University of Southern California Dissertations and Theses

Studies on the biology of zelleriella (protozoa, Opalinidae)

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Studies on the biology of zelleriella (protozoa, Opalinidae)

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Content STUDIES OH THE BIOLOGY OP ZELLERIELLA (PROTOZOA, OPALIHIDAB) *7 John Arthur Brookes A Dissertation Presented to the FACULTY OP THE GRADUATE SCHOOL UNIVERSITY OP SOUTHHIN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OP PHILOSOPHY (Biology) January 1< ) 6$ UNIVERSITY O F S O U T H E R N CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES. CALIFORNIA 9 0 0 0 7 T his dissertation,'written by ........ Jolm...Arthur ...Brookes u nder the direction of Dissertation C o m mittee, and a p p ro ve d by all its m em bers, has been presented to and a ccep ted by the G raduate School, in partial fulfillm ent of requirements fo r the degree of D O C T O R O F P H I L O S O P H Y j .... ' * Dean D ate ‘ rarjuaI’ I .............. DISSERTATION COMMITTEE .... TABLE OF CONTENTS Page LIST OF ILLUSTRATIONS.................... v LIST OF TABLES.................................. vii LIST OF GRAPHS....................................vlii Chapter I. INTRODUCTION AND STATEMENT OF THE PROBLEM . 1 Introduction Statement of the Problem Aim of the Study Acknowledgments II. MATERIALS AND METHODS Anur&ns Examined Zellerlella Species Studied Obtaining the Opalinlds Permanent Preparations Silver Impregnation Electron Micrographs III. MORPHOLOGY OF THE TROPHOZOITE........... II4 . General Appearance Orientation and Terminology Pelllele Cilia - Infracillature Falx Fibrils Cytoplasm Nuclei Chromosomes and Nucleoli Gigantism ii Chapter Page IV. FISSION AND GROWTH...................... 60 Fission Dimorphism Morphologloal Axes Growth Budding Reduction of Basal Granules V. LIFE CYCLE................... 81 Summary of Life Cycle of Opaline Stnmtary of the Life Cycle of Zeilerlella hirsute Enoysiment Infection Cysts Development of Larval Opallnlds Development of Larval Zeilerlella hirsute Infeetlon Experiments— Materialsand Methods Development of Infection Cysts in Young Tadpoles Development of Infection Cysts in Older Tadpoles Fate of Secondary Cysts Gametes and Syngamy in Zeilerlella Morphogenesis of the Infraolllature Discussion and Conclusions VI. PHYSIOLOGY............................... 103 Influenoe of Host Hormones Cyst Induction In Zeilerlella Cyst Induction— Materials ancT Methods Cyst Induction— Results Cyst Induction— Discussion and Conclusions Bile Pigment Absorption Gradients In Vitro Cultivation VII. ECOLOGY.................... 119 Habitat Associates Host Specificity Geographic Distribution 111 Chapter Page VIII. TAXONOMY ................... U+l Relationship to Other Protozoa Relationships vlthln the Opalinidae Taxonomy of Zeilerlella Inapplicability of non-morphologlcal characters Morphological species characters In opallnlds Morphological Characters in Zeilerlella hlrsuta TBysToTogloally variable characters Ontogenetlcally variable characters Ontogenetically constant characters Conclusions Zeilerlella in the United States APPENDIX I. Regional Check List of Zellerielles and Hosts......................... 177 APPENDIX II, A List of Papers Containing Original Observations on Opallnlds alnoe 1930 and Not Summarized by Metcalf (I94O) . 188 LITERATURE CITED................................ 201 iv TABLE OP ILLUSTRATIONS Figure Page 1* Zeilerlella hlrsuta, Adfalcular Surfaee View T T .......................... 15 2. Zeilerlella hirsute, Anterior Cross Section • ....................... 17 3* Zeilerlella hirsute. Cross Seotion at Level of Posterior Nucleus . . . ............ 17 1|. Zeilerlella hlrsuta. Posterior Cross Section . ........... ........ 17 5. Zeilerlella hlrsuta. Electron Micrograph, Cross Section of Pellicle.............. 21 6. Zeilerlella hlrsuta Showing Anterior and Longitudinal Pellicular Furrows........ 2lf 7* Zeilerlella hlrsuta. Electron Micrograph, transverse Section of Pellicle ........ 27 8* Zeilerlella hlrsuta, Ciliary Rows . . • . . 31 9* Zeilerlella hlrsuta, Detail of Falx .... 31 10, Zeilerlella hlrsuta. Electron Micrograph, Cross Section through Nucleus and Surrounding Cytoplasm................... I 4 . 6 11. Zeilerlella hlrsuta, Abnormal Multlnucleate BlanFTT 7 T T 7 ..................... 57 12* Zeilerlella hlrsuta, Large Normal TropKozoIte ...... ............... 57 13* Zeilerlella hlrsuta. Longitudinal Fission • 66 lif. Zeilerlella hlrsuta^ Budding......... 63 v ^ ■ Figure Page 15-2lf. Polymorphism in Zellerlelle hlrsuta .... 70 25. Morphological Axes in Zelleriella hirsute ............. ......... 70 26. Opaline. Life Cycle •••••••••••• 82 27. Zelleriella hirsute. Life Cyole ...... 82 28. Opaline Gamonts, Gametes and Syngamy ... 87 29. Larval Zelleriella antunea1 ......... .. 87 30. Larval Zelleriella braaillensis ...... 87 31. Zellerlelle hlrsuta. Infection Cyst .... 87 32. Zelleriella hlrsuta. Recently Excysted . * 87 33* Zelleriella hlrsuta. Binucleate Larvae • . 87 3I 4 .- 38. Zelleriella hirsute from Metcalf, I923 . • 173 39* Cyats of Zelleriella hirsute, from Metcalf (1923)................................ 173 vi LIST OP TABLES Table Page 1. Fate of Infection Cysts of Zelleriella hlrsuta ............. . . 97 2* Influence of Chorionic Gonadotropin on Ehcystment of Zelleriella hlrsuta......... 110 3* Distribution of Opalinld Genera among Anur&n Families ....................... 136 If* Distribution of Opalinld Genera among Anuran Suborders ...................... 138 $. Distribution of Opallna and Zelleriella in New World Anurans....................... l£8 6. Characters Used by Metcalf (I923) for the Separation of Species and Subspecies of Zelleriella from Five United States Anurans........................ . 172 vii ♦ LIST OF GRAPHS Graph Page 1* Variation In Rectilinear Ciliary Row Number in Zelleriella hlrsuta.............. 3^ 2. Variation in nuclear diameter In Zelleriella hlrsuta.................................. 51 3. Proportional Variation in Nuclear Diameter in Zeilerlella hlrsuta..................... 52 Vili CHAPTER I INTRODUCTION AND STATEMENT OP THE PROBUBM Introduction The opallnlds constitute a natural group of mouth- less olliated Protoioa endocommensal In the large Intestine of eotothermlc vertebrates,^ mainly anurans. Known slnoe Leeuwenhoek (Dobell, 1932), opallnlds have been studied for quite diverse purposes by investigators In many parts of the world* Physiologists have Investigated eell membrane characteristics and the meohanloal and eleotro- ehemieal phenomena assooiated with the olliary beat* The physical nature of eilla and related elements has been explored through study of the fine structure of pellicular components by eleotron mioroscoplsts* The distribution of opalinld genera in their anuran hosts has led to theories on host phylogeny and migration routes and even past connections between continents. Finally, systematlsts intrigued with the phylogeny of this aberrant group have ^Opallnlds have been reported from a fresh water peleoypod (Yagiu, 1939) * gastropod (Lavler, 1936). In the former they were found in the mantle cavity, in the latter in the intestine. Such infestations are probably facultative, attributable to the feeding of the mollusks on anuran feces containing opalinld infection cysts* 1 expressed «nd debated vigorously a variety of notions on the relationship of opallnlds to other Protozoa* Statenent of the Problast Of the hundreds of publications relating in whole or in part to the opallnlds, the great majority have dealt with two oosmopolltan genera, Onallna and Protoopallna. Apart frost a few species descriptions, a series of papers by Chen (1932a, 1932b, I9I 4 . 8) on nuolear phenomena, and a short paper by Mohr (19^0) on orientation, little has been written on the third genus, Zelleriella sinoe the mono graphic works of Metealf (1923, I9I 4 .O). Knowledge of the biology of Zelleriella Is needed not merely because the gap exists, but because auoh knowledge may aid In the resolution of certain fundamental problems applying to the opallnlds as a whole. Forty years ago, Metoalf established oertaln precedents In taxonomic methodology for the opallnlds whioh have been accepted more or less uncritically by the majority of later workers* Of particular significance for the modern student of the group was Metcalf's practice of describing opallnlds from preserved hosts* Opallnlds so obtained, while usually sufficiently intaot to permit Identification of genus, lack the structural details needed for the separation of speoles. In spite of the drawbaoks, Metoalf persistently named speoles and even subspecies fro* museum specimens* some of which had boon in preservative for over eighty years* Coaqparlaon of proparly flxad opallnida fro* fraah hoata with tha litera- tnra daaeriptiona ahowa thaaa lattar to range fro* Inadaquata to unreoognlsable. Such da tail a aa r amain in preserved opallnlds* for axampla approximata body dimanaiona9 broad outline* nuolaar aiia and nuelaar ahapaf were* largely by default* hold to ba of value for aeparating apeoles* Inoludad alao in this eatagory were auoh artifaeta of fixation aa plioa- tion and toraion of tha body and certain anomaloua inolualona of tha cytoplasm* A oritioal review of thaaa methoda haa been given by Mohr (19ifla)« Implicit in Metoalf*a uaa of museum materials was a aeoond souroe of error* The number of hoata of a given speoies examined* limited by tha extant of tha oollecticm, waa often am all* Many opallnida have bean named from a single host specimen* Sinoe tha observation by Karaahaimer <1907) that opallnida undergo diminution In sise and form oyata in tha spring* numerous workers* inoluding Metoalf* have pointed out that the morphology of any opalinld population is* to a considerable degree* dependant on tha season of the year* Reoent studies by Bleniars (1950)* MoConnachle (i960)* and El Mofty and Smyth (I96I 1) have shown that fission frequency and growth potential in opalinld* refleots tha endoorine atata of tha host* A proper approach to understanding either host- lnduoed variation or normal lntraapeclflo Tarlatlon in opallnlds would saan to ba ona based on comparative norphologloal studies of a given spaeias frost numerous host Individuals* In most oases, Metoalf was In no posi tion to undertake eoaq>aratlve studies of opallnlds from several Individuals of a single host speoles due to the paucity of museum material available* However, he and his followers failed to do so even when live hosts were avail able In abundance* Por this reason, norphologloal varia tion In opallnlds is poorly understood* The reoent study by Vessenberg (19^1) on morphogenesis In Ooallna represents a start toward suoh understanding but, In Its emphasis on olarifleatlon of taxonomic problems at high levels, falls short of resolving speoles difficulties* The Impaot of this uncertain taxonomy has been transmitted both to protozoan systematists and to workers in other areas* Many of the former, alert to the defi ciencies or bewildered by the vagaries of opalinld morphology and reluotant to add to the confusion, have refused to have anything to do with the group at all* With the exception of Chen*s careful study of the opallnlds of Bufo valllosps no taxonomic work has been undertaken on opallnlds from Worth American anurans for the past quarter century. Calrna (1953) In a major study on transfaunation and boat apaalflolty of enterlo Protoaoa of anurans, frequently transferred opallnida with other protista but refused to inaluda raaulta In hi a report baaauaa of tha oonfualon axlatlng relative to tha relationship of larval to adult forma. Thla omission la partleularlj unfortunata alnea comprehensive data on tha experimental tranafar of opallnida la lacking and tha ganaral qua»tIon of hoat apaalflolty la atlll somewhat unsattied. McArthur (1955) for tha aana reason, and beoauaa ha vaa unabla to ldantlfy opalinld apaolaa with certainty, laft than out of hla study on antarle Protoaoa of Rana plplens during larval davalopnant and metamorphosis. Plainly, tha major problem within tha opallnida la an unrallabla taxonomlo literature brought about by unerltloal acceptance of faulty methods. Its solution depends first upon tha establishment of dependable apeeles charactera for eaoh genua, and second upon tha redeaorlp- tion of most. If not all, opalinld apaolaa. Identification of valid oharaeters can coma only through testing tha oonstanoy or variability of daaorlbabla features in properly fixed and stained preparations from populations obtained from an adequate number of hosts* Lass urgent, but rather more Intriguing, la tha problem of opalinld phylogeny. Following Metoalf (1918a, 1918b and several later papers), It has been assumed that Interfamlllal relationships ara reflected by s Haeckelian recapitulation during ontogsny in which all gansra begin development in tbs oondltlon of tbs most primitive sub genus, (bis "Protooeallna. Subganus I"), and prograss through larval stagss sorrsspondlng to tbs phylogsny of tbs group until tbs definitive oharaotsr Is reaohed* Metoalf supported his phylogenstlo soheae with evidence adduced from the gsographlo distribution of his various opalinld genera and subgenera anong their anuran hosts* From these data ha developed an elaborate theory of anuran evolution and Migration routes which, despite severe orltlelsa, persists In nuoh of the protozoological liter ature* At higher taxonomic levels nuoh debate has been and is still carried on with respeet to tha relationship of opallnlds to other protosoan groups* Long classed as dilates, sons recent workers have aligned then with the flagellates while others hold to dilate affinities or place than in various intermediate positions* Evldenoe for intergroup relationships as well as for relationships to other Protoaoa has been based chiefly on the study of comparative morphology together with resemblances or differences in life cycles* Detailed morphological Investigations have been limited to Qpallna and Protoopallna. Certain characters considered trust worthy in establishing relationships by earlier workers are nov known to bo unreliable. Other characters, regarded by modern protoxoan systematlsta as highly useful hare been neglected. Foremost among these is the arrange ment of the meridional rears of olllary basal granules at various stages of the life cycle, With the ezoeptlon of Wessenberg (1961), students of the opallnlds have paid soant attention to this norphologloal unit. Studies on opalinld life eyoles have similarly been limited to Opaline and Protoopallna. thorough investigation being restrieted to the former. The life oyole of Zelleriella la completely unknown. It la apparent that evidence for relationships adduced from the morphology, morphogenesis, and life oyeles of opallnlds Is Incomplete, Extension of knowledge In these areas to all representative members of the family would seem requisite to the establishment of acceptable phylogenles. Aim of the Study It is the primary purpose of this work to extend general knowledge of the biology of the Opalinldae through the presentation of new information gathered from the study of the little known genus Zelleriella. Raphaels is plaoed on comparisons within the family. The adequacy of existing and potential taxonomic characters is examined in terms of constancy or variability both in the adult and in 8 the larval host* It le hoped that this study