,63,70,84).

EXPERIMENTAL MORPHOGENESIS

Finally, in the last topic to be considered here, perhap: stomatogenesis will prove most valuable, or at least most exciting, in providing the developmental or molecular biolo- gist with highly favorable material for solution of 2 ol today’s most intriguing problems in cell biology, namely. the origin of new kinetosomes or centrioles, and their morphogenetic role in the life cycle of a cell or unicellular organism. Modes of replication, which may very well be identical for all kinetosomes and,’or centrioles, as well not a.( the specific functions in which these important intracellular organelles may be involved in various developmental phe- nomena taking place in their micro-environment, still remain ill-understood, although some progress has been made. The rapidity and enormity of events during stomatogenesis in many ciliates-consider, for example, that in less than 4 hours’ time over 15,000 new membranellar basal bodie? are formed, aligned, and organized in Stentor during new- mouth formation(94)-provide the investigator with a beautiful, and practically (to date) untouched, opportunity to catch the kinetosome in the act of doing what it does!

I n this approach to study of stomatogenesis the technics will often differ from those mentioned above in discussion‘- of other aspects of stomatogenesis, even as the goals are different. The process is viewed primarily as a dynamic one and the researcher must attempt to find his answers through the use of the most sophisticated appropriate experimental methods available. An invaluable adjunct to physiologic and cytochemical approaches is, of course, electron microscopy, since an understanding of the macromolecular anatomy of the “kinetosomal territory” ( to use a term created, J believe, by D. R. Pitelka) is indispensable for interpretation of its morphogenetic, “inductive,” or organizational role. I t is possible that extirpation or in situ irradiation or somec other kind of experimental manipulation of all or part of the sltomatogenous area of a ciliate will have to be carried out (more extensively than has been done by anyone to date) to learn the whole story. I t would be ideal, of course, to discover an ingenious method by which basal bodies or entire kinetasomal territories could be grown in a chemically de- finable medium, free from the body; but such a success would appear to be far beyond the limit of our present biochemical abilities, great though they are. However, someone may surprise us in this intriguing area of research.

In stating that the experimental approach to the morphogenetic potential of the kinetosomes involved in stomatogenesis has been practically untried, I do not wish to dis- parage data, directly or a t least indirectly related to the over- all problem, that have already been obtained in a goodly number of works on several ciliates.$ Important contribu- tions in this area include both the experiments and the theoretical considerations published by such workers as Tartar (see especially 94) , Randall and associates (80-52), L‘hlip

.4nd. obviously, research carried out on basal bodies and/or ccn- trioles oi flagellates, other non-ciliate protozoa. algae. and various metazoan and metaphytan cells (in vivo or i n v i t r o ) cannot be ignored by the investigator of kinetosomes in ciliates. But it is beyond the scope of the present paper to cite even major ivorks accomplished with such (other) species as the experimental organism.

A~OHPHOGENESIS IN CILIATES 5

(106,107), and b’eisz (e.g., 110,111) on Stcntor and fol- liculinids: Gliddon(54), Roth(86), Tuffrau( 102,105), and Wise(116,117) on Euplotcs; Ehret(20-22), I’itelka( 72-74), de Haller(21), l’usa( 118), Hanson(57), and Dippell(l8) on Paranzeciuin; Bradbury( 3,4) on the apostomes; Noirot- ‘Timothle( 69), Bretschneider ( 5 ) , and Roth (87) on ophryoscolecids: Faurl-Fremiet and colleagues (e.g., 17,3 1,33,34, 34a,36-38,83,88,103,105), and de Puytorac and students ( e g , 55,77,77a,78,84,85) on diverse ciliates: and the Co- penhagen and Iowa schools on Tetrahynzena (e.g., 6,42-47, ’51,52,65,68,112-115,119). Still others could be cited-such as the exquisite work on synchronization by Scherbaum (see 88a and many papers before and since) and the biophysical investigations in several laboratories on cilia and ciliary movement (see summaries in 75 and 89, for example): the preceding references have been singled out arbitrarily to +serve as examples only, But such researches, for the most part, represent just the beginning and have often only led, ’as might be expected, to new, more sophisticated questions rather than to simple, straightforward answers to the origi- nal queries which were posed.

I t seems to me that the type of ciliate stomatogenesis which could most profitably be investigated to provide answers concerning both replication or neoformation of kinetosomes and their possible role in fibrillogenesis is the one which I have described as de novo kinetosomal. I n these cases-and various ophryoscolecid species of the spirotrich ciliates will serve as perhaps Ithe neatest examplesll-it may be recalled that the new buccal infraciliature arises from what appears to be a spontaneously appearing group or field of kinetosomes(69). I t is reasonable to suppose that these primordial basal bodies cannot have had any previous con- nection, direct or indirect, with the parental buccal ciliature of the organism because of the great spatial separation of the 2 areas; and, certainly in the ophryoscolecids, they cannot have been under any influence of somatic, meridional kinetosomes for there are no organized body kineties in these ciliates. The possibility of heretofore undetected barren basal bodies cannot be excluded, admittedly, as mentioned earlier. But here, with proper material properly handled- plus an enormous amount of luck-ne might thus be en- abled to observe what happens with respect to three principal stages in the entire process:

(1 ) The origin or neoformation of the first kinetosomes comprising the primordial field. What is really their source? What are their precursors in that particular cytoplasmic environment at the macromolecular or even molecular levels, assuming it is not simply a matter of already available but undetected basal bodies scattered erratically thruout the area some distance below the cell surface? When do cilia appear; or are these kinetosomes always barren?

