The Opalinid Infusorians: Flagellates or Ciliates?

THE OPALINID TXFUSORIANS 107

25. Polge, C., Smith. Audrey L-. & Parkes, A. S. (1949). Re- vival of spermatozoa after vitrification and dehydration at low temperatures. .Ynf?trr, 161, 666.

26. Saunders, G. M. & Scott, Y. (1947). Preservation of Plasnzodiirni t h a x by freezing. Science, 106, 300-301.

2 i . Scharf. J. F. (1954). Survival of an amoeho-flagellate after freezing. Biodyizaiirka, 7, 225-228.

2s. Smith. .\udrey U. (1954). Effects of low temperatures on living cells and tissues, in Harris, R. J . C., Biological A p p l i - cn t iom of Frrezing aud Drying, Academic Press. Inc., S e w I’ork, 1-62.

29. Smith. Audrey U. 6r Polge, C. ( 1950). Survival of spermatozoa a t l a w temperatures. S a t w e , 166, 668-6619,

30. Smith, Audrey U., Polge, C. 6r Smiles, J . (1951). Micro- scopic observation of living cells during freezing and thawing. J . Roy. Microscop. Soc., 71, 186.195.

31. Weinman, D. (1953). African sleeping sickness trypano- eomes: Cultivation and properties of the culture forms. Afziz. X . Y . Acad. Sci., 56, 995-1003.

3 2 . Weinman, D. 8. McAllister, Joan (1917). Prolonged storage of human pathogenic protozoa with conservation of viru- lencc: Observations on the storage of helminths and lepto- spiras. A m . J . H y g . , 45, 102-121.

3.3. Wolfzon, Fruma (1945). Effect of preservation by freezing upon the virulence of Plnsniodirim for ducks. A J J I . J . Hyg., 42, 15 j - l bb .

J . PRUTOZOCJI.., 2, 107-114 (1955)

The Opalinid Infusorians : Flagellates or Ciliates? JOHN 0. CORLISS

Department of Zoology, University of Zlliizois, Uubuna, Illinois

SUMM.4RP. The controversial question of the affinities of lthe enigmatic opalinids is discussed in the light o i recent findings and with regard for modern ideas concerning characters of most significance in the phylogeny of various major protozoan groups. Metcalf’s “protocilia’te” hypothesis still retains a high popularity although many of the factors supporting it have been adequately disproven during the past 20 years. The “ciliate-” and “flagellate-like” characteristics of the Qpal- inidae are listed and reviewed.

Considered to he one of the most significant of mastigophoran features is the “symmetrigenic” mode of fission, a character generally disregarded in the past. Coupled with nuclear monomorphism and a syngamous reproductive cycle ithe type of division presents a very strong argument in favor of closer affinities with the zooflagellates than with the ciliates. I t is stressed that the opalinids are highly differentiated forms, not likely primitive or ancestral for any major group of protozoa, which have had a long evolutionary history of their own. Grasst’s recent erection of a new super-order in the Zooniastigophora to contain the family Opalinidae is defended and commended.

K VIE\Y of the current revived interest in the phyl- graphs Rletcalf (51) separated the family Opalinidae I ogenj- and systematics of various seemingly aber- from the “regular” astomes, a move which received rant groups of animal organisms, the classical problem immediate and strong support from most biologists. of the enigmatic “opalinid jnfusorians” and their Subsequently Rletcalf (52,53) , by then already the taxonomic position in the phylum Protozoa may ap- recognized authority on the group, erected the category propriately be aired once again. Such consideration “Protociliata” and set it apart from all the other Cilio- seems particularly in order because of recent pertinent phora which were named the “Euciliata.” His views. findings and the modern shifting of emphasis regarding or a t least his taxonomic arrangement. were widely characters held to be of value in the “new systematics” accepted and are the ones most commonly followed to- of protozoa in general; reevaluation of the pros and day. In very recent years rebellion against his sys- cons of the alleged affinities of the Opalinidae will be tematic conclusions has arisen, instigated particularly attempted keeping these factors in mind. by French protozoologists.