contributes to a better understanding of biologleaX relationships within the opallnlds and to other protosoan groups as well* Acknowledgments Gratitude is expressed to Professor J* L* Mohr for his enoouragemant, direction and counsel throughout the course of my study and for making available his extensive collection of opalinld slides, record books and reprints* Particular thanks are aooorded Professor R* F* Bils, who prepared the thin sections of Zelleriella and took the eleotron micrographs used in this paper* Appreciation is expressed to the remaining members of my dissertation committee, Professors Robert Chew, Horman Fertlg, and Kenneth Stager, for their continued Interest and advice* Professor T* T. Chen kindly allowed me to view his exeel- lent slides of Zelleriella and offered several useful suggestions on microtechnique* Mrs. A* E* Hoare has assisted me in numerous ways, among them the translation of the lengthy papers of Dimas Fernandes-Galiano and Juan Carlos Otamendl* Mr. Ira Pearlman contributed his artistio talent to the drawing of Z. hirsute shown in Figure 1* I wish, finally, to express gratitude to my wife, Betty Brookes, for help in typing the manuscript and, more importantly, for her patience, understanding and enthu siasm during the course of these studies* CHAPTER II MATERIALS AHD METHODS Anurans Examined Zeilerlella were obtained from representatives of 17 different anuran speoles in the course of the present investigation. Critical study vas limited to sellerlelles from 3 Berth American hosts. By speoles and number of individuals examined these were: Bufo Say, 11 adults and 2lf$ tadpoles; Bufo woodhousll Girard, 18 adults and 116 tadpoles; Bufo alvarlus Girard, 12 adults. Larval sellerlelles were also studied In tadpoles of Bufo oanorus Camp. Other species examined but whose sellerlelles were not studied critically are listed in Appendix I, Zelleriella Species Studied Metcalf (1923) described Zelleriella hlrsuta from Bufo cognatus and an unnamed sellerlelle from Bufo wood" houall. His remarkable description of Z. hlrsuta. from a preserved host, Is Inadequate for comparison with my material from B. cognatus. Zelleriella obtained by me from B. cognatus. B. woodhousll. and B. alvarlus are identical. They are here treated, for convenience but 9 10 without certainty of their equivalence,- as Zollsrlolla hlrsuta, Metcalf’s taxonomlo treatment of the Zeilerlella of forth American anurans is considered in groatsr detail In Chapter VIII. Adult anurans vsro pithed and the rectum was removed together with about one half Inoh of Ileum. Tad poles were killed by severing the spinal cord with a dlsseetlng needle and the entire Intestine was removed. Intestines were plaeed In a Syraouse dish oontalnlng either the buffered saline solution developed by Yang (i960) for In vitro oultlvatlon of opallnlds or the buffer solution used by Vessenberg (1961) In his study on the life history of Opallna. Yang’s proved the more suitable in Its osmotle characteristics, the other solution produolng slight swelling In Zeilerlella left In It for more than a few minutes. Yang's buffer (pH 7*5) Is prepared as follows s Obtaining the Opallnlds Had If grams Na2HP0jf KH2P0Jj. HaHCO^ KC1 . 1.1 grams 0.3 grams O.lf grams 0.2 grams 0.05 grams trace CaCl2 MgSOlj. Enough distilled water to make one liter 11 The gut vail of adult anurans or oldsr tadpolaa vaa opanad by making a longitudinal inolalon aorosa tha lleo-reetal Juncture. In young tadpolaa with undiffsr- antlatad raeta tha gut vaa opanad in several plaeas with a needle* A avail amount of fluid from tha araa immadlataly adjaoant to tha inolalon vaa vlthdravn and transferred to a slide for observation under the mioroaoope* If opallnida vere present and lire observations only desired* the remainder of the intestine vaa left Intact, nev material being vlthdravn aa needed. Permanent Preparations Altogether 830 smear preparations vere made during the oourse of this study* Moat smears vere stained aooording to either the hematoxylin method of Chen (19Ut-) or a modification of Wessenberg's (1961) silver carbonate method* The rectal oontenta vere spread thinly on oover- slipa together vlth a small amount of buffer solution and fixed vhlle vet in either (1) warn (lfO°C) Schaudlnn'a fixative containing 5 par cent acetic acid if for hema toxylin or (2) 15 par oant neutral formalin containing 2 per oent HH^Br if for ailver. Silver Impregnation Claasioal ailver methods Including those of Bodian (1937)» Klein (1927) and Chatton and Lwoff (1930) failed to stain the cilia and basal granules of Zelleriella satisfactorily* The technique used successfully by Wessenberg for staining basal granulas in Ooallna was found to ba quite satisfaetory in ay stadias of this ganas bat ganarally unsatisfactory for usa with Zallarialla. After oonsldarabla axparinantation a Modification was found which, while not quite as reliable for Zallarialla as for Qpallna, did give reasonably satisfaetory results with tbs forner* This Modification differs from Wessenberg's basic Method In tha following particulars: specimens ware not killed in osmlc acid but ware dropped alive onto tha fixative; gelatin imbedding was not found necessary; preliminary bleaching of saears in $ per cent KMnOj^ after the Method of Honlgberg (191*7) end terminal gold toning in 1 per cent aqueous gold chloride were employed* The amount of pyridine was Increased fourfold. Results obtained with ailver impregnation are at best oaprlolous* Occasionally all individuals on a slide will impregnate well while at other times none or only a few are satisfactory. Drawbaoks notwithstanding, the clarity with whloh the basal granules may be shown renders ailver impregnation an Invaluable aid to the taxonomio study of opallnlds. Detailed study of living and stained sellerlelles was made with a Zeiss Wlnkel GPL microscope equipped with Zeiss achromatic objectives* Drawings included here were made with the aid of a binocular drawing tube mounted on a 13 Wild Model 20 mlorosoope* Electron Micrographs Electron micrographs of Zeilerlella are presented for the first time In this paper* The Protoaoa vere fixed in veronal-acetate-buffered (pH 8*5) one per oent osmium tetroxlde for thirty minutes* Dehydration was aooompllshed by passing through a graded series of ethyl aloohols to absolute alcohol* Zellerlelles vere Infiltrated with a three to one mixture of n-butyl and methyl methacrylate catalysed by two-tenths per oent bensoyl peroxide* The Infiltration mixture was used to Imbed sellerlelles In gelatin eapsules* Thin seotlons vere cut vlth a diamond knife mounted on a Porter-Blum microtome* Seotlons vere mounted on collodion-coated grids and examined vlth an RCA - 3F eleotron mierosoope* CHAPTER III MORPHOLOGY OF THE TROPHOZOITE The genus Zeilerlella was ereoted by Metcalf (1923) aa an expression of his view that among blnueleate opallnida tha flattenad forma stood apart fro* thoaa which wara cylindrical* Earlier, Metcalf (1918a) grouped all blnuoleata opallnida together In tha single genua Proto- opallna* Metoalf distinguished Zallarialla froai Protoopallna on the basis of Its flattened body, spherical nuclei (as opposed to elliptical or dunbbell-shaped nuclei In Protoopallna) and absence of diversity in mltotlo condi tion, regarded as oharaoteristlo of resting nuclei in Protoopallna* Metcalf cited the lack of transitional forms between Zelleriella and Protoopallna as further justification for their separation into distinct genera* General Appearance Zelleriella is a morphologically compact genus. If outlines of the 60 species and subspecies were to be drawn to the same scale, most of these outlines would be nearly congruent* A generalized trophozoite is shown in Plates I and II, Figures 1 through The body is Ik PLATE I Figure 1* Zelleriella hirsute trophozoite Yiewed from the adfaleular surface. 16 PLATE I PLATE II Figure 2 Figure 3 Figure I j . Zelleriella hirsute. eross seotion at a level just anterior to the nuclei, Zellerlella hirsute, eross seotion at the level of the more posterior nuoleus, Zelleriella hirsute, eross seotion at a level two-thirds of the way between posterior nuoleus and rear margin. 4 flattened, being thiekest at the level of the nuclei, and tapers aondWhat toward the margins. Length and braadth are variable, tha latter commonly exceeding the former* On tha average, the dimensions of a trophosolte approaoh 100 microns In breadth by 90 mlorons In length. Two oval nuolel lie slightly ahead of the mld-polnt of the body* Zelleriella swims spirally through the coordinated aotlon of numerous oilla lying In parallel rows on the body surface• A second component of the eiliary system, termed tb* by Mohr (19^0) oonsists of a double row of cilia along most of the anterior margin* Orientation and Terminology Mohr (I9I 4 .O) pointed out that the oillary rows on either surface in opalinlds follow different oourses, and that the falx Is located subterminally on one surfaoe* Zelleriella. because of its shape and flattened body, shows these patterns particularly well* Mohr termed the surface on which the falx lies adfalcular. the other abfalcular. He naned the adfalcular surfaoe ventral and thus treated the opalinlds as dorsoventr&lly flattened. Vessenberg (1961) disagreed, holding that opalinlds should be considered flattened laterally* It has been shown by Okajlma (1953)* Naltoh (I96I) and others that the ciliary beat in opalinlds originates in the vlolnity of the posterior-most end of the falx* Wessenberg oompared this 20 region with the oral area of atonatous dilates and con sidered the adjaoent margin a more likely homolog for the rentral surface* Consideration of the probable mode of flattening by anoestral opalinlds Is speculative* There seems little reason to depart from the original view expressed by Mohr* In this paper, the tvo broad surfaces will be referred to by the non-committal terms adfalcular and abfaloular* The terms right and left (upper and lower respectively In Figure 1) are here used In Identifying the margins, nuolel, and the daughters produced during fission* The anterior- most portion of the body, (at the right In Figure 1), is termed the apex in accordance with prior usage by several authors, among them Mohr (19lj-0), Cosgrove (19^7) and Vessenberg (1961). Pellicle Electron micrographs of the pellicle of Zelleri ella hlrsuta have permitted comparison of Its fine structure with that of Qpallna obtrlgonoldea studied by Pltelka (1956) &nd Qpallna ranarum studied by Nolrot- Timothee (1958, 1959)* As in these speoles of Qpallna. the pelliole of Z* hlrsuta consists of numerous parallel ribs lying erect on the oell surfaoe ('Plate III, Figure $)• Pellicular ribs in Z. hlrsuta are about 250 to 300 milli microns In height* These structures have been desorlbed PLATE III Figure 5« Zelleriella hiriutt, electron mi01* 0graph of orosa section showing pellicular ribs, cilia, and granules of the eyto- piasm. Magnification X 12,550. PLATE III 23 as rldgss or crests by light mlorosooplsts, most recently Cosgrore (19^7) and Pernandez-Oallano (19^7)• Ribs and eotoplasm between appear to bs covered by a oommon mem- brans. Similar psllleular ribs ars known slsswhars among tbs Protosoa only in gr agar ins sporosoans (Pltelka, 1963). Ciliary rows arlss in tbs depressions between tbs ribs, snob depressions being broader and shallower than those without olliary rows. While no thin sections were out through the anterior end of Zelleriella. I hare observed in oertain silver preparations an anterior depression from which arise the ellla of the falx. This t depression, here termed the anterior furrow, is consider ably broader than those from whieh the somatlo ollla emerge. An anterior depression has been desoribed by Hara (193l+» 1936, 1937) in Protoopallna axonuolcata lata. Qpallna obtrlgonoldsa iaponioa and Cepedea (- Qpallna) dimidiate orlentalls. by Cosgrove (I9I 4 . 7) In Opaline 1 obtrlgonoldsa and by Wessenberg (1961) in Qpallna obtrl- gonoldea and 0. virguloidea. Hara oonsldered the anterior furrow of opalinlds a eytostoae. Cosgrove found no satisfactory evidence for such an interpretation but suggested that it might represent the vestige of a former cytostome, a view with whieh I am in agreement. The anterior furrow and somatic olliary furrows of Z. hlrsuta are shown in Plate IV, Figure 6. PLATE IV Figaro 6* Zelleriella hlrauta. adfalcular aur- faco wiew ahowing anterior furrow and pellieular furrowa froa whioh ariae the oilia of the falx and body, reapeetirely. 25 PLATE IV 6 •a 26 Cilia in Z* hirauta emerge froai depressions in tha call membrane (Plata V, Figure 7). Adjacent rows of oilia ara separated by several pellicular ribs. My material ahowa fro* 13 to 16 riba batwean rows. Pitalka found up to 15 in Onalina obtrlgonoldea. Presumably tha ntnabar would wary according to whara along tha anterlo-posterior axia tha aaetiona wara out* Cilia in Z. hirauta ara of uniform length but ara not uniformly diatributed* Oreateat olliary denaity oooura along the falx* Posteriorly*, oilia beoome inoreaalngly aparae* Decrease in olliary denaity ia attributable to the termination of aome rows before they reach tha rear of the animal, and to a progressive increase posteriorly in the distance between basal granules. Hara (193^4-) reported that in Protoopallna axo- nuolaata lata the left marginal oilia were about 2 miorons longer than those of the right margin but found no differ ence in length between the oilia at the anterior and posterior ends* Chan (19^8) observed that in the several speoiea of Zelleriella from Bufo vailloops oilia were of uniform length and distribution* I found no differenoe in olliary length or denaity between Z. hirauta and the many aelleriellea examined from hosts other than the 3 Arizonan Bufoa* Contrary to tha view axpraaaed by Metoalf (1923) that ciliary length in opalinlds inoreaaea with body alee, PLATE V Figure 7* Zelleriella hlrsuta. alaotron micrograph nearly tangential to the pelllele show ing olliary basal bodies, klnetodesmata (a) and transverse vesicular bundles (b). Depressions In the eell membrane from whieh ollla arise may be seen In the middle of the photograph. The vertioal striatlons are pellloular ribs. Magnification X 12,550* PLATE V 28 7 29 ellla in all sellerlelles studied by hara bean between 10 and 11 microns long irrespective of the life eyele stage encountered. Infraolllature Within the pelliole of oiliated protosoans lie the basal grannies of the oilia, frequently arranged in meridional rows. Each basal granule is usually Joined to its neighbor in the row by a fibril or fibrils. Following the terminology of Chatton and Lwoff (1935) the basal granules are klnetosomes. a single row of kinetosomes a kins tv and the fibrils oonneeting one basal granule to another the klnetodesmata. The entire assemblage of rows and fibrils constitutes the infraolllature. The charaoter of the infraolllature has, in the past twenty-fire years, eome to be widely used by students of olllate systematica in developing ideas on relationships between members of the Clllophora. Ciliary rows in Z. hlrsuta proceed posterlad from the falx in nearly parallel rows. Those on the adfalcular surfaoe may lie either along or somewhat oblique to the apparent long body axis. Such rows, termed rectilinear by Mohr (I9I 4 .O), exhibit greater or lesser degrees of curvature and are more or less oblique depending on their location. Those toward the left margin are more