( 2 ) The method of organization or means of replication of these first basal bodies to produce the great mass of kinetosornes soon visible, even with the light microscope, in

,+

l l It is (also) perhaps not generally realized that important ad- vances in the refined laboratory cultivation of ophryoscolecid ciliates are being made in recent years; for example. see the series of physiological papers by Coleman(9, and shorter works preceding and following this 1963 citation).

such an area. Is there solely a one-to-one relationship, kinetosome for kinetosome? \\’hen is the new kinetosome rec- ognizable as such? What role does the surrounding cytoplasm play? \\’hen do cilia appear?

(3) The subsequent organization of the anarchic field of erratic (possibly barren?) kinetosomes into the well-defined rows of membranellar ciliature; and the production of the complex fibrillar systems associated with the definitive, ma- ture buccal organelles. Are the ciliary rootlets, kinetodes- mata, microtubules, or fibrils of any other kind of greater direct significance a t this stage than the kinetosomes themselves?

\\’hether the answers obtained are expected or full of sur- prises, they will be of value. Such basic information would also lay the groundwork for various additional refined experimental approaches. Is successful transplantation of the early field possible? \\’hat happens if chemical or mechani- cal disruption is applied to the area, a t any of the 3 stages described briefly above? Are physico-chemical or even ana- tomic fields or gradients involved in repair and regeneration? And perhaps light could be thrown on one of the most important questions of all: exactly how is the message concerning the kind of “genetic continuity’’ attributable to the non-dividing parental cell’s cortical and buccal infraciliary patterns transmitted during (or preceding) stomatogenesis to the developing filial products? Many more questions could be added to this very short list. Partial answers to some of them are already emerging from the experimental approaches of investigators such as Ehret ( 2 1 ) , Frankel (42), Hanson(57), Tar ta r (94) , and LVise( 116).

If members of the order Entodinimorphida are not the most attractive species to be studied in this experimental- molecular approach to stomatogenesis, forms from various other groups of the subclass Spirotrichia might serve as well or better as the laboratory organism.7 As should be well known from many studies (e.g., 7,8a,l1,23,29,32,69.109, 1 17), quite a number of spirotrich ciliates are characterized by the pre-fission appearance of an intracytoplasmic vacuole or pouch which contains the early anlage(n) of the new buccal organelles. Such highly evolved species may all belong in the same category with respect to their mode of stomatogenesis. Although some specialists may not be in agreement with me, a t the present time I should include under the broad dc novo kinetosoinal type of stomatogenesis such genera as-to nam? only a few well-known ones-Stcntor, Blcpha- r i s w i , Folliciillna, Syctotherus, and Licnophora among the heterotrichs: Euplotcs among the hypotrichs: Strom- bidiuin among the oligotrichs : and Tintinnopsis and lintin- nus among the tintinnids. Species of such genera, and others not named, might be more easily available to the experimen- talist than forms belonging to the entodiniomorphid genera Diplodinium, Epidinbm, Entodinimum, Ophryoscoler, etc.

Thus the choice of material is wide and waiting; all that needs to be done is the work!

7 But recall, as pointed out in / I footnote, that considerable progress is being made in the culturing of species of ophrgoscolecids under controlled laboratory conditions.

6 hIORPHOCENESIS I N CILIATES

REFERESCES

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2 . Beisson, J. & Sonneborn, T. M. 1965. Cytoplasmic inheritance of the organization of the cell cortex in Paramecium aurelia. Proc. No t . Acad. Sci. U S . 53, 275-82.

3 . Bradbury, P. C. 1966. The organization of somatic kineties of an apostome tomite and observations on neoformation of kinetosomes. J . Protozool. 13 (Suppl.), 9.

4. Bradbury, P. C. & Pitelka, D. R. 1965. Observations on kinetosome formation in an apostome ciliate. 1. Micr. 4, 805-10.

5 . Bretschneider, L. 1962. Das Paralabialorgan der Ophryosco- leciden. Kon. Xed . Akad. Welensch. Amsterdam 65, 423-37.

6. Buhse, H. E. 1966. Oral morphogenesis during transformation from microstome to macrostome and macrostome to microstome in Tetrahynrena vorax, strain V2 type S. Trans. Amer. Micros. SOC. 85, 305-13.