First seen by Leeuwenhoek in 1683’, the opalinids, Hundreds of Papers have been Published dealing in almost without exception inhabitants of the large in- whole or in Part with these fascinating protozoa: only testine of tailless Amphibia, were placed by early tax- a few of them will be directly cited here. Details of onomists with the now well-known astolnatous ciliates, historical interest are available in Metcalf ( 5 1.54,56) : also exclusively symbiotic organisms. Calkins ( 7 ) excellent references to more recent studies, particularly maintained them in this group of holotrichous ciliates of a cytological nature, will be found in GrassC(33). long after other protozoologists had removed them Ha11(35) and Klldo(46). from it. In the first of a series of classical mono-

The date of Leeuwenhoek‘s discovery of these protozoa is still generally given as 1685 although Dobell(2-l) has pointed out clearly that the earlier date is the correct one. The generic name O p t d i ~ i ~ was created 1 5 2 years later, by Purkinje & l-alentin(62).

POPULARITY OF THE PROTOCTLTATE HYPOTHESIS

Before turning to the characteristics of these controversial animalcules it may be worthwhile to con- sider, albeit very briefly, why hlelcalf’s “protociliate“

10% THE OPALINID INFUSORIANS

hypothesis has gained and held such favor in spite of the fact that “opalinids differ from the typical ciliates in almost every respect except the possession of cilia” (39 ) , a statement very few protozoologists would dis- pute.

( 1 ) The theory does allow the opalinids to fill the popular role of the “missing link” between flagellates and ciliates; historically this may have influenced thought on the subject, consciously or unconsciously. The superficial “ciliate-look” is obvious and cannot be denied.

( 2 ) A number of assumptions or supposed facts which support Metcalf’s theory of the “primitive” nature of the opalinids and which are often still accepted actually have been or can be shown to be er- roneous, in part or in full: such as the beliefs that only these protozoa undergo acentric mitoses or show a dis- tinctive pleurinuclear condition ; that each opalinid nucleus contains distinct L‘tropho”’ and “idiochro- matin”; that the nuclei “rest” in different mitotic stages; that a primitive excretory system is present; that their type of fission is sometimes “longitudinal.” sometimes “transverse.” Metcalf insisted that the Opalinidae are “pivotal forms, instructive . . . as to the origin of the Euciliata,” and one of the strongest bits of inferred evidence was the alleged presence of .‘micro-” and “niacrochromosomes” presumably homologous with ciliate micro- and macronuclei.

( 3 ) The once popular interpretation of an “amphi- nucleus” in certain homokaryote examples among the true ciliates ( E . K . , cf. m’oodruff, 7 1 ) lent indirect support to ciliate affinities for the likewise homokaryote opalinids. Some support even came from an opposite direction, a number of investigators insisting that certain cytoplasmic inclusions, the endoplasnlic spherules or “endosarc bodies,” were homologous with one or the other of the ciliate nuclei.

(4 ) A significant influence, not to be overlooked, in perpetuating an “anti-flagellate,” even if not an ardent .po-ciliate,” viewpoint regarding the affinities of the opalinids may well be related to the rather weak or sometimes even fallacious arguments which have been proposed from time to time purporting to prove their mastigophoran nature. The earliest proponents of the notion that the Opalinidae are flagellates tended to base their reasoning on single facts, such as Hartog (38) who emphasized the inonomorphic nature of the nuclei. or Neresheimer (60) who early pointed out the flagellate nature of the life cycle. Upon Metcalf’s en- trance into the fray such arguments became minimized or reinterpreted. Of no help a t all to the cause were the rather dogmatic assertions by Gatenby and King (32) and Kofoid and L)odds(45), in preliminary no- tices of work never published in full, that these or- qanisms should be classed among the flagellates on the

basis of certain anatomical features. These claims. never corroborated by subsequent workers, involved the relationship of the locomotor organelles to various cytoplasmic inclusions in the body ; the .4nierican team even described extranuclear paradesmoses. A number of workers have homologized certain structures with the flagellate parabasal apparatus. conclusions no longer held to be valid today.