oblique and curve more toward this margin than those on the right side. On 30 the opposite (abfaleular) surfaoe, ciliary rows describe a shallow S, a condition found in approximately the sane degree regardless of their location. Mohr has tensed these *ho eiwete rows. The asymmetry of reotllinear and slg- mate ciliary rows Is shown In Plate VI, Figure 8. Ciliary rows in Zelleriella terminate at the margins. In Qpallna and Protoopallna they continue around to the opposite surface, frequently appearing again on the original surfaoe. When viewed along the anterio-posterior axis from the front, olliary rows in Qpallna and Proto opallna therefore follow a spiral course over the body. Occasionally in large Zelleriella a few rows bifurcate near the posterior margin. The bifurcated ends extend away from each other, parallel to the margin, toward the ciliary rows on either side. This gives the appearanoe of a posterior marginal ciliary row similar to a portion of the falx. Metcalf (1923» 19^°) suggested that the ratio of Incomplete to complete ciliary rows be used In making species distinctions, though in practice he did not use this character for any species. In Z. hlrsuta. each surfaoe bears an equal number of olliary rows, about half of which fail to reach the posterior or lateral margins. Incomplete sigmate rows are longer than incomplete recti linear rows. The interval between ciliary rows, frequently used PLATE VI Figure 8, Zelleriella hirauta. olliary rowa. Rectilinear rowa are indicated by a dotted line, alga at* rowa by a aolid line. Figure 9* Zellerlella hirauta. anterior end of a alightly tipped individual ahowlng details of the falx. The apex Ilea at the right. PLATE VI 33 by Metcalf aa a apeolaa character, la variable In Z. hlrsuta. Rowa are oloaer together toward the apical or right end of the falx than they are toward the left and* Metcalf (194-0) propoaed that the distance between basal granules night be of value In distinguishing opal ini d apeeles but did not use suoh measurements In practice* In Z* hlrsuta. the dlstsnoe between adjacent baaal granules Increases progressively from anterior to posterior. At the front, basal granules are about 200 millimicrons apart, at the middle, I 4 .OO millimicrons apart, at the rear, 600 millimicrons apart. Gaps much wider than these average figures occur, particularly in the terminal portions of a row. The number of ciliary rows, used by Uttangl (1951) as an aid to the separation of several species of opalinlds in India, increases with body size in Z. hlrsuta. but not altogether regularly. Variation in number of rectilinear ciliary rows In SO individuals from a single population of Z. hlrsuta Is shown In Graph 1. Falx Fatten (1932) desoribed a lateral fibril In Qpallna ranarum connecting the anterior-most basal granules of each row which she termed the primary anterior fibril. From this appesred to arise longitudinal fibrils which joined together the basal granules In a single row. Hara GRAPH 1 Number of Ciliary Rows VARIATION IN RECTILINEAR CILIARY ROW NUMBER AMONG FIFTY INDIVIDUALS FROM A SINGLE POPULATION OF Zelleriella hirsuta Metcalf r4 3 -41 -39 -37 -35 -33 -31 -29 -27 -25 -23 • • • •• -21 -19 •• 4 -17 -15 -13 40 50 20 30 60 70 80 Breadth of Zelleriella (microns) (1934* 193^# 1937) described paired fibrils running sub- terminal ly along the anterior margin In Protoopallna axonueleata lata. Qpallna obtrlgonoldsa laponloa and Copodea (* Qpallna) dlmldlata orlontalls. the nore poste rior of «hleh was thicker. Hara termed this component the oral ring and considered It capable of opening and olosing about an anterior cytostome. Chatton and Braehon (1938) pointed out that at their posterior ends the somatic olliary rows of Qpallna ranarum lie in parallel without continuity or contact with each other while along the anterior margin the gap between rows is oooupied by a series of more or less regular ohains of basal granules linking adjacent somatic rows together. These were termed primary or generative kinetics, indicative of Chatton and Braehon*s view that somatlo rows arose as a result of multiplication of specialized anterior basal granules. Mohr (194°) based on studies of several opalinid species, oonflrmed Hara’s finding of a subterminal double fibril but failed to find any difference between them with respeet to size or the presence of an interposed oytostoma. He termed this element the falx because of its curved siekle- blade shape. Cosgrove (1947) described the falx in 0. obtrlgonoldsa as a double, (single more posteriorly) row of heavy flagella near the anterlo-lateral margin, under lain by heavy faleular fibers in the ectoplasm. Wessenberg found the falx in the same speeies and in Qpallna 36 vlrguloldea to oonslst of a single row of basal granules at its extremities but as It progressed anteriorly the single row was widened by additional basal granules until along most of the anterior margin there was a dense field of from 1 ^ . to 6 basal granules In width. Pltelka (1956) attempted to Identify the falx In her electron micrographs of Qpallna obtrlgonoldea but was unable to do so with oertalnty. One seotion* presumably through the falx at a level where it was single showed a basal granule ". . • perhaps heavier than oooqparable sections of others but not markedly so." She summarised her findings in the negative* writing that if the falx is represented In her material there are no fundamental differences separating it from somatic ciliary rows. It should be mentioned that one of the methods used by Pltelka In separating the pellicle from the oytoplasm was sonloation* which produced random pellicular fragments whose relationship to the whole organism was obscure. For this reason* Identification of the falx is questioned, k second* less random method for Isolation of the pellicle used by Pltelka involved solu tion of the oytoplasm by dlgitonln* which left intact pellioular "ghosts." She did not Indicate whether the seotion of presumed falx was from a preparation of this latter kind or from one subjeoted to fragmentation by sonloation. I have studied the falx of numerous Zelleriella and Qpallna In silver preparations and agraa with Waasanbarg on tha presenoe of a median faloular fiald in Qpallna* In Zallarlalla tha falx (Plata VI, Figure 9) consists of a double row of basal granules for most of Its length* As noted bj Mohr, It Is situated Just subterml- nally on tha surfaoe bearing the reotilinear ciliary rows. It is oharaoteristlo of the falx in all sellerielles I hare studied orltieally that the double row of basal granules terminates abruptly at the apioal extremity and that from the point of termination arises a single, usually incomplete olliary row whieh lies along the right margin. As the falx Is followed to its opposite end the double row is seen to give way to a single row. Somatic oillary rows in this region appear to arise from basal granules of adjaoent somatic rows* I have been unable to distinguish any si as difference between basal granules of the falx and those of somatlo rows, nor are the ellla in the falx heawler than those of the rest of the body. I believe that Cosgrove*a interpretation is attributable to the differences in oillary density encountered in the falx and in more posterior regions of the body* Wessenberg stated that lateral displacement some times 00ours in a somatlo oillary row In Qpallna giving the impression that a new row has arisen at some point posterior to the falx* I have observed lateral displace ment in somatlo rows of Zelleriella rarely but find it to oocur cowmonly la the falx* la suoh instances tha inter posed faloular row of from It to 6 basal granulas axtandlng batvaan two ad jaoent soaatle rows aay prooaod from tha anterior-most granule of ona rov to tha saoond or third of tha othar* This situation seems at Tarlsnoa with tha Tlew of Chatton and Braehon* aohoad by latar authors* that tha antarlor or "generative" basal granulas give rlsa to thosa situatad behind than In a row. Lwoff (1950) writing of tha olllata Infraolllature states* "Ona klnatosona is always ganaratad by dlrlslon of another* Wa saa kina to- somes dividing and have no evidenoe whatever of thair fornation da novo." However* Grlastone (1961) has pointed out that tha evidence for division of basal granules In olllates has been developed frost light micro- soopa studies of stained Material and that In suoh static preparations division can only be inferred* For example* groups of basal granules lying within a restricted part of tha oytoplasm or 2 granulas lying in elosa proximity have bean interpreted as avldanea for division* Studies of basal granulas in living calls have not bean possible* A large and growing literature on eleotron microaooplo studies of olllata fine structure in which oonvlnolng pictures of basal granule division might be expeoted have failed to reveal suoh behavior* Qrlmstone suggests that* "Tha cytoplasm!o environment in whloh a basal body already exists is likely to be a favorable one* oonoeivably tha only possible ona, for tha ganaala of a new baaal body, and tha postulated ganatlo continuity of thaaa organallaa might inrolre no mora than tha development of nav onaa In eloaa proximity to old.1 * Elaboration of auoh apaeulationa la bayond tha aoopa of thia paper* However, tha irregularities notad In intarpoaad faloular baaal granules In Zallarlalla and tha apparant origin of tha laat fav somatlo rowa in tha neighborhood of lntermedlataly positioned baaal granules of the next ad jaoent row suggests that tha oonoept of olliary row origin by raplioatlon of spaolaliaed anterior olliary basal granules be re-examined. Thia matter la oonsidered again In the final ohapter as It relates to the generalisation of Chatton and Braohon that tha falx and Its specialised baaal granules hare homologies with tha eantrlolea and ao-ealled paradesmose In flagellates. Plbrlla Intraoellular fibrils in opalinlds hare been deserlbed by sereral investigators, among them Konauloff (1922), Orerbeek de Meyer (1929) and Hara (193^* 193&). Fernandes-Qallano (19^7) and Cosgrore (19^7) hare summa rised tha studies of earlier light mioroseoplata on opallnid fibrils. Obserratlons on fibrils In opalinlds with the eleotron microscope hara been reported by Pltelka (19#) and Nolrot-Timothae (1958, 1959). ko Bara (1934* 193&) distinguished four kinds of fibrils in Protoopallna axonuoleata lsts and Oepedea (* Opallna) dinidlsts orlentalla. eonsldering then ss component* of a neurosMtor system. Inoluded were? (1) paired oral ring fibers? (2) longitudinal fibers paralleling the rows of basal granules; (3) inner fibers, passing from basal granules to the interior where they eonneoted with (4) the deep fiber net, an Irregular net** work oorerlng the whole endoplasmlo surfaoe. Cosgrove (1947) desorlbed in Opallna obtrlgonoldea a pair of fibrils along the anterior end fron whloh arose the ollla of the falx, (the anterior faleular fibrils of Mohr); oblique fibrils eonneetlng basal granules of one row to those of adjaeent rows; and stout doreo-watral fibrils whioh extended fron basal granules through the eytoplasm to others on the opposite surfaoe• Fernandes-Gallano (1947) desorlbed in Cspedea (« Opallna) dinldlata: (1) a robust single anterior subsutural fiber? (2) primary aubpellloular fibers parallel to the basal granules; (3) secondary aubpellloular fibers consisting of a fine fibrillar network throughout the endoplasm; (4) elemental fibers whioh passed from basal granules into the interior and (5) bulky transverse fibers extending between basal granules of adjaoent rows. Vessenberg (1961) observed in a few preparations a network of extremely fine, branching and anastomosing fibrils throughout the endoplasm. In in 5 slides out of 600 ho sow what appeared to bo a bundle of fibrils parallol to tho falx* Pltolka (1956) and Voirot-Timothoo (1959) have dosoribod as tho klnstodaamata in Onalina obtrlgonoldca and 0. ranarun pairod fibrils arising froai tho antoro- 1 at oral curve of oaeh basal granulo whioh join and oxtond toward tho noxt basal granulo anteriorly. This presumably corresponds to tho longitudinal fibers of Hara and tho primary subpellicular fibers of Fernandes-Gallano* At about the snao level as the klnetodesmata, in the material of Pitolka and Voirot-Tlmothee, appeared groups of vesicles organised into transverse rows* These workers found no evldenee for transverse or dorso-ventral fibrils or a fine fibrillar network within the endoplasm* Electron mlorographs of Zellerlolla show kineto- desmata similar to that described by Pltelka and Voirot- Tlmothee for Opallna* (Plate V, Figure 7)* Klnetodesmata do not quite meet the next basal granules. Pitelka found the same to be true in 0. obtrlgonoldea and Voirot-Tlmothee reports that only rarely did a kinetodesma reach the next basal granule forward in 0* ranarum. Transverse bands of vesicles are also apparent in my material (Figure 6) at about the same level as the klnetodesmata. It is likely that these are the transverse fibrils of earlier light microscopists. Voirot-Tlmothee regards these vesicles as tubular, extending Inward for some distance toward the 1 * 2 orator of tho tody. This is not shown In ay ©rose sectional photographs of Zsllsrlella. If Holrot-Tlnothee*e Tlow is eorroet those strueturos nay bo homologous to tho bulky dorso-ventral fibrils of Hors, Fernandes-Galiano, rad Cosgroro. Tho number rad spaelng of those transverse vesicles does not correspond to tho number and spaelng of basal granules in Z. hirsute, as was similarly pointed out for 0. obtrlgonoldoa by Pitelka. I found no evidenoe for an endoplasmic net in my material. However, the oytoplasm of opallnlds contains a great many membrane-limited bodies of unknown constitution which often lie in contaot with each other. Seotions out tangentlally to the flat surfaces of Opallna or longi tudinally through the body of ProtoopalIna could show the out edges of the membranes as a fibrillar reticulum. Cytoplasm In stained preparations, a relatively non- granular, sometimes vaouolated sons, the ectoplasm, is observed immediately beneath the pellicle. Toward the interior, this area merges with the more granular and vaouolated endoplasm. The relative proportions of ecto plasm rad endoplasm in correspondingly sized opallnlds rad the size and shape of oertaln inclusion bodies, termed "endospherules” were regarded by Metcalf (1923, 19^0) as rather oonstant species characters. Chen (19i*8) observed what he oailed "eetosere alveoles" 1a the outer oytoplaa- mio layer of Zellerlella and used the also of these alveoles as an aid In the saparation of tba various spaolas dasoribad froai Bufo valllceps. Fernandes-Gallano (19^7) in his study of Canadaa (■ Opallna) dimidiate observed vacuo las, of tan with spharieal Inclusions, In tha eoto- plasm. Ha believed that tha spharulaa rapresantad drops of liquid ooagulated through tha aotion of tha fixative into solid granulations. Huntar (1955) in * review of the literature of cytochemloal investigations of granular complexes in opallnlds rsauurked that two types of granules, large and snail, eould be identified. Tha eonpoaltion of tha latter vas disputed, though Huntar found than to be positive for tests for mitochondria. Tha large granules vara considered largely proteinaceous. Bretsehneider (1950) found in electron nlorographs a variety of vacuoles and granules ranging in alsa from 160 mlllimlorons to 2 microns. He agreed vlth the earlier findings