6a. Canella, M. F. 1965. Buccal apparatus, stomatogenesis and phylogeny of Ciliata. (Ahstr.) Excerpta Medica, Int. Cong. Ser. S o . 91, 2nd Int. Conf. Protozool., London, Aug. 1965, Progress in Protozoology, pp. 209-10.

7. Chatton, E. 1942. Le prohlkme de la continuite du cinetome chez les hypotriches. Nouvelles recherches sur 1’Euplotes crassus. Bull. Biol. France Belg. 76, 314-35.

7a. Chatton, E . 8; Lwoff, A. 1950. Recherches sur les ciliCs thig- mctriches. 11. Arch. Zool. ExP. G i n . 86, 393-485.

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Hexose and Glycerol Absorption by Some Trypanosomatidae

THEODOR VON BRAND, ELEANOR JOHNSON TOBIE, and H A R R I E T HIGGINS*

U . S. Department of Health, Education and Welfare, Public Health Service, National Institutes of Health, h’ational Institute of Allergy and Infectious Diseases, Laboratory of

Parasitic Diseases, Bethesda, Maryland 20014

SYSOPSIS. Culture forms of 9 species of Trypanosomatidae were studied. Of these, Crithidia fasciculata, Lpishmania brasiliensis, L . donovani, L . tropica, Trypanosoma conorhini, T . cruzi and T . rangeli consumed significantly less glycerol than glucose, both when the 2 substrates were offered singly or simultaneously. On the other hand, T . Ranibiense and T . rhodesiense consumed as much glycerol as glucose when the 2 substrates were given separately. When both substrates were offered simultaneously, more glycerol than glucose was consumed, but the sum of glucose + glycerol carbon taken up ap- proximated closely that taken from glucose alone. This held for species consuming only little glycerol, indicating that in all cases some mutual inhibitiqn took place. Atttempts to adapt T . cruzi to glycerol

YLEY ( 5 ) reported the very interesting observation that the culture form of Trypanosoma rhodesiense by pref-

erence consumed glycerol from a mixture of equal amounts of glucose and glycerol. I n fact, under aerobic conditions the flagellates withdrew no glucose from the solution, while under anaerobic conditions a small amount of glucose was consumed. It seemed of interest to extend these studies to other Trypanosomatidae and to investigate the influence of inhibitors and changes in some environmental factors on the aerobic rates of glucose and glycerol absorption. Some data on the absorption of hexoses besides glucose are also in- cluded.

MATERIAL AND METHODS

Parasitological material. Nine species were studied. All species ex- cept Crithidia fasciculata have been maintained in our laboratory for a number of years and have been used by us in previous studies. Crithidia fasciculata was obtained from Dr. Louis S. Diamond, who isolated it from Aedes solicitans. The media on which the parasites were grown are outlined below and the specific variation employed is associated with each species by number and letter.

*With the technical assistance of Mrs. Flora C. Gilliam.

consumption were unsuccessful. When T . gambiense and T . rhodi siense were cultivated in the presence of glycerol, their normall rapid glycerol consumption remained also unchanged but, curiousl! they consumed more glucose than before “adaptation.” Iodoacetamil‘ and NaF strongly inhibited glucose and glycerol absorption of 1 gambiense ; phloridzin, deoxygalactose and deoxyglucose gave no sik nificant inhibition. KCN slightly stimulated glucose absorption, hu weakly inhibited glycerol absorption. Omission of sodium from t l medium was without effect, while lack of C02 markedly inhibite, glucose and glycerol uptake. Galactose was not a satisfactory sul! strate for T . gambiense and T . rhodesiense, nor were deoxygalacttrs and deoxyglucose. These 3 hexoses were consumed in small amouni only and did not allow maintenance of motility.

The following species were used: Crithidia fasciculata MG-1 strain ( l a ) Leishmania brasiliensis Guatemala strain ( l a ) Leishmania donovani Khartoum strain (lc) Leishmania tropica Tehran strain ( la ) Trypanosoma conorhini ( lc ) Trypanosoma cruzi Brazil strain ( l a ; 1b)

Culbertson strain ( l a ) Tulahuen strain ( l a )

Trypanosoma rangeli El Tocuyo strain ( l c ) Trypanosoma gambiense Cheick strain (2a; 2b) Trypanosoma rhodesiense Wellcome CT strain (2a; 2b) The media used were as follows: la: Diphasic blood agar with 10% blood and Locke’s solution over-

lb: Same as la, but with glycerol replacing glucose in the overlay. Ic: Same as la, but with 30% blood. 2a: Diphasic blood agar with Locke’s solution overlay containing

2b: Same as 2a, but wi?h glycerol replacing glucose in the overlac All species were grown in 200-ml flasks containing 25 ml base a r ?

15 ml overlay. After 7 days growth, harvests were made by pooling the liquid phase from the number of flasks needed to yield enough material for the experiment planned. The flagellates were concen- trated by centrifugation. The packed organisms were washed twicb in Tyrode’s solution and after the final centrifugation were resus-

lay containing glucose(2).

glucose(7).

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