The most powerful “pro-flagellate” evidence was published some years ago(9) but the paper was brief and, until quite recently, received very little attention. Also these investigators conservatively left the opalinids in Metcalf’s taxonomic category, althouqh point- ing out how certain of their characteristics ( to be discussed below) would allow them to be considered as forms intermediate between true flagellates and ciliates. This was not to be interpreted as meaning that the Opalinidae fill a direct-line “missing link” role. Had this significant work followed rather than pre- ceded the next paper by the same workers( 13: also discussed in section I1 below) its taxonomic implications might have received niore emphasis at their hands. Nevertheless the first paper has had a strong influence in the recent decisions by French and certain other protozoologists to include the Opalinidae as a family of higher zooflagellates(e.g., 26.28.29.33.41, 5 0 1 ) .

VALID CHARACTERISTICS OF THE OPALINIDS

From the point of view of alleged ta\onomic affinities the valid characteristics of the opalinids may be considered to fall into three groups: those features which appear to be very ciliate-like. those which are strongly flagellate-like, and those of much less significance which might be argued as suppx t f o r either side in the controversy. Curiously enough. this highly differentiated group of symbiotic protozoa appears to have no absolutely unique features, Metcalf notwith- standing; their distinctiveness lies principally in their unusual combination of characters.

I. Ciliatc-like Charactus. 1 . Although generally not listed by the propments

of the RIetcalf school of thought, the most clear-cut ciliate feature of the opalinids is their exhibition of an acentric nuclear division ; that is, no centrioles have ever been detected at any stage of the life history. Tt is generally accepted that the true ciliates likewise lack centrosomes or centrioles although intranuclear ones occasionally have been described in the older literature. Metcalf actually considered the lack of this

2 In this work, incidentally, success was first achieved in cu!- turing certain opalinids free from their host in an awnic med- ium.

THE OPALINID INFUSORIANS 109

type of ‘.kinetic center’‘ to be a unique character for the opalinids.

2. The presence of numerous, short locomotor organelles arranged in more or less longitudinal rows covering the entire body, functioning like the cilia of good holotrichous ciliates and presumably related to a typical infraciliature or silverline system, has always been employed a s the strongest evidence for the opalinids’ ciliophoran affinities. This cannot or need not be en- tirely refuted but can be considered to have been weakened by the realization that flagella and cilia (and sperm tails! ) ultrastructurally may be identical [there are dozens of recent good investigations employing the electron microscope which now attest to this] and that all such organelles originate from or are in intimate association with a subpellicularly located basal granule, whether the latter be called a blepharo- plast. kinetosome, or something else. Furthermore, the role of the rows of “cilia” in the morphogenetics of fission (see section IT, below) is quite different from that shown by the holotrichous ciliates which super- ficially the opalinids resemble so closely. The ciliary rows or, more precisely, their infraciliary counter- parts. the kineties, of true ciliates are fundamentally independent, autonomous, asymmetrical, bipolar structures: most of these properties are not shown by the somatic ciliary rows of the opalinids, as shall be discussed i n more detail below. The presence or absence of a longitudinal fibril (= the kinetodesma?) joining the basal granules of the infraciliature is still a dis- puted matter in the literature; the matter is perhaps of limited taxonomic value, anyway, as it or its homolopue may be present in members of both protozoan groups.

TI. Flagdlatc-like Characters. 1. The possession of monomorphic nuclei, two or

many, depending upon the genus, is an undeniably strong mastigophoran character. At one time the dis- cover!’ of various strains of ciliates possessing but a single nucleus. the niacronucleus, was thought to be of great significance(e.g., cf. 2,64,71); it is now realized that amicronucleate races are of value in showing the important role of the macronucleus in the life history of ciliates but that such strains do not possess specialized “amphinuclei”( cf. 1,20,28). Cer- tain cases of honiokaryote ciliates do remain enigmatic, for elample that of Stephnnopogon nzesnili described by Lwoff (4s ) .