of Horning (1925) and Kedrowsky (1931) that tha abundance, structure and position of these inclusions varied vlth the nutri tional condition of the opallnld. Pltelka found no clear desmrkation between ectoplasm and endoplasm in her electron micrographs of Opallna obtrlgonoldea. She tenta tively distinguished three sorts of oytoplasmlo bodies, all clearly defined by membranes, on tha basis of aise and location but vas unable to relate them to inclusions described by light nlorosooplsts. Hoirot-Tinothee (195&* 1959) interpreted oar tain granule a in tha eotoplasmlo layer of 0. ranarwa aa Golgl bodies while other Irregu larly oral bodlea ware Identified as mitochondria, similar to those of other protozoans but vlth much finer nioro- tubules. Pltelka (1963) In her recent nonograph on eleotron Microscopic struoture of Protozoa, has accepted Hoirot-Tlmotbee's Interpretations with respaot to these two Inclusions. On the basis of their size and shape, the nltoohondrla appear to correspond nore olosely to the ”endospherule s" of Metcalf than do other endooytoplasnlc bodies. The ectoplasmio sons in Zellerlella hirsute is distinct In individuals stained with hematoxlyn, sometimes but not always so in silver preparations, and, as Pltelka found in 0. obtrigonoldea. not particularly distinot in electron mlorograph sections. The width of this layer appears to vary slightly vlth size, being a little wider in large individuals than in snail ones, the difference often only a micron or so. However, within a single population of Z. hirsute, variation of eotoplasmlc width in Individuals of the sans size nay be of approximately the same magnitude. Vacuoles oocur oonaonly in the eotoplasn of Zellerlella. often containing granular inclusions that stain weakly with hematoxylin. Such vacuoles are seen ks vlth particular frequenoy in selleriellea fron tadpoles. They rofloot, In nj opinion* tho physlologled state of tho anlnal oh an fixed. Zolloriolloa from starved or aiok hoata or in artlfiolal eultnro nodi a exhibit than nor a of ton than thoao fron apparently nornal adult hoata* Electron miorographs of tho cytoplasm of Zeller- lolla hlrauta ahov It to ho similar to that of 0. obtrlxonoldea and 0. ranarun. Humorous Irrogularly oval bodioa In tho ondoplaan, (Plato VII, Figure 10) appoar to be Identical vlth thoao identified aa Mitochondria by Noirot-Tlmothee • Certain granular inolualona eorreapondlng in also and position to tho Oolgi bodioa of Holrot- Tlmothee ara present In tho photographs of Zellerlella but « tholr struoture Is not sufficiently dear to bo oertaln that they are tho sane* Present also are numerous large and small vacuoles and granules vhoae nature Is unknovn. Several vorkers, including Metoalf (1909), Konsuloff (1922) and Hara (193&) have deserlbed an exore- tory system In opallnlds oonslatlng of an elongated vaouole or series of eonneetlng vacuoles opening to the exterior through a posterior pore. Other investigators have failed to oonflm the presenee of such structures. I find no evidence for an excretory aysten in Zellerlella hlrauta. PLATE VII Figure 10* Zellerlella hirsute, electron micrograph of a transverse oblique seotlon shoving nucleus, fragments of the nucleoli (n), mitochondria (m) and endoplasmic vacuoles (v). Magnification X 12,550. 11A 31VHd Mu old At lnterphase , the two nuolel of Zellerlella exhibit a veil-defined membrane, finely granular ehromatin, and a masber of large irregular ohromophile bodies typi- oally, but not invariably, lying at tha periphery. These last are tha "macrochronosomes” of Metcalf, correctly identified by Chan (1932a, 1932b) as nucleoli. Interphase nuelai in Z. hirsute are subspherloal, being somewhat oonpresaed dorso-ventrally, (Plata VII, Figure 10). They appear broadly oval in eross section. Metcalf (1923) held several Misleading notions about opallnld nuclei vhioh, because of the bulk of hie work, became widely circulated among students of the opalinldae. He olalned that two seta of chromosomes were present, the "macroohromosoaes," (actually nucleoli), and the "microohromoaomes" (the real chromosomes). This unique "amphlnucleus" was used by Metoalf as presumptive evidence for hia NprotoelllateN hypothesis aocordlng to whioh opallnlds are transitional forms between flagellates (with monomorphio nuclei) on one hand and true dilates (with macronuoleus and mioronuoleus) on the other. Metcalf further held that in certain binucleate opallnlds the nuolel "came to rest” in some mid-mitotie stage. He used this oharaeter in distinguishing several species of Protoonalina and Zellerlella. among them P. mi totlea. P. papuensls, P. montana, P. flllformla. Z. atelopyxena. k9 Z. magna. and Z. Interiaedla. A third nuclear oharaotar, considered by Matealf to ba speolfloally distlnot, was tha presence or absanea of a eytoplasmio thraad linking one nucleus to another* Metcalf's views ware severely orlti- olsad by Chan (1932a, 1932b# 19^8). In a farias of extremely oarafnl investigations on tha nuclei of Zallar- ialla, Chan demonstrated that: (1) "maoroohrouosomes" wara in faot nuoleoll similar in composition to tha nucleoli of many met&zoan cells; (2) apparent mid-mitotic resting nuclei were illusionary, being based on observing nuoleoli, mistakenly identified as chromosomes, at opposite poles of a resting nucleus Cresting in anaphase") or at the equator ("resting in metaphase"); and (3) the oyto- plasmio thread between nuclei represented the remnants of the parental nuolear membrane which persisted during mitosis* Chen further demonstrated that nuclei in Zellerlella were diploid and underwent mitosis in a manner similar to oells of metasoa* A majority of students of the sellerielles, notably Metcalf (1923. 19^0), Carini (1933b, 1933«. 1938), Otamendi (191*5) and Uttangi (1951# 19&1) have used the sise and position of the nuclei in describing species* Implicit in this practice was the assumption that corres pondence exists between the sise of the body and the diameter of the nucleus* For example, Metoalf pointed out that in comparing nuolel it is neoessary to do so with 50 animals of "corresponding sixes." In fact, no worker has attomptod to relate nuclear diaaster to body alio in any apaoiss of opalinid. Otamendi, in a study of tho seller- iollos of Bufo arenarum and Leptodactvlus ooollatus in Argentina oomparad tho ratio of nueloar width to nuoloar longth and tho ratio of body width to longth but failed to indioato how body dimensions wore determined. As will be shown in the following chapter, length and breadth in the highly asTmmetrioal sellerielles have not been clearly defined with the result that one worker’s Interpretation has not necessarily corresponded to another’s. In Zellerlella hlrauta. the nuclear diameter varies with body sise but not altogether regularly. Suoh variation has not been reported. Graph 2 illustrates the relationship between nuclear diameter and one parameter of sise* breadth, in a single population of Z. hlrauta. Measurement of breadth was made on a line between the nuolear mid-points. Justification for this method is given in the following chapter. It will be noted (Graph 2) that the nuclear diameters in individuals of the same sise are not necessarily identical but may vary somewhat. Moreover, as smaller individuals are examined, the ratio of nuclear diameter to body width decreases (Graph 3). The position of the nuclei in the body of Z. hirsute is also variable. The distance from the more anterior (right) nucleus to the adjacent margin, measured GRAPH 2 VARIATION IN NUCLEAR DIAMETER AMONG FIFTY INDIVIDUALS FROM A SINGLE POPULATION OF Zelleriella hirsuta Nuclear Diameter (;i) -15 -14 -13 -12 -11 -10 • •• • 60 70 80 90 20 30 40 50 Breadth of Zelleriella (p) GRAPH 3 Breadth Nuclear Diameter PROPORTIONAL VARIATION IN NUCLEAR DIAMETER AMONG FIFTY INDIVIDUALS FROM A SINGLE POPULATION OF Zelleriella hirsuta -11. 5 -11 -10.5 -10 • • • • • 30 40 60 70 80 50 20 Breadth of Zelleriella ( j u ) vn r \ j on a line through tha nuolear mid-points, increases more or less regularly vlth also. However, tho dlatanoo batvaan tho too nuolel, measured on tha same lino, oar lea Irregularly. Tho eonoopt that growth in Zollorlolla la unequal, largoly dlraotad laterally toward tho loft margin, to bo developed In the next chapter, holda that In a now daughter tho loft nucleua norea away laterally fron Ita nato until flaalon oooura. Tho longer tho period between flaalona the greater will be tho dlatanoo between nuolel. For thia reason, populations of zellerielles undergoing rapid division In the aprlng tend to have nuolel oloaer together than In membera of oorreapondlng populations In non-reproduotlve hosts. Chronoaonoa and Huolooll Tha number of nuolaoli, their manner of attaohment to ohromosomea and the partioular chromosome with which they associated were regarded by Chen (19^8) as specifically distinct and served as primary characters In distinguishing 5 species of zellerlella from a single host, Bufo valll- oopa. Chen's views have been aooepted by moat recent workers on opallnlds. Hunter (1955)» for example, expresses the opinion that, "If auoh characters as chromo some number, the number of nuoleoli, the distribution of the nucleoli and the attaohment of the nuoleoli were known (they) would be of aid in the Identlfloatlon of species.” Z have bean privileged to look briefly at some of Professor Chon1a original proparatlona, now thirty or more yeara old, and find than to compare favorably with hi a illustrations. I have boon unable to duplicate hia results, however, even when following hla nethod with extreme care. Nucleoli atain readily and nay be counted, but the identity of the chronoaonea and the preoiae nature of the nuoleolus-chromosome association has not been clear In any of ny material. In part, these difficulties nay be attributable to the smaller also of the nuclei in Z. hlrauta. Chen also used apoohronatlo objectlvea in hla work while I have had to depend on achromatic lenses. It should be mentioned that I have not studied nuolear oharaoterl8tloa In Zellerlella as intensely as did Chen, whose Investigations oovered a span of over fifteen years. There la soma doubt whether, baaed on the descrip tions given by Chen, the nuoleolus-ohromoaomea of one species of Zellerlella oan be separated from those of another with complete oertainty. In each of the speoiea studied critically by Chen (Z. elllptloa. Z. Intermedia, and Z. loulalancnala) the ohromosome number (twelve pair) and configuration were the same. Distinctions between Individual nueleolus-chromosomes were minor with the exception of a single ohromosome (ohromoaome 1) which was longer than the rest and which, in Z. Intermedia, lacked 55 an associated nucleolus • Tha remaining nucelolus- chromosomes vara distinguished fron ona anothar solely on ttaa langth of tha ahortar arm in proportion to that of tha longer* Total variation vaa from one-third to one-half tha langth of tha longar ana* It doaa not sesm impossible that any of thaaa chromosomes (mashers 5, 6, and 8) oould ba oonfuaad vlth any other in tha series* Dr* Chan (paraonal communication) ha a informed me that tha various apeolaa of xellerielle fron B* vallloepa vara alvaya anoountarad in aaparata infaetlona* It la poaaibla that dlffaranoaa in tha appearance of tha nucleoli observed in thaaa aevaral apaelaa raflooted, in part, different environmental statea attendant to nutri tional or other variationa in the host animals* Vineent (1955) haa remarked that vhlle the view that nuoleoli are formed by apeolallzed heteroohromatle regions of definite ohromoaonsa la generally aooaptad by oytologlata, tha nature of this association with reapeot to alae and compo sition of tha nucleolus may ba variable depending on tha physiological state of the cell* Chan's observation that the apparent number and the real number of nuoleoli are not alvaya tha same, due apparently to fusion of two or more adjacent hypertrophic nucleoli, would seam to support Vincent's view* Thera ia a further considerations Chen's approaoh requires dividing individuals with metaphase nuclei in perfect condition and optimally fixed and stained, Fur- thor it la useful only in speoles with Tory largo nuolel, and so can bo applied only to spools s of Protoonalina and Zellerlolla, Aetually it laolcs general usefulness for ordinary systematic works. Gigantism Large multlnueleate sellerlelles in whioh nuolear dlylsion has not been aoeompanied by division of the cyto- plasm hare been reported by several workers including Raff (1912), Mohr (19lf0)r Chen (19^8) and Otamendi (1945)* Dr, Mohr (personal communication), in an effort to dls- oover whether giant sellerielles from Bufo peltooeohalus were a true genetie entity, lnnooulated suoh Individuals into the reotum of Bufo marlnus. They failed to beeoaie established, I have found giants (Plate VIII, Figure 11) in sellerielles from several host speoles. In eaoh oase rapid division of the general population was in progress* The oapaolty to swim spirally seems to be lost or greatly reduoed, looomotlon being most frequently In a plane tangential to the surfaoe of the slide. It is possible that spirallng was restricted due to pressure of the oover slip on these extremely large individuals, I have never observed giant sellerielles In fission. They seem to be anomalous forms ineapable of reproduetlon. Gigantism In Zellerlella appears to be more coaaon than has heretofore PLATE VIII Figure Figure 11* Giant, abnormal Zellerlella hlrauta with fire nuclei, showing tha eouraa followed by the oiliary rows on one (adfalcular) surfaoe. 12. Large, normal Zelleriella hlrauta trophozoite, showing similarity in course followed by oiliary rows to abnormal giants (Figure 11). 58 PLATE VIII 11 12 been suggested• CHAPTER IV FIS3I0H AID GROWTH Pinion Following tho terminology of Chotton and Villonouro (1937)» fission in flagellates Is sjmmetrlgenlo. producing two fundamentally symmetrical mirror-image daughters while fission In olliates Is homo the tlaenlc1 giving rise to an anterior daughter or proter and a poste- rior oplsthe. Corliss (1955) h&s held that these patterns conform rigidly to taxonomic boundaries and that supposed aberrant forms, as the "obliquely" dividing dlnoflageHates and thigmotrich dilates and the "longitudinally" dividing perltrlehs may be fit with precision Into their appropriate category. It Is characteristic of symmetrigenio fission that the division plane passes between oiliary meridians when such are present, that is, In hypermastigote flagel lates. Such division has been oalled lnterklnetal by Chatton and Braehon, while the pattern seen In dilates In which oiliary meridians are transected is perkinetal; the ^Corliss (1955) defines homothety as a condition realised by two forms which are similar and similarly placed with pairs of points in a one to one correspondence, in contrast to the mirror image oondltlon seen in symmetry. 