The hypothesis of macro- and microchromosomes and other misinterpretations of the nuclear picture in opalinids has been completely overthrown by the beautiful work of Chen( 14,15,16). The “macrochro- niosonies” turned out to be true nucleoli, structures, incidentally, much like those found in various meta-

zoa and in certain flagellates. particularly in the symbiotic higher zooflagellates so intensively investigated by Cleveland during the past fifteen years. ”Nucleoli” have begun to be reported in the macronuclei of ciliates(cf. 28) but they are practically submicroscopic in size and their relation there to “chromosomes” can only be a matter of conjecture a t the present time.

2. By the existence of true sexual reproduction in their life cycle, through complete fusion of anisogam- ous gametes, the opalinids show a closer relationship to any of the other groups of protozoa than to the Cili- ophora. The sexual phenomenon of conjugation, a tried and true ciliate characteristic, may be considered quite different from true syngamy, apart from the meiotic process, in spite of the fact that in some specialized groups the conjugants secondarily have come to resemble “gametes.”

For a long time evidences of sex have not been found among the zooflagellates; in the ”lower” forms, such as the Trypanosomatidae, clear-cut, irrefutable evidence is still lacking but in the “higher” orders the recent brilliant researches of Cleveland have clearly demonstrated its presence(l8,19; and cf. 70 for a concise review of much of this work). The sexual cycles of these zooflagellates are under hormonal con- trol of the insect host; in the case of the opalinids there is also suggested some intimate relationship to the life cycle of the amphibian host although future investigation of a refined nature will be necessary to determine its exact nature. Syngamy has been reported in scattered cases among the true ciliates: most of these require reinvestigation and,‘or reinterpre- tation in light of our modern knowledge of sexual phenomena among the protozoa.

3 . The mode of fission in opalinids is strictly flagellate-like; this is perhaps the most important of all characteristics linking the opalinids with the Mastigo- phora although it has been little emphasized or discussed in the literature. hletcalf(e.g., 51,54) has insisted that the Opalinidae exhibit two kinds of binarj- fission, “transverse” and “longitudinal.” But his few figures are not convincing, details are lacking. and no other investigator, to my knowledge, has found other than “longitudinal” division. What is basically most important in this whole matter is an understanding of the fundamental modes of fission possible among protozoa; consideration of these will require a brief di- gression here.

Chatton and Villeneuve( 13), in a paper generally as neglected as their earlier one(9; vidc supra) , present a careful although brief critique of modes of division and rightly conclude that the commonly employed terms “transverse” and “longitudinal” are in- adequate, occasionally misleading, and lacking in both precision and general application. Although most

110 THE OPALINID INFUSORIANS

flagellates divide in such a way that the plane of fission passes through the longitudinal or antero-posterior axis of the organism some entire groups show modi- fications of this, for example, the Dinoflagellida which commonly exhibit an b*oblique” type of fission. And there is at least one example in which species belong- ing to the same genus exhibit what seem to be strik- ingly opposed modes of division, one showing the ”longitudinal” type, the other, a “transverse” type:< ( 1 7 ) . In the less highly differentiated ciliates the plane of fission typically passes “across” the body at an equatorial level, a t right angles to the longitudinal axis of the body. But certain groups show ‘boblique” (e.g., the Thigmotrichida) or “longitudinal” ( c . g . , the F’eritrichida) division.

Chatton and Villeneuve introduce as more precise, and therefore more useful, terminology the descriptive words synmetrigcnic and homothetigenic: no matter what the angle of the plane of fission, two fundamentally symmetrical, mirror-image filial organisms are produced in the case of flagellates, two homothetic4 daughters, the so-called proter and opisthe, result in the case of ciliates. Examples formerly considered aberrant among both groups, some of which are men- tioned above, fit perfectly into one or the other of these two mathematically defined categories without crossing taxonomic boundaries. In the more complex forms considerable morphogenetic movements may have to occur in the parental organism before symmetrigenic or homothetigenic fissions can be success- fully realized. And regular de- and redifferentiation of certain cytoplasmic structures are often involved. I n the less specialized holotrichous ciliates the homothetigenic division cuts through the ciliary rows or kineties (srission percine‘tienne“) , each daughter organism receiving half of each of the parental kineties

Returning to the morphogenesis of fission in the opalinids, from the papers describing it with accuracy (e.g., 9 ) , it can be seen that it is of the symmetrigenic type. The plane of division is parallel to the longitudinal, or oblique, rows of locomotor organelles, the latter being separated into two groups in the result- ing daughter organisms. Thus although the Opalini. dae strongly resemble typical holotrichous ciliates in their superficial covering of “cilia,” the somatic kineties of the parent are not a t all transected as they are

3 Kirby(42) was aware of the essential simllarity of division in these two flagellates in spite of the apparent great dissimil- arit?.

-I 4 condition of homothety is realized by two forms which are similar and similarly placed, with pairs of points in a one- to-one correspondence This may be contrasted with the mirror-image condition seen in symmetry. j For escellent accounts of the views of the Chatton school

on these and rclated matters the reader is referred to Chatton ( 8 ) or, for a more accesaib!e source, to the summarized ireat- ment in Yilleneuve-Brachon( OS, pages 6-12).

in the ciliates. This is a difference of such a funcla- mental nature that it alone might be considered to set this enigmatic family far apart from the ciliated protozoa.

Whereas the somatic kineties of ciliates are autonomous, self-duplicating structures ( v ide strpra, page 109), those of the opalinids appear to be of a secondary nature completely dependent upon certain so-called generative kineties which are located in the apico-ven- tral region or margin of the body, more or less a t right angles to the roNs of somatic ”ciliature.“ This mul- tiple “kinetic center,” the falx in the terminology of Mohr( 5 7 ) , Cosgrove( 23) and others, is composed of basal granules larger in size than those elsewhere on the body and bearing heavier .‘falcular’‘ cilia; it is bisected in the symmetrigenic division of the organism. When an opalinid has completed its plasmotomous fission the somatic rows which each daughter is wanting. in comparison with the number found in the mature parental organism, appear to be proliferated from basal granules of the halves of the generative or pri- mary kineties with which each daughter is equipped. The association or juxtaposition of “head“ granules of the somatic or secondary kineties with the larqe grar: ules of the falx has been described(36) and qood indirect evidence of the relationship is seen in the vary- ing lengths of the rows of locomotor organelles cours- ing the body surfaces; presumably the shorter (incorn- plete) ones grow longer in the newly produced daughter organisms with intercalation of new incomplete secondary kineties as the body widens with growth.

Precise studies of fission in this group of protozoa. employing the most rewarding techniques. such as that of silver impregnation, have been very few in number. Further details of the phenomenon are greatly needed and investigations should be extended to a number of species in the family. It seems unlikely, however, that the basic, if unpublicized. observations of Chatton and Brachon will be refuted by the findings of future workers.

The reports of finding such specialized mastigophoran characters as a paradesmose active in mitosis and a typical parabasal apparatus have never been confirmed and thus may be considered as completely discredited.

111. Ciliate or Flagellate Chararters 1 . Are the locomotor organelles of opalinids to be

known as cilia or flagella? Perhaps it does not matter; we have already noted the striking similarity in the fundamental ultrastructure of them both. their ax- onemes in common being composed of some nine to eleven fibrils(e.g., cf. 4,5.30.61). But it seems there may be at least two pxsible bases for distinction b e tween typical cilia and flagella regarding which only

THE OPALINID INFUSORIANS 111

future work of the greatest precision can provide the answers:

(1 ) The nature of the enveloping plasma membrane or sheath-is the helical structure reported for simple Hagella(4. and other workers) and for the organelles of Opulinn(3) present in true cilia? T o date some investigators have said no, some, yes.

( 2 ) And are the mechanics of motion fundamentally different, as it would seem? Ultimately such differences might be expected to be reflected in the exact anatomical make-up of the organelles, ultrastructurally and molecularly speaking( 30). Locomo- tion in the opalinids appears to be very much like that of typical holotrichous ciliates: but detailed studies of flagellar movement to date have been re- stricted, as far as the writer is aware. to free-living species possessing very few flagella. Do the long flagella of the various multiflagellated symbiotic &.higher” Zoomastigophora function exactly like those of Euglena?