60 6l more conventional terms longitudinal and transverse will ba uaad in tha diaouaaion of fiaaion developed hera. Virtually, all workers on opallnida hare raportad longi tudinal fiaaion* Several, including Lagar and Duboaoq (1901).), Hereaheimer (1907)» Matealf (19<>9» 1923), Konsuloff (1922) and Vaaaanbarg (1961) hava daacribad transverse fiaaion in apaolea of Opallna and Protoopalina* Chatton and Braehon (193&) daniad that transverse fiaaion oeourred in opallnida, baaed on their atudy of Opallna ranarua. Thla view haa bean adopted by recant worker a •caking to align opallnida with flagellatea, most notably Gr&sae (1952) and Corllaa (1955)* Little haa been written of fiaaion in Zollerlolla. Matealf (1923* 19^0) preaenta several unoonvlneing outline drawlnga of aellerlellea purported to be undergoing tranaverse fiaaion. Otamendi (19U5) «nd Chan (19218) refer only to longitudinal fiaaion in their worka on Zellerlella* In Z. hlrauta fiaaion la invariably longitudinal, the fiaaion plane paaalng between eillary rows. I hava found no cvidenoa for tranaverse fiaaion in aellerlellea fron hosts other than B. oognatua. B. woodhouall. and B. alvarlua. Longitudinal fiaaion in Opallna. described by Veaaenberg, la aa follows: The falx increases in length with an accompanying increase in number of somatic ciliary rows, producing broad individuals. An indentation appears in the falx, usually at the center but sometimes 62 "displaced to the side," which proceeds to the posterior as a fission cleft* In adTanoe of the cleft such ciliary rows as cross the body obliquely straighten and are thus not transeoted* Just prior to final separation, daughters are observed attached at their posterior ends by a thin strsnd of cytoplasm* They swim In opposite directions until separation Is effected* As In Opallna. growth preoeding fission In Zellerl ella hirsute Is accompanied by elongation of the falx and an increase in number of somatio ciliary rows* The ini- tlal sign of impending fission Is an indentation, usually seen first in the anterior margin but sometimes developing earlier in the rear margin. In trophozoites these indentations are typically medial while in rapidly repro ducing populations undergoing diminution in sise they may be displaced toward the left margin (Plate X, Figure llf). The fission cleft extends across the body between marginal indentations* In silver preparations the pelli cular ribs within the fission deft appear flattened* I attribute this to stretohlng of the pellicle away from the cleft oenter* The pellicle adjacent to the cleft is compressed resulting in crowding together of several ciliary rows on either side (Plate X, Figure Ilf)* For most of its langth the fission deft follows the course of the rectilinear ciliary rows. Anteriorly, slgaate ciliary rows bend paralleling the cleft. PLATE X Figure l l j . * Budding In Zellerlella hlrauta. Compression of slgmate oiliary rows adjacent to fiaslon oleft Is shown* 6k PLATE X 14 6$ As diriiion proceeds the falx is Interrupted by the anterior Indentation. The right daughter retains one half of the parental falx and aasoelated somatic elliary rows* the left daughter the other half. In the right daughter* a somatic row lying close to the fission cleft appears to beoome the nev Marginal falx* (Plats IX* Fig ure 13). Longitudinal fission in Zeller lei la does not pro ceed exclusively from anterior to posterior. As division progresses the fission deft deepens* the anterior end more rapidly than the posterior* until separation is ef fee ted. There is thus some resemblance to the "pinching- in* of cytoplasm observed in transversely dividing Cili- ophora* though in Zsllerlslla it is unequal. Dimorphism As a result of the original parental form and the course taken by the fission cleft the products of a single division differ in appearance (Plate IX* Figure 13 and Plate X* Figure l i j . ) . In division of trophosoites in a non-reproduotive host the difference is mainly one of shape while in zellerielles undergoing reduotion in size prior to encystment the difference is both in shape and size. The right daughter in either ease is broadly oomma- shaped while the left daughter is narrower at the front than at the rear. PLATE IX Figure 13* Snail Zelleris11a hirsute undergoing equal fission* The dissimilarity In shape of daughters and relationship of rectilinear oiliary rows to * fission plane is shown* 67 PLATE IX 13 68 It is obvious that if saoh of the daughters grew proportionally in all directions, or failed to grow at all, a single population of sellerlelles would eonslst of individuals of two distinot sorts. Sueh populations have been reported or Illustrated in the literature* Metoalf (1923), describing Z. oouehll. writes, "Two shapes of the body nay be noted, one rather wedge-shaped • • • the other more rounded*" With respeet to Z. atelonodos the same author noted, "This snail Zellerlella has two distinguish able forns which, however, intergrade. In one form the individuals are broadly wedge-shaped, usually with the posterior end distinctly pointed* The broad round individ uals are not usually pointed posteriorly*" Carinl (1938) referring to the wedge-shaped Z. truncate, found this species "... present in Leptodactvlua ocellatus only rarely and always with other species that arc regularly oval in shape." Chen (19^-8) did not remark on dissimilar ity in any of the sellerlelles he named from B. valllceps but clearly figures it in his drawing of Z* elllptioa in fission. Otamendi (19^5)> describing Z. cuneata. noted that in some individuals the anterior extremity of the body was broad while in others it was out off obliquely* It is apparent that while these principal students of Zellerlella taxonomy observed dimorphism, its significance as a fundamental source of variation went unappreciated. With time, differences in form resulting from , 69 fission tend to bs neutralised by growth* thus accounting for the similarity in shape seen in sellerielle popula tions in nan-reproductive hosts. In populations undergoing diminution in else the differences remain. A series of such individual s from a single infection is shown in Plate XI* Figures 1£ to 2l * . Morphological Axes Historically* determination of length and breadth in opalinlds has been an individualistic matter. Metcalf usually indicated* by appropriately placed dots* where on a specimen dimensions were taken (Plate XIV* Figures 3 l j . to 39)* However* it maybe readily noted from his illus trations that no consistent pattern was followed. Breadth* for example* was sometimes measured approximately along the falx; at other times some place quite removed from the falx. The greatest linear dimension was usually considered as length. Other workers* almost without exception* have given no indication how length and breadth were determined despite wide use of such measurements in taxonomy. Mohr (unpublished MS* 1939) observed that in multlnucleate giant sellerlelles from Bufo peltocophalus the falx extended nearly the length of the apparent left margin. Functionally* this margin was anterior. That the apparent long body axis may be in fact more nearly the breadth is not unique to giant zellerielles but occurs in PLATE XI Figure* 15 to 2 1 * . . Outline drawings of several member8 of a single population of Zellerlella hirsute. Comma-shaped Individuals are derived from the right half of the parent* ovoid individuals from the left. Figure 25* Semi-dlagramatlc drawing of a large ovoid Zellerlella hirsute. The simi larity in angles between the reotl- 1inear ciliary rows and the falx and axis of nuclear oenters is shown. PLATE XI O O 20 22 o ^ ° y 24 72 varying degree in normal individuala• Whether the true anterior la eolnoldent with the apparent anterior or the apparent left margin dependa on the eourae followed by the reotillnear eiliary rowa, the d la similarity in daughters from a single fission, and the period of growth uninter rupted by flaalon* Reotillnear eiliary rowa in Z* hlrauta maintain a rather constant relationship to the falx* Toward the apex they Interaeot the falx at an angle of about ninety degrees* Aa the falx la followed to Its opposite or left end, the angle Increases gradually. Due to theae angular relationships and the oourse taken by the fission plane, (parallel to the reotillnear rows), ciliary rowa In daughtera derived from the left half of the parent appear to cross the body more obliquely than do those of Its mate (Plate IX, Figure 13). In left daughtera the falx lies along much of the apparent left margin, while in right daughtera the falx lies more nearly along the appar ent anterior margin* In giant Zellerlella hlrauta. eiliary rowa lie obliquely to the apparent long body axis* As in giant sellerlelles from 6* peltooephalua. mentioned above, the falx extends along most of the apparent left margin* Funotlonally this margin is anterior, movement being in a direction at right angles to It* It la apparent that growth In such individuals has occurred laterally* 73 When giant sellerlelles are compared with large normal Individual* (Plate VIII) it la noted that the location of the falx and the orientation of eiliary rowa are similar, indicating that growth in normal selleri- ellea aleo oooura laterally. The true functional anterior in Zellerlella. as indicated bj the position of the falx, lies approximately at right angles to the reotillnear ciliary rowa. Body length should therefore be determined by measuring from the mid-point of the falx to the opposite margin parallel to the rectilinear rows. In Z. hlrauta. a line drawn through the nuclear mid-points interseots the margins about equidistantly from the ends of the falx (Plate XI, Figure 25)* The angles formed between this line and the reotillnear eiliary rows are nearly the same as the angles between rectilinear oiliary rows and the anterior margin (falx), The distance between margins, measured along the axis of nuclear mid points, is therefore a measure of breadth. Based on my study of Z, hirsute from the three Arlsonan Bufos, sellerlelles from 1I 4 . other host species, and the illustrations of sellerlelles in the literature, the relationships between falx, rectilinear eiliary rows and the axis of nuolear mid-points appear to be the same throughout the genus. 74 Growth The hypo thesis on growth In Zellerlella developed here prediets that, with tine, dissimilarity doe to fission variation will be neutralized. It also predicts that, subsequent to this, the body form characteristic of a single population will change in aoeord with the interval between fissions* These prediotlons are consonant with ay observations* Finally, the same hypothesis offers an explanation of how, in the absence of transverse fission, the number of basal granules may be reduoed prior to enoystaent. It has been pointed out that the axis of nuclear centers in Zellerlella is approximately parallel to the anterior aargln and at right angles to the long body axis* This eanditlon obtains in both large and small zellerl- elles* It is thus apparent that as the nuolei move apart after fission they do so laterally. Such movement, if uninterrupted by fission, is aooompanied by an Increase in nuclear and oytoplasmio m s s * It is suggested here that growth is unequal, characterised by a higher rate in the region of the left end of the falx. This view is based on several lines of evidence. First, the number of seoondary or incomplete ciliary rows, which are apparently proliferated subsequent to the formation of primary rows, is greater noar the apical end of the falx than near the left end. This suggests that the falx has more recently extended into this latter region* Second, tho morphology of tho falx itaolf, in which tho double row of boool grannloo terminates abruptly ot tho oploal and but gives way gradually to a slnglo row at tho oppoaito and, auggoata that now faloular and aonatio baaal granuloa develop at tho loft end. Third, whon broad outllnoa aro eompared, for example botwoon a wedge-shaped right daughtor and ita ovoid mate, tho groator difforonooa aro found to rolato to variation in tho left margin* In tho right daughtor this margin ia Initially eonoavo while in tho loft it ia oonvox* Addition of now cytoplasm In tho loft marginal area of tho former would produce a form more nearly like that of tho loft daughtor while in tho lattor tho addition of now oytoplasm in tho same region would alnply maintain ita already broad eharaoter* I have indicated that growth in Zollorlolla la mainly in breadth* If growth oontlnued uninterrupted by flaalon an individual would boeomo inoreaaingly broad, eventually reaembllng in form tho abnormal giant seller- iolloa* Ciliary rowa in those individuals would oroas tho body obliquely to tho apparent long axis and tho nuclei would lie one behind the other along tho same apparent axis* Should fission occur at some point before this extreme la reached, intermediate individuals would result. Unusually large (broad) sellerlelles together with such "intermediate" individuals are commonly encountered in 76 populations of Z. hirsute during tbs Initial period of rapid dlrlsion In tho spring. From the foregoing it Is obvious that the sise and body font exhibited by a given population of sellerlelles is highly variable in aocord with the reproductive condi tion of its host* On this basis may be explained the oeourrenoe of dissimilar populations in different host individuals of the same speolss from the same locale, a oossaon oondltlon but recognisable only when adequate numbers of hosts are examined. Since also and shape have served more than any other character for the separation of speoies of Zellerlella. these observations are of signifi cance for the taxonomy of the group. It is relevant and of considerable interest to note the following remark made by Metoalf in the final part of his monograph, published posthumously in 19^- 0* On the occasion of naming his tvsnty-eighth speoies of Zellerlella. appropriately Z. dubla. Metoalf wrote: Studying whole Infeotlons, shape, sise, and measurements, I am Impressed that it is a distinct speoies, if Indeed there be a distinct speoies in this exasperating genus that refuses to play the Llnnaean game, but it is difficult to give a diagnostic des cription. Yet, on the basis of the strong impression from the study of whole infections of this and other Zelleriellas it resembles, I am giving it a distinctive name. ... The variation in size and shape typical of zeller- ielles in reproductive hosts is not encountered in sexually immature anurans, adults about to hibernate, or adults kept a long tin* In captivity 77 Bedding Ents (1901) remarked that Opalina ranarum vara all of a similar also through most of tbs year and dividad only infrequently* Toward tha last of April or the first of May rapid division 00ourrad producing grsat number• of small Individuals with ona or two nuclei which encysted and passed out with tha faces* Sise reduction and cyst formation has been reported by virtually all later students of tha opallnlds* Chat ton (192£) in a paper on morphogegesis in apostome dilates ooined the term palintomy for tha life cycle stage during which smaller and smaller individuals are produced* The term has since been employed for similar phenomena In other dilates by numerous workers, and Wessenberg (19&1) has used it to characterize the pre-oystic phase in tha life cycle of Opalina* Palintomy Involves repeated transverse fissions and the daughters of a single division are of the same size* The similar life cycle phase in Zellerlella involves repeated longitudinal fissions and the daughters of a single fission are sub- equal in size* Application of the term palintomy seems inappropriate*^ Fission in Zellerlella in which the ^Observetlans on fission in Opalina made during the present study suggest that in most cases it is similar to that in Zellerlella* Transverse fission ooours only 78 offspring are smaller than the parent and in which the parent way continue to survive and produce additional daughters for a tine seems better designated as lateral budding. It is a characteristic of zellerielles that due to the shape and course taken by the fission plane the left daughter is somewhat smaller than the right even in normal division of the trophozoite. In budding populations, these differences may be aooentuated only slightly or may reach the extreme shown in Plate X, Figure lit. Reduction of Basal Granules A large Zellerlella trophozoite may exoeed 150 microns in breadth and contain, in a medial ciliary row, upwards of 300 basal granules. A small pre-cystic individual may be only 20 microns in breadth with if 5 basal granules in a medial row. The question arises, how, if fission is invariably between ciliary rows, may the differences in number of basal granules be reconciled. A similar problem does not exist with the Clliophora where reduotian in number may be attributed simply to repeated transverse fissions with no or little intervening growth. It has been demonstrated that growth in Zellerlella infrequently in Opalina and seems no more requisite to size reduction in zh'is genus than It does in Zellerlella. 