2. Lack of a mouth or cytostome could be considered either flagellate-like, primitively missing, or ciliate-like, secondarily lost as an adaptation to a saprozoic existence. But this is a negative characteristic of opalinids and probably one of no phylogenetic value. Calkins( 7 ) , however, apparently was heavily influenced by this feature in his persistent inclusion of the Opalinidae in the Astomata group of holotrichous ciliates. A furrow in the pellicle between the falcular fibrils has been interpreted as a cytostome by one worker(37) but this view has not met with acceptance among other students of the opalinids.

3. The saprozoic existence of the Opalinidae as synibionts in poikilotherniic vertebrate hosts is of no phylogenetic significance, of course, as far as deter- mining affinities a t the higher taxonomic levels. Met- calf(cf. citations in 54 and 56) has stressed the real and potential values of the Opalinidae in testing pale- ogeographic hypotheses by critical study of distribu- tional data of the parasites and their anuran hosts. There is no doubt that such lines are worthy of pur- suit although hfohr(58) has pointed out some of the pitfalls which must be avoided in obtaining significant results in such investigations. Systematic implications in the “host-parasite” relationships of the zooflagellates associated with termites and roaches have been most profitably discussed by such leaders in that field as Kirby. Cleveland, and GrassC(cj. G r a d , 33, for an up-to-date bibliography on this subject).

SYSTEMATIC POSITIOK O F T H E OPALINIDS Searly 2 50 opalinid species, distributed among four

genera in a single family, have been described. over seventy-five per cent of them by Metcalf, foremost student of the group. Since many of the species, not

to mention subspecies and formae, were characterized from study of not ideally preserved materials and a number were distinguished on the basis of features now known to be unreliable, i t is possible. as some protozoologists have inferred, that the actual number of species known to date should be considerably lower. Nevertheless the highly differentiated opalinids form a distinct group and need to be placed somewhere in the hierarchy of protozoan forms: should they be associated with the subphylum Mastigophora or the subphylum Ciliophora or located in a newly created .‘intermediate” category?

From the discussion presented on preceding pages I believe one can justifiably conclude that the thesis maintaining the opalinids are primitive ciliates or an early offshoot from such forms is quite untenable. On the contrary, their organization seems to show a very high degree of differentiation and specialization in- dicating a long evolutionary history of their own far removed from the main line of development of any other protozoan group. The detailed cytological studies of van Overbeek de Meyer(67) and Fernan- dez-Galiano(31) alone would attest to the great internal complexity of these organisms. The category of “Protociliata” thus becomes inappropriate both taxonomically and in its etymological connotation.

In the “new systematics” of protozoa information gained from study of the processes of differentiation and morphogenesis is beginning to be considered more and more as being of significant value. For the zooflagellates the works of Kirby(e.,a., 42.43.34), unfor- tunately terminated by his most untimely passing in 1952, should be cited in this regard. For the ciliates the investigations and hypotheses of the French schools of Chatton and Fad-Fremiet(c.g. , cf. 5,lO- 12,25-29,34,49,59,68) have been of utmost importance. The role of the ciliophoran infraciliature (roughly synonymous, a t least in part, with the silverline system) in morphogenesis of the body and of the mouth (stomatogenesis) has been stressed : a nearly indispensable cytological technique has been the method of silver impregnation (cf, references just cited above: also 21,22.69). Many precise data have been accumulated of value in redetermining taxonomic affinities of, for example, such more or less enigmatic groups of holotrichous ciliates as the Suctorida. Chono- trichida, Peritrichida, and Thigmotrichida. Critical examination of various stages in the ontopeny of a species, when the complete life cycle of the protozoon. whether the organism leads a free or symbiotic life, is studied, casts considerable light on evolution within the group being investigated and can sugrest phylogenetic relationships with neighboring groups.