79 is, with raspaot to its linear dimension, largely lateral. It has been suggested that a differential growth rate exists, new cytoplasm being added more rapidly in the neighborhood of the left end of the falx than in other parts of the body. It is my belief that the falx elongates by proliferation of new somatic ciliary rows In this region. This is supported by the morphology of the falx as mentioned above and by the morphology of the somatic rows. If these latter be compared, it will be noted that those originating toward the apioal portions of the falx are longer than those at the opposite extremity, which extend but a short distance before terminating at the margin. The basal granule number in the short rows is considerably less than that in others. In order to explain how the basal granule number may be reduoed it is necessary to reject the oonoept that no increase in cytoplasmic mass occurs during the period of size reduction and concede that some Intermediate growth occurs but in a smaller increment than is typical for trophozoites in non-reproductive hosts. Budding and reduction in number of basal granules may be character ized as follows: a large Zellerlella divides sub-equally; moderate growth of both mashers occurs, accompanied by an lnorease in cytoplasm and the proliferation of a few new somatlo olllary rows in the left marginal region; parent and offspring again divide, each sub-equally, the former giving rice to a daughtor of alnllar also to tho original bud, tho lattor producing a amaller bud* Repetition of the aequenee would roault in tho production of amaller and amaller progeny with charter and ahortor ciliary rowa* Thua, each aueeeaaivo bud itaolf beoomea a parent and continuea to produoe aub-oqual offaprlng* CHAPTER V LIFE CYCLE Summary of Lift Cycle of Opalina Obaervations on tha life oycle of tha multl- nueleate ganua Opalina have baan reported by Hereahalmer (1907), Brumpt (1915)* Konauloff (1922), Metcalf (1926, 19^0), Overback da Mayor (I929), Hara (1938) and Waaaanbarg (1961). Tha Ufa cycle of Opalina reported by Weaaenberg agrees In general with that deacrlbed by earlier workers. Such differences as do axlat will be noted In later parts of this chapter. A summary diagram of the life eyele of Opalina la given in Plate XII, Figure 26. The letters In parentheses refer to the stage Illustrated in the figure. Large multlnucleate Individuals (a) designated trophonts, are present In the anuran rectum during most of the year. In the spring, correlated with the hosts* breeding time, trophonts undergo a series of oonseoutlve divisions (b - g) resulting in the production of paucinu- eleate oysts (h). These pass into the water with the feces and are Ingested by tadpoles, where exeystment occurs* Excysted Opalina. termed gamonts. (J) divide and 81 PLATE XII Figure 26. Llf« cycle of Opalina. (a) trophozoite; (b-g) palintomy; (h) Infection oyat; (1) ezeyatment; (J) gamont; (1) maoro- gamete; (1’) dividing miorogamete; (m) ayngamy; (n) sygote; (o) cygooyat; (s-t) aeoondary gamont formation; (q-r) prototrophonta; (u-y) aeoondary cyat formation. Stagea (a) through (h) occur in the adult ho8t, the remainder occur in the tadpole. From Veaaenberg (1961). Figure 27. Life oycle of Zellerlella hlrauta. (a) trophozoite; (b-g) budding; (h) infeotion oyat; (1-k) developing larvae; (m-o) aec- ondary oyat formation; (p) aeoondary oyat. 83 PLATE XII c 27 eventually beoome uninucleate anisogametea (1, I1). Syn- gamy (a) follows, after which ths sygots (n) forms a uninucleate cyst (o). Zygoeysts ara shad with tha feces, ingested by a tadpola nearing metamorphosis, excyst and develop into trophonts with tha transforming host (p to r). Tha pattern may wary depending on tha age of tha tadpola Infeoted. Zygoeysts Ingastad by a young tadpola may repeat tha sequence gamont, gamete, sygoeyst (s to o). In older tadpoles development may be direct from sygoeysts to trophont (p to r), or may Involve formation of asexual secondary oysts (u-y). Observations on the life cycle of blnucleate opallnlda have been limited. Cohn ( 1 9 0 i | . ) described conjugation In Protoopallna. an event denied by Metoalf (1909) whose description of the life oyole of Proto- onallna Is similar to that of later workers for Opalina. The life oyole of Zellerlella has not until the present been reported. Summary of the Life Cycle of Zellerlella hirsute The letters In parentheses refer to Plate XII, Figure 27* Trophic zellerlelles (a) are present in the rectum of the adult host during most of the year. Corre lated with the hosts* reproductive activity occurs a period of rapid and unequal division (b to f) giving rise to small uninucleate Individuals (g) which encyst (h) and are voided with the feces. Cysts lags*ted by a tadpole hatoh, exoysted Individuals (1) closely resembling saall pre-eystle fonas in tho adult host. Initial development is characterised by synchronous nuolear mad oytoplasmlo divisions producing numerous uninucleate Individuals similar to (i). With time, division of the oytoplaam lags behind nuolear division, giving rise to blnuoleate daughters (J). In hosts nearing metamorphosis sellerlelles oontlnue growth through the period of transformation (k to 1), In younger tadpoles, initial development is followed by a period of rapid division with little inter vening growth similar to that seen in the adult (m to o). Secondary cysts, similar in appearance to infeotion cysts are formed (p) and voided with the feces. Secondary cysts ingested by tadpoles without limbs exoyst and repeat the cyole. Secondary cysts ingested by older limbed tad poles hatoh and develop directly into trophozoites with the transforming host. Bncvstment Enoystment occurs in Z. hirsute when the body length is reduced to 20 to 2$ microns. Such individuals are uninucleate. Encystment is similar to that in Opalina obtrlgonoldea and 0. vlrguloldea. as described by Wessenberg. The small sellerielle doubles back upon Itself and rotates in place. Within ten to thirty minutes a transparent membrane appears on the outer surfaoe which, as it Inoreases In thlokness, stops the beating of the ollia. Infection Cysts Following the terminology of Wessenberg and earlier workers, InfsotIon ovst Is here used to distinguish cysts produced In the adult host from morphologloally Identical cysts produced In tadpoles* Infection oysts of Z. hlrsuta range from 18 to 25 microns In diameter (Plate XIII, Figure 31). The transparent oyst wall measures from one to one and a half microns In thickness. Wessen berg found Infection cysts In 0. obtrlgonoldea and 0. ▼lrguloldea to wary In diameter from 20 to I j J j . mlorons. The transparent oyst wall In these species was of the same thlokness as In Z. hlrsuta. The oyst lnfraelliature is not shown clearly in any of my material* The falx appears to remain but the orientation of the somatic clliature is obscure. Metcalf (I909) found one or two nuclei In Infeotlon a oysts of Protoopallna caudata and P. lntestlnalla. Various workers have referred to infeotlon oysts of Opalina as pauolnuoleate. Wessenberg observed from 3 to 6 nuclei In cysts of 0* obtrlgonoldea and 0. ▼lrguloldea. The infec tion cysts examined by me from Z. hlrsuta have been uninucleate, the broadly oval nucleus measuring 6 to 7 PLATE XIII Figure Figure 29 Figure 30 Figure 31 Figure 32 Figure 33 • Opalina gamont s (a-e), gametes (d-e), and syngamy (f-h), From Wessenberg, (1961). • Larval Zellerlella antunesl. from Metoalf, 19^0. • Larval Zellerlella braalllensls. from Metoalf, 1 9 l | . 0 . * Zellerlella hlrsuta, infection oyst, * Zellerlella hlrsuta. reoently exoysted larvae. * Zellerlella hlrsuta, typical broad blnueleate larvae, about thirty hours after exoystment. Figures 31, 32 and 33 are to the same aoale. 88 PL A TE XIII 28 29 30 31 32 33 micron* in Its longer dimension. Wessenberg found infection oysts of Opalins viable for thrso weeks in the laboratory. He regarded this tine as ninlnal. I hare not investigated oyst viability in Zelleriella. Development of Larval Opallnlds Aooording to Wessenberg, small exoysted Opalina. or gamonts, (Plate XIII, Figure 28), divide and redivide until uninuoleate anisogametes are formed. Only one sort of gamete is produoed by a gamont. Wessenberg states that maerogametes are similar in sise and appearsnoe to gamonts. Microgametes are of similar length but very slender with a tapering tall. Syngamy in Opalina is characterised by the attachment of the mierogamete by its tall to the posterior end of the macrogamete. The miorogamete fuses with the macrogamete, forming a uninuoleate sygote. Gametes and syngamy in Opalina are illustrated in Plate XIII, Figure 28. Wessenberg*s desorlptlon of syngamy is similar to that reported for Protoopaljna intestinalis and P. eaudata by Metcalf (1909), for P. inteatlnalia and Opalina ranarum by Brumpt (191£) and for 0. ranarum by Konsuloff (1922). Aooording to Wessenberg, the pear-shaped Opalina sygote encysts. Zygocysts may be distinguished from infection cysts by having a single nucleus or, rarely, two 90 nuolel* Zygocysts have also been reported by Nereshelmer. Metoalf and Brumpt danlad that aygooyata were formed, holding that development was diroot from sygota to tropho- solte. Konsuloff statad that Onallna sygotaa anoyatad and tha oyats vara voided, necessitating a aaoond or third tadpola to complete tha cycle, a Tlav aharad by Overbeek da Meyer (1929) and Hara (1938). Waaaanbarg infootod a aarlaa of tadpolaa with faoea containing aygooyata and eorralatad tha natura of tha product a with tha aga of tha hoat. Raaulta auggaatad that aygooyata Ingaatad by amall tadpolaa fora gamonta and rapaat tha aazual eyola while aygooyata ingaatad by oldar tadpolaa nay althor form gamonta or develop dirootly Into immature adulta, termed prototrophonta♦ Waaaanbarg offered In aupport of thla view hi a observation that amall tad polaa collected from natural aouroaa eommonly oontaln gamonta and gamataa while in larger tadpolaa prototrophonta are more common. Ha tentatively propoaed that hormonal ohangaa in the tadpolaa attendant to impending metanorpho- aia may be tha reaponaibla faotor in determining tha fata of aygooyata. Subsequent development of prototrophonta involved inoreaae In nuclear number and cytoplasmic mas a without division, produolng small trophonta with 30 to lfO nuclei. Soma of these individuals continued growth without fission during host metamorphosis, passing through a broad 91 "zellerlella-form” stage Just prior to absorption of tha tail. The typical elongate shape was achieved at about the time tadpoles left the water. Other small proto- trophonts had a different fate. These underwent a period of rapid division without growth eventually forming pauelnucleate cysts identical with infection oysta. A similar event was described by Brumpt, who observed an ". . . epidemic of division in the anuran larvae . . some weeks after infection paralleling that in Opallna in adult anurans in the spring. Metcalf (1909* 1923) also observed late tadpole cysts identical with those produced in adult frogs. Wessenberg found the pattern of development in Opaline from prototrophont through trophont stages reminiscent of Metcalf*s view on ontogenetic recapitula tion of the family history. Young prototrophonts were fusiform with one, then two nuclei, similar to Protoopalina. the presumed primitive opalinid. Inorease in nuolear number produced "cepedean" individuals which flattened, became broad, and finally assumed the definitive narrow shape regarded by Metcalf as the most highly evolved body form within the family. Development of Larval Zellerlella hirsuta Apart from two reports by Metoalf (I9I 1O), nothing has been written on zellerlelles in tadpoles. During a » 92 half year stay In Rio da Janeiro, Matealf twice collected tadpoles containing "... Zellerlella larvae in tha protoopaline stage of development•" In tha first instance, * several tadpoles were taken from a pond harboring a few adnlt Bufo crucifer. All but one tadpole had typical Zellerlella trophozoites. The exception had slender opallnids with a long tapering tall (Plate XIII, Figure 29 a to c). These last were assigned to Z. antunesi. one of four species of Zellerlella described from B. crucifer. On the seoond oocasion, a single tadpole, taken from a pond harboring some adult Crossodactylus gaudlchaudll. was found infected with blnueleate opallnids (Plate XIII, Figure 30, d to e), resembling narrow zellerlelles in body form. Metcalf assigned these to Z. braslllensls, a species reported from several South American anurans including C. gaudlchaudll. Infection Experlments~-Mater1ala -----------a M Befeoda---------- Three infection experiments were carried out during the spring and summer of 19&3* In the first, development of larval Z.. hirsute from infeotlon cysts was studied in young tadpoles. In the second, development from infeotlon cysts was studied in older limbed tadpoles. In the third, the fate of secondary cysts, produoed in the tadpole, was studied in limbed and limbless tadpoles. Infection oysts were obtained from the feces of 93 adult hosts. The recta of the donors were examined prior to exposing tadpoles to the contents In order to be certain that cysts were abundant and to assure that Zeller lella was the only opallnid present, (Opallna also oocurs In these hosts). Feces containing large numbers of flagellates or nematode worms were discarded. Seoondary cysts were obtained from the debris at the bottom of the bowls In which tadpoles, exposed earlier to infection cysts, had been kept* Zn order to Insure that these were not unlngested Infection cysts, donor tad poles were netted and removed to fresh water daily during the Initial three days of secondary cyst formation. Tadpoles to be infected were placed in shallow bowls and allowed to feed for four or five hours on feoal debris containing cysts. Infeoted and uninfeoted oontrol tadpoles were subsequently maintained In groups of 1$ to 20 individuals In eight lnoh glass finger bowls containing spring water. The water was aerated throughout the course of the experiments. Boiled, dried lettuce was provided as food every third day. Tadpoles were killed and exemlned for zellerielles every four hours during the first twenty-four hours follow ing Infeotlon. Subsequently, tadpoles were killed and examined once or twloe daily. 9lf Development of Infection Cysts In foung ¥a<IpoIsI Excystment of sellerlelles was observed three hours aftar exposure of young, limbless tadpolaa to Infec tion oysts. Reeently axoyated sellerielles (Plata XIII, Figure 32) are similar to amall pre-eystio forms encoun tered In the adult boat* The body la ovoid and eontalna a aIngle nucleus. Division of the nucleus oceurred within five hours. Completion of nuclear division was synchronous with fission, resultant daughters remaining unlnuoleate. Twenty- four hours after exeystment most individuals were broad with a single elliptical nucleus, characteristic of early mitosis. At thirty hours, broad binuoleate Individuals were oommon (Plate XIII, Figure 33). These closely resembled smaller trophosoltes In the adult host. Wessenberg found reeently excysted gamonta of Opallna to have a narrow, flexible anterior end and to show quick darting movements, "... probing Into crevices," turning and reversing. Recently exoysted sellerielles do not exhibit these characteristics. Their movement is similar to that of typical trophozoites. Forty-five hours after exposure to cysts, broad binuoleate forms were dominant. Very broad quadrlnuoleate individuals were occasionally seen. These were invariably undergoing fission and are not here regarded as anomalous. 95 They resemble abnormal giant selleriellea in their swim ming characteristics* Spiral movement* whan it occurred* was slow* Loeomotlon was typioally along a plana tangen tial to tha surfaoe of tha slide* For tha next several days development was charac terised by growth and malntenanoe of binnelearity* Fission in a few individuals eontlnued to anticipate nuclear division with the result that uninucleate forms were found occasionally throughout the period* On the fourteenth day after exposure to oysts* small dividing forms were noted* They became increasingly numerous in the population during the next five days* Large broad individuals continued to be represented though in ever decreasing numbers. Cysts* here termed secondary cysts to distinguish them from those produced In the adult host* were observed from the seventeenth through the twenty-second day. I found no obvious morphological differences between secondary oysts produoed in the tadpole and infection oysts produced in the adult toad* On the twenty-third day only seven tadpoles remained in the experimental group* Examination on this date shewed secondary cysts present in all tadpoles* small trophic forms present in four* No large trophosoltes were present in any of these tadpoles* Devslopmentof Infeotlon Crate in Older Tadpoles In an attempt to determine whether host aga Influenced tha development of larval sellerielles, two groups of tadpolaa* ona with raar limbs only* tha othar with all limbs evident, vara axposad to infaatlon oysts* Sxoystment and aarly development of.sallarlallaa In both groups was similar to that obaarvad aarllar In young tadpolaa axpoaad to eysts bafora tha development of limbs* Small dividing forma and oyata vara obaarvad in aavaral tadpolaa infected vhile in tha raar limb ataga. Two tad poles from tha group infected vhile fully limbed vare found to contain small dividing forma. No oyata vara noted in this group. Three recently transformed tadpoles, infected vhile fully limbed* contained amall trophoxoltea tvo days after metamorphosis. Results of this experiment are summarised in Table 1. Pate of Secondary Cyats Secondary eyata* Ingested by amall limbless tad polaa, underwent development In the same manner aa infection oyata ingested by tadpoles of similar age* Initial growth phases were followed by a period of oyat production. The number of larger individuals present bafora tha onset of cyst formation decreased aa numbers of oysts increased* It was not possible to investigate tha development TABLE 1 97 FA TE OF INGESTED INFECTION CYSTS OF ZELLERLELLA HIRSUTA (D= p o stcy stic d e v e lo p m e n ta l, B= budding fo r m s, C= c y s ts , T= trop h ozoites; each le tte r or com bination r ep re se n ts the z e lle r ie lle sta g e or sta g e s found in a sin g le tadpole) REAR LIMBS PRESENT WHEN INFECTED Tadpole sn o u t-ta il length (mm ) fection 19 20 21 22 23 24 25 26 27 1 D D 2 D D D 3 D 4 D D 5 D D 6 D D, B D,B D 7 D ,B D B,C 8 D B,C B 9 D ,B D ,B D 10 C ALL LIMBS EVIDENT WHEN INFECTED D ays after infection 1 2 3 4 5 6 T adpole sn o u t-ta il length (mm ) 22 23 24 25 26 27 28 29 30 T* D D, B D D, B D D D D D D D D D D * M etam orphosed h o sts 98 of seoondary oysts In older limbed tadpoles from any of the hosts harboring Z. hirsute. However, opallnld-free tadpoles of Bufo canorus In various stages of maturity were available. About 50 of these tadpoles, all with front limbs or front limb buds evident, were exposed to eysts from B. canorus tadpoles. Dally examination showed only Immature trophosoltes present. Five of 7 individuals examined after transformation harbored trophle zellerl- elles. The other 2 tadpoles were unlnfeoted. Gametes and Svngamy In Zellerlella On a single ocoaslon I observed In a tadpole lnfeoted with secondary oysts what appeared to be a small zellerlella attached to the posterior end of a larger Individual. It was not possible to see the nuclei as the cytoplasm was extremely granular and refractlle. I followed this individual for a period of an hour and a half under the mloroaoope. During this time the apparent small zellerlella became less and less distinct, finally merging with the body of the larger. While this may have been gametic fusion, I consider It unlikely for several reasons. The apparent "mlorogamete" was smaller than any individual I have ever seen In hundreds of examinations of larval zellerlelles, being less than half the size of a recently exoysted form. The apparent "macrogamete" was large, about two-thirds the size of a mature trophozoite. 99 Distorted, fragmenting Individuals are frequently encoun tered in populations of Zellerlella. The medium used in this particular ease was slightly hypotonic and vhat appeared to be fusion may have simply been masking of a fragmented portion by swelling of the entire individual. % Morphogenesis of the Infraclllature Vlth the exception of Wessenberg's (19&1) report* nothing has been written of morphogenetic ohange in the infraoiliature of opallnids. Wessenberg noted that in trophosoltes of Qpallna obtrlgonoldea and 0. vlrguloldea the ciliary rows splraled posterlad in a clockwise manner when the protozoans are viewed from the front. He termed this dextral torsion. During the life cycle phase characterised by reduction in size preparatory to cyst formation, detoraion occurred, the ciliary rows becoming meridional. In the small sexual stages found in tadpoles, the meridional condition gave way to anticlockwise or slnlatral torsion. Growth from zygote to trophozoite was accompanied by detorsion and the return of dextral torsion. Wessenberg suggested that torsion involved twisting of the entire organism and that direction of torsion was related to the number of faloular ciliary rows, it being slnlatral when the number was low, dextral when the number was high. Ciliary rows in Zellerlella hirsuta do not oontinue 100 on to the opposite surface but terminate at the Margins. For this reason a strlet ooaq>arison with the rovs of Opallna Is not possible. Ciliary rows in snail seller- lelles are nore nearly Meridional then in large tropho- soltes. Within a few hours after excystment, the orientation of eillary rows in larval sellerielles is sinilar to that in nature trophozoites. I have found no evidenoe that the eiliary rows In Zelloriolla eross the body in the opposite direction from that seen in tropho zoites at any stage In the life oyole. Discussion and Conclusions Preliminary studies suggest that the tadpole phase of the Zellerlella life oycle differs from that of other opalinld genera. No forms were seen that could be inter preted as gametes nor were "protoopallne-llke" larvae similar to that Metoalf described as occurring in Z. antunesl observed. Development of sellerielles in young tadpoles appears to Involve an Initial period of growth followed by budding, terminating in the production of asexual seoondary oysts at about the time rear limbs are formed by the host. Several authors have directly or lndlreotly linked the course of opalinld development with the age of the tadpole. Brumpt (191*>), Metoalf ( 1 9 l {- 0) and Wessenberg (1961) observed oyst formation only in late tadpole 101 lnfeotlons* Brunpt believed that if Infaotad tadpolaa in vhloh opallnids had undargona thair sexual prooaaaaa vara reinfeoted, tha oyata vauld hatoh bat davalop directly into adults, attributing this to an inunity raaponaa by tha host* It ahould ba polntad out that at tha time of Brumpt's publication, little vaa understood of tha eomplex- itlaa of inaninity re action*. It vould a a an renarkable indeed to find antlbodlaa produced in raaponaa to tha presence of opalinlda, whose relationship to thair boats la that of benign guests* It la obvioua, however, that tadpoles in whieh opallnids had undergone sexual repro duction would be at a more advanced stage of development than whan first infected* Konauloff (1928), Overbeek da Mayer (193&) And Hara (1938) all agreed that direct development of infeotlon oyata was poasibla if they were ingested by an older tad pole* Hegner (1931) following a different line of investigation, observed that in Rana olarltana trophlo opalines were lost from tadpoles aoate tine after the appearanoe of rear limbs* He suggested that unfavorable changes in the rectal milieu attendant to metamorphosis were responsible* Metoalf (1923, 19^0) held similar views on the disappearance of opallnids from Asoaphus truel and Rana catesbelana* Both infection and secondary cysts of Zellerlella. if Ingested by older tadpoles, appear to develop directly 102 into trophozoites with the metamorphosing host* Beoause the host used in the final experiment was not the natural one for Z. hirsute, the possibility that zellerielles did not undergo normal development eannot be overlooked. Whether the fate of the produets of either lnfeetion oysts or seeondary eysts is influenced by the endocrine state of the tadpole, as suggested by Wessenberg, has not been determined, though it seems a reasonable possibility. The reduetlon of loss of trophozoites during the later stages of seeondary eyst formation suggests that an additional host is needed to complete the cycle. In view of the Influence of tadpole age on the fate of seeondary cysts, it appears that the second host must be either an older tadpole or the same tadpole at a later stage of development. Sexual reproduction, if it occurs, must proceed very rapidly. The possibility that maintenance of tadpole hosts under artificial conditions may have acted in some way to prevent sexual reproduction in the zellerielles cannot be ruled out. CHAPTER VI PHYSIOLOGY Influence of Host Hormones It has been known for over half a century that the breeding period of anurans was synchronous with eyst produotlon by opallnids. This oolnoldenee has been observed by most students of the opallnids and was remarked upon In some detail by Hereshelaer (1907)* Metoalf (1909, 1923) and Brumpt (1915)* Details of hormonal control of amiran reproduction and metamorphosis beoame known during roughly the same period. Gundernatsoh (1913) described the effect of thyroid feeding on the growth and differen tiation of tadpoles, while Allen (1927) demonstrated that the anterior lobe of the pituitary gland exerts an import ant oontrol over metamorphosis. Houssay and Glustl (1929) showed that the breeding season rhytbm of amphibians Is under the direct control of gonadotropin from the anterior pituitary. Hazard (1935) was the first to suggest that frog hormones could be responsible for cyst produotlon in opallnids. Lwoff and Valentinl (I9I 4.8), In the course of studies on In vitro cultivation of Cepedea (■ Opallna) 103 dimidiate observed that at one time Many eyata appeared in the cultures, They suggested that thla may have been brought about by hormonal change, during the breeding aeaaon, in frog liver uaed in the medium, Blenlars (195> 0) found it peaalble to lnduoe eyat produotlon in Opallna ap, from "frogs" by injecting human pregnaney urine, Thla ocourred only during the non-reproduetive aeaaon of the hoat and vaa taken aa an indication of the aotion of ohorlonic gonadotropin in the urine. No difference In numbera of oyata vaa found during the breeding aeaaon of the hoat, presumably through the overriding effect of the frog’s own pituitary gonadotropin, Cehovic (195> 6) repeated Bieniarz's experiment and, in addition, injected frog pituitary extract. In both oases cysts vere formed. He concluded that the coincidence between cyst production in opallnids and reproduction in froga was due to pituitary activity, probably the proliferation of gonadotropin, El Mofty and Smyth (19&0) in a brief report, described results obtained in a series of experiments on normal, hypophyseotomlzed and gonadeotomlzed Rana temporarla Injected with several gonadotropic and gonadlo substances. This study is described in greater detail by Smyth (19&2) and El Mofty and Smyth (19&l|-)« They found that gonado tropins induoed cyst production in 0. ran arum only during the period November through February while testosterone would lnduoe it during any part of the year. In gonadeotomixed animals only testosterone vaa effective* Tha addition of gonadotropins to opallnids in vitro failad to stinulata oyst produotlon* Soma eulturas rsspondsd positively to pragnaney urine however. A fev oultures, treated with estrone or testis extract produoed dividing stages and aaall forms but no oysts* The authors suggested that oyst produotlon in opallnids is directly related to gonadlc hormone levels rather than to gonadotropin. A similar view was expressed by McConnachle (i960), who studied oyst induction in the same host using chorionle gonadotropin and whole frog pituitarles. Wessenberg (1961) found it possible in a limited number of experiments (two host individuals) to induce cyst formation in Opaline obtrlgonoldea during the non-breeding season by injeoting frog pituitary extract. Cyst Induction in Zellerlella During the summer of 19&3 1 undertook a small number of experiments on the influence of chorionic gonado tropin on enoystment of Z. hirsute from Bufo woodhousll and B, alvarlus. Before eonsidering the experimental results it is necessary to review the natural pattern of reproduction in these hosts and their zellerielles* Much remains to be learned of the reproductive behavior of Bufo alvarlus. Acoounta by Musgrove and Cochran (1930) and by Stebblns (195^) indicate that this 106 species In the neighborhood of Tuoson, Arizona breed* dtiring May and June, apparently in responge to early summer rains. Dr. David Make (personal communication) tells me that he has observed B. alvarlus in the sane area breeding in August. In the oourse of a series of collect ing trips to Yuma, Arlsona over a three year period, I have observed that B. alvarlus may breed during muoh of the spring and summer. I have found members of this species in amplexus as early as mid-April and as late as mid-August. Rains, rare in Yuma, do not seam to play an important role in the initiation or duration of breeding aotivity in local populations of B. alvarlus. Reproductive behavior in B. woodhousll in the vicinity of Yuma begins and ends somevhat earlier than does