As discussed above (c f . section 11, part 3 ) . the mode of fission seen in the Opalinidae is typically symmetri-

112 THE OPALINID INFUSORIANS

genic. that is, of the mastigophoran type. Additional observations on the morphogenesis of division in a variety of opalinids should be made. Yet the inforina- tion already available, principally through the work of C‘hatton and Brachon(9), seems sufficient to sug- gest strong ancestral bonds between the Opalinidae and the higher zooflagellates. This is on the basis of mode of fission alone: the nuclear condition and type of life cycle in their own right also argue very strongly for flagellate affinities of the group. The akaryomasti- gont system of certain zooflagellates, so ably studied by Kirby, is paralleled in some ways by the conditions found in the opalinids. The kinetic center involving centrioles and often some sort of paradesmose in flagellates may be represented by the generative kineties and their specialized basal granules in the falcular region of opalinids where acentric mitoses prevail. That the systems in the two groups of organisms are far from being identical could he considered evidence of long separation in an unknown history of parallel evolution. The division of certain multinucleate zooflagellates of the order Trichomonadida, where the separation of two groups of flagella and two groups of nuclei results in production of symmetrical daughter organisms. i h . in the last analysis, not unlike the outcome of the symmetrigenic fission of the opalinids. Much more study, incidentally, is needed on the mechanics of fission in the highly evolved multinucleate. multi- flagellate Mastigophora.

I n recent years the Opalinidae have been placed formally in the Zoomastigophora by a number of sys- tematists(c.g., 33,41,63). Generally they have been considered to represent an ordinal group located alongside the orders of higher zooflagellates. GrassC( 33), in the most estensive and most thorough modern treat- ment of the matter, has raised the opalinids to the rank of a super-order (Opalinina), in his class Zoo- flagellata, a t an equivalent level with the lower zooflagellates (Protomonadina) and the higher zooflagellates ( Metamonadina) . This decision, presumably made after considerable thought on the subject, allows the opalinids to be considered as “good” zooflagellates although only distantly related to any of the other eyisting nroups of Rfastigophora. I t is suggested that the Opalinidae have independently acquired their few ciliate-like characteristics by virtue of a long history involving convergent evolution.

In general in parts of the Trait6 de Zoologic written by himself Grass6 has proposed iconoclastic and, in the opinion of some protozoologists, rather radical revi- sions of pre-existing taxonomic schemes. He is a *‘splitter“ par excellence and has created many new cateqories at the higher levels, often by elevating for- mer families or suborders to ordinal or supra-ordinal ranks. In the case of the opalinids I believe Grass6

has performed a worthwhile service to protozoology by publicizing their flagellate affinities, by removing them bodily from the Ciliophora, and by dropping the name “Protociliata” with its misleading connotations. His erection of a separate super-order for the opalinids among the zooflagellates would appear to be a sensible and commendable act. Removal of the opalinids from the subphylum Ciliophora, incidentally, with atten- dant abolition of the category Protociliata, obviates the necessity of retention of hletcalf’s other term: Euciliata.

The name of Grasse’s new super-order, Opalinina, would appear to be a somewhat unfortunate choice. although legalistically it probably must be considered acceptable. He has claimed it to be a noiiwtz noz~u11z; actually only its super-order position is new. “Opal- inina” was commonly employed as a familial name in the late 19th and occasionally in the 20th century, fol- lowing the terminology of Stein(cf. 6,47,65,66). J’ irovec et ah.(41) have used the same name a t the ordinal level. Jahn & Jahn(40) have considered the order under the heading “Opalinida,” and if one fol- lows the uniformity in tasonomic style suggested by these workers and by Hall(35) then the name Opal- inina implies a subordinal rank. Grass6 suggests no name to be used if one should wish to say that his super-order“ contains a single order, the -?-.

N o taxonomic case can probably ever be considered “closed.” In the opalinid problem many aspects 3i significance to the systematically minded protozoolo- gist still remain to be investigated or reinvestigated and reinterpreted. In his monumental works Metcalf has laid a thorough foundation for future students of these curious protozoa, but many questions asked by Metcalf ( 5 5 ) himself in an heuristic paper entitied ‘,Research Problems in the Opalinidae” still remain unanswered. If in the minds of many biologists the matter of the true taxonomic affinities of the opalinids still remains controversial this should have the good effect of stimulating new and much needed research on these forms employing the latest techniques and most precise approaches possible. The theoretical significance of the problem alone would make such efforts well worthwhile.

REFERENCES 1. Beers, C. I). (1916). Tillina nzagna: Micronuclcar num-

ber, encystment and vitality in diverse clones; capabilities of amicronuc!eate races. B i d . B d l . , 91. 2.36-271.

2 . Bishop, E . L., J r . (1933). Studies on the cytology of the

(i The writer cannot help but feel that some descriptire supra-ordinal name such as “Paratlagellida” (alongside the flagellates), as contrasted with ‘lEuflagellida” (based on .i name originally proposed by Claus) for all other zooflagellate;, would be more appropriate than “Opalinina.” The Paraflagel- lida would contain the single order Opalinida ; the Euflagellida, the five orders Rhizomastigida, Protomastigida, Polymastigida, Trichomonadida, and Hypermastigida.

THE OPALINID INFUSOKIANS 113

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pp. 983-1004.]

114 ELECTRON h”1cRoscoPY OF Tricho.monas

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The Electron Microscopy of Trichomonas mzcl-is*+

E V E R E T T A N D E R S O N Department o j Zoology, State Zfniversity o j Iowa, Iowa City, Io.ula

SUMMARY. Thin sections of Trichoinonas muris, from the caecum of Mesocricetits auratus, were observed with the aid of the electron microscope in order to determine structural details of organelles of this species.

The blepharoplas,t appears to he limited by a mem’brane. What seem to be basal granules occur in the hlepharoplastic area for the axostyle and the lamellae of the undulating membrane and meas- urc approximately 500 A in diameter.

In cross section the anterior flagella and posterior flagellum are composed of eleven fibrils, two centrally and nine peripherally located. Each fibril measures about 380 2% in diameter.

The so-called “accessory filament” of the undulating membrane is devoid of any filamentous structure and a p p e a s to be composed of two differentiated mesh-a.ork areas. The undulating membrane is composed of a series of lamellae ranging from 300 to 400 in thickness. This organelle is attached t o the external body surface by fine fibers ranging from 167 to 300 -c in thickness.

The costa consists of a series of disks embedded in a matrix. The costa is attached to the internal body surface by what appear to he extensions of the costal disks. These disks are approsi- matelv 370 A in thickness and spaced 490

The axostyle is limited by a double, corrugated membrane. The corrugations are about 150 A in thi’cknes and spaced 110 .k apart. The so-called chromatic ring is composed of a series of rods approximately 640 A in thickness.

The parahasal body consists of a series n l filaments approximately 190 A in thickness. Mitochondria, spheroidal in shape, are limited by a membrane about 1SO A in thickness. Thc

internal structure consists of a varyinz number of projections which make this organelle appear in section as a series of compartments.

Chromatic granules (paracostal, para-asostylar, endo-asostylar and those sca,ttered in the cyto- plasm) are irregular in shape and limited bv a membrane approximately 200 -1 in thickncss. These granules typically display a vacuolated internal structure.

apart.

richomonas nrzrsis (Grassi. 1879) has been found T to inhabit the caeca of many rodents(20,39). In the genus Trichomonas there exists a variety of different kinds of organelles of which the structure, division and reorganization have attracted the attention of protozoologists and cytologists. (For a detailed

* . A revision of a dissertation in zoology presented to the Faculty oi the Graduate School of the State University of Iowa in partial fulfillment of the requirement for the degree of Doctor of Philosophy (1955).

t Grants to the Radiation Research Laboratory from the Iowa Division o i the iZmerican Cancer Society have made possible the purchase and maintenance of the electron microscope.

description of Trichomonas mzrris see Kirby and Hon- igberg( 20) .)

Differences of opinion regarding the details of structure of the organelles of trichomonads exist in the literature. For a Letter understanding of the morphology of these organelles, a detailed picture of their fine structure would appear to be essential. The electron microscope, with its resolving power and magnification far beyond the limits of the light microscope, and the technique of ultra-thin sectioning provide a new ap- proach to the study of these structures.

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The Opalinid Infusorians: Flagellates or Ciliates?