that of B. alvarlus. I have observed amplexus in thla species from early March through late July. Bragg (19^0a) found most breeding by B. woodhousll in Oklahoma to oecur from March through July. Contrasting breeding behavior in these two toads with that in Rana temporaria, the experimental host used by El Mofty and Smyth, shams marked differences. Rana temporarla is, in the term of Goln and Goln (1962), an explosive breeder, all breeding occurring within a matter of two weeks or so. All adult B. woodhousll and B. alvarlus on the other hand, do not appear to reach breeding condition at the same time. Similar reproductive behavior 107 has been observed in other species of Bufo In the south- 4 we• torn United States Including B* oognatus (Bragg, 19i|.0b) and members of the Bufo boreas species oosiplex (Karlstrom, 1962) . El Mofty and Smyth (I96I 4 .) report that In Rana tomporarla only trophosoitea of Opallna ranarum were present during the period late April to early February* Small diriding forms appeared in early February, oysts in mid-February* At the time of ovoposltlon, (mid-February) 70 per oent of 0* ranarum were trophozoites, 15 per oent were small dividing forms, and 15 per oent oysts. Subse quent to ovoposltlon (mid-February to early April) the proportion of oysts increased to 1^0 per .oent, small forms to 30 per oent. From mid to late April, numbers of oysts and small forms declined until by late April they disap peared altogether, which they attributed to a decrease in gonadal hormone levels in the host. Because of the protracted breeding period In the anuran species studied by me, It was not possible to make a clear-cut distinction between pre- and post-reproductive hosts in the manner of El Mofty and Smyth. I have found cysts and small dividing zellerielles in B. woodhousll and B. alvarlus throughout most of the spring and summer, but not in every individual. Populations oonslstlng entirely of trophozoites are encountered during the same period. Whether these hosts had already completed breeding la unknown* Bufo woodhousll and B* alvarlus in the Tuna area hibernate around the Middle of October* I hare been unable to aecure theae toads subsequent to hibernation and eannot oomment direotly on the oonditlon of their opalin- ida. Indirect evidence suggests that, like the opalines of R. tenporarla during the non-breeding season, Z. hlrauta does not fora cysts subsequent to hibernation by the host. Bufo woodhousll and B. alvarlus collected by ne in late sunuer and early fall typloally contained tropic sellerielles only. Small dividing forms and oysts are seldom encountered in these toads after several weeks in oaptivlty under conditions analogous to those encoun tered in hibernation (restricted physical activity, reduced light, temperature and food)* Cyst Induetlon--Materials and Methods Twelve hosts were used in this experiment. Six (2 B, woodhousll. l \ . B. alvarlus) were Injected intra- peritoneally with 1,000 International Units (IU) of ohorionic gonadotropin, ("Antuitrin S,N Parke Davis and Company), dry powder dissolved in 2 milliliters of sterile distilled water. Another 6 hosts were injected with 2 milliliters of sterile distilled water only and served as controls. Toads were maintained in captivity in the dark for a minimum of three weeks before injection in an effort 109 to reduoe lerels of pituitary and gonadlo hormones* Cyst Induction— Boaulta Experimental results are summarised In Table 2• Thirty-six hours after injeetlon, 1 fmuale B. alrariua, In oaptlrlty one month, orulated* All test and oontrol B. alrarlus were killed and examined for opallnids at this time* Small diriding forms and oysts were present in the recently orulated female and in a test male in oaptlrlty for a similar time* One oontrol animal in oaptlrlty one month contained large trophozoites only, another large trophozoites and a few small diriding forms* A test male in oaptlrlty three months was negatlre for opallnids; a test female in oaptlrlty for a similar time contained trophozoites and small dlridlng forms but no cysts* One control B, alrarlus in oaptlrlty three months eontalned trophozoites only, the other was negatlre for opallnids* Forty-fire hours after Injection, one female B. woodhousll. in oaptlrlty twenty days, orulated. This toad and a female oontrol animal in oaptlrlty a similar time were killed and examined. The test female was negatlre; the control female contained many trophozoites, a few small dlridlng forms and a few oysts* The remaining test female B* woodhousll was glren a seoond Injection of "Antuitrin 3" at this time; the oontrol a seoond injection of distilled water* The test TABLE 2 110 INFLUENCE OF CHORIONIC GONADOTROPIN ON ENCYSTMENT OF ZELLERIELLA HIRSUTA FROM BUFO ALVARIUS AND BUFO w o o d h o u s ii't (A = alvariu s; W = w oodhousii) Host Sex Tim e in Captivity TEST GROUP Large Trophozoites Sm all Dividing Form s C ysts N egative for Opalinids A* ? 1 month + ++ + A* d ii ii ++ ++ A* ? 3 months + A * d ii ii + + w** ? 20 days + w*** 9 2 months + ++ ++ Host Sex T im e in C aptivity CONTROL GROUP Sm all L a rg e D ividing T rop h ozoites F o rm s C ysts N egative for O palinids A* ? 1 month + A* c f it i i + + A* d 3 m onths + A* d i i i t + w ** ? 20 days ++ + + w*## 9 2 m onths + * R esp on se 36 hours a fter in fection ** R esp on se 45 h ou rs a fter in fection *** R esp on se 96 h ou rs after in fection 1. E x p erim en ts p erform ed during m id -J u ly Ill host ovulated fifty-one hour* after tha seeond injeotlon of hormone. Examination at thla tlaa showed a larga number of small dlridlng sallariallaa and nvnaaroua oyata. Tha oontrol hoat eontainad trophlo *ellerlellea only. Crat Induction— Discussion and Conclusions On tha baala of thla aaall aample it appeara that ohorlonla gonadotropin induoaa oyat production in Zeller- lalla hlrauta in a manner alallar to that daaerlbad for othar opalinlda In thair hoata by Bienlara, Cehovic, El Mofty and Smyth, MoConnaehle and Waaaanbarg. Rugh (1951) polnta out that tha activity of tha anterior pituitary in anurans la known to be influenced by auoh external factora aa light and temperature and that direct corraapondenoe axiats between aecretion of pituitary gonadotropin and gonadic hormonea. The decline in numbers of small dividing farms and oyata of Z. hlrauta in B. woodhousll and B. alvarlus Juat before hibernation and after some time in captivity may reflect a decrease in gonadotropic or gonadic hormone levels in these hoata attendant to changes in the environment. There la aome indication that a direct relation- ahip exlsta between the time in captivity and the time required for gonadotropin to exerolse ita influence over the zelleriellea. If the views of El Mofty and Smyth and MoConnaehle that gonadal hormonea are reaponalble for eyat 112 induction in opalinld* are correct, the apparent delay In Initiation of eyet formation seen in B« alTarlna in captivity three month* may be due to greater gonadal regreaalon in theae individuals* Whether inorease in levels of gonadal hormone Is dlreetlv responsible has not been demonstrated for any opalinld. Results obtained by El Mofty and Smyth when treating opalinld cultures with gonad extracts suggest this may be the oase* MoConnaehle remarked that, while little Is known of amphibian gonadal hormones, those of menials are known to stimulate mitosis In several differ ent types of oell* They are also known to be present in mammalian feces. If anuran gonadal hormones behave similarly, their increase during the reproductive season of the host would seem a likely factor in the stimulation of rapid division in opalinids during the period when oysts are formed* The possibility that cyst formation in opalinids may be only indirectly related to host hormones cannot, on the basis of present evidence, be completely ruled out* Bile Pigment Absorption Kedrowsky (1931) reported finding reddish rhomboidal crystals in the endoplasm of Opallna ranarum. Endoplasmic granules in these individuals were colored green. He identified the crystals as bilirubin and the green of tha granules aa blliverdin. Fanthan (1931) described a protoopallna fro* South Afrloa aa P* vlrldls* % on tha baala of lta green coloration. Lavler (1937) reported on cryatala In 0. ranarum similar to those described earlier by Kedrovsky. He observed that orystal development vaa preeeded by diffusion into the opalinids of a green pigment present in quantity in the feces of the host* Crystal growth occurred secondarily at the expense of this pigment* Lavler agreed vith Kedrowsky that the orystals vere bilirubin and the green pigment biliverdin. Mohr (19l|.8) found orystals in several speoies of Horth American opalinids associated vith a greenish suffusion of the intestinal contents. Growth of orystals vas attended by deorease in greenness of the ambient fluid. Brookes and Mohr (19&3) reported on the composition of crystals in Zellerlella sp. from Bufo vallloepa. condi tions for absorption of bile pigments and the persistence of crystals in zellerielles cultured in vitro. They suggested that bile pigments may be absorbed by opallnids vhen pigments are concentrated in the large intestine at elevated pH, pigment concentration being dependent on Intestinal stasis due to inactivity and irregular feeding on the part of the host* Elevated rectal pH presumably aoted both to enhance solubility of bile pigments and to soften the pellicle of opalinids by forming soluble soaps through a reaction between sodium ion and lipids of the outer oell layer*. Cry*tale were analysed and found to be oomposed of the reduoed aloohol soluble billrubinoid steroobllln rather than bilirubin as had been previously supposed. Two types of crystals, single large rhomboids and dusters of small rhomboidal needles were noted* Reduction of biliwerdln to stereobllin and growth of orystals was attributed to a shift in eytoplasmie pH toward neutrality. Crystals were observed to persist unohanged in Zellerlella eultured in vitro for fourteen days. No apparent interference with the normal activities of the Protozoa was observed. The absence of crystals in Opallna from the Bame infection was tentatively considered due to their larger size; the level of alkalinity in the gut being sufficient to inorease permeability in the smaller, thinner zellerielles but Inadequate to effeot a similar change in the more robust Opallna. Gradients Child (I91I 4 .) was the first to demonstrate con vincingly that physiological gradients exist along major protozoan axes. Taylor (1928) found regeneration in the hypotrlch Gronychia unclnata to proceed more rapidly near the center of physiological dominanoe. A similar observa tion was made on another hypotrioh, Groleptus mobllla by Tlttler (1933)* Balamuth (I9I 4 -O) in a comprehensive study on regeneration in Protozoa, suggested that the basic 1X5 mechanism operative In regeneration was no dlffarant from that acting in normal reorganisation and growth. Balamath further pointed out that while the nucleus may be largely responsible far the faot of reorganisation, the aotlvlty of physiological gradients might explain the directional movements lnTolred. Evidence indicates that in opallnlds the region around the left end of the falx is dominant physiologi cally. Okajlma (1953) and Kinoslta (1952) hare shown the olllary beat to originate here. In moribund opallnlds, the ollla of the left anterior margin are the last to cease beating. Becker (1926) reported that Opallna, stained intravltally with Jsnus Green B, reduced the dye to pink diethyl safranin, attributing this to the presence of a reductase in the cytoplasm. Mohr (1939* unpublished notes) found that in Opallna vlrguloldea reduction of Janus Green B proceeded more rapidly in the region of the left margin than elsewhere. Mohr (19l|.0) also observed that in abnormal giant sellerlelles with many nuclei the nuclei closer to the left margin were farther advanced In mitosis than those to the right. I have found that, in normal Zellerlella. the left nucleus ooomenoes mitosis somewhat in advance of its mate. Growth in Zellerlella and other opallnlds proceeds most rapidly in the neighborhood of the left end of the falx and new primary oiliary rows appear to be proliferated only in tills region. 116 In Vitro Cultivation P&tter (1905) was able to maintain Opallna ranaruat in dlluta sodium chloride solution containing small amounts of sodium and potassium tartrate for from three to seven days* Addition of boiled egg white and simple sugars permitted survival of opalines for up to three weeks* Konsuloff (1922) bj frequently changing the medium so as to avoid excessive bacterial growth, was able to maintain Cepodea (* Opallna) dlmidlata in Putters solution for two months* Larson and oo-workers (1925, 1928) and Larson (1928) cultivated Opallna obtrlgonoldea in several media including P&tters solution, Locke's fluid, sea water, sodium oltrate and tap water* Best results (sur vival for four days without subculturing) were obtained with media to which blood serum or a small piece of the host's cloaoal wall were added* Kedrowsky (1931) studied lipid reserves in Opallna ranarum kept in a variety of saline solutions* He found that hypotonlo (0*05 molar) solutions of several salts favored longevity of opalines over solutions of' a single salt* Isroff and Valentlni (191*8) attempted bacterial free cultivation of Copcdea (* Opallna) dlmidlata in a complex salt solution to whloh was added frog liver extract and gum arablo* In some instances cultures were maintained for twenty-six days* The authors were not certain of the absence of bacteria. The first truly successful culture of opallnlds 117 was established by Tang and Bamberger (1953)* Using a diphasic medium containing a solution of several salts over a slsnt of coagulated vhole egg these workers main tained Opallna sp. from Bufo wall loops in culture for four months* Tang (19&1) reported on the continuous cultivation of Opallna (from the original oultures of Tang and Bamberger) for a period of over six years in which the original medium had been replaced by a more convenient monophasio medium containing liver extract* The same culture is now maintained in its tenth year by Professor Tang* I have attempted to oultlvate Opallna and Zeller- lclla but with little sucoess. My cultures have been killed after a few days by exoesalve bacterial contamina tion or by large numbers of flagellates. I have been able, using antibiotics in combination with serial washing, to control the numbers of bacteria, but have not been able to eliminate the flagellates* The cultivation of opallnlds does not, as suggested by Metcalf (1930) require the replacement of atmospheric oxygen in the environment by a reducing gas* Yang main tains his cultures in a small amount of medium in the bottom of a cotton-stoppered test tube. Success in separating opallnlds from undesirable associates, particularly the ubiquitous small flagellates, appears to be the greatest single requisite for thea 118 establishment of permanent oultures. Opallna. freed of flagellates nay ba naintalnad satisfactorily in several nedla. Several cultures of flagellate-free Opallna. made available through the generosity of Professor Tang, vara naintalnad by ne for several months in Tang’s (1961) nediun, a modification of this medium to vhich gun arabic was added, and a modification of the nediun of Lvoff and Yalentlni (19^8) in which frog liver was replaced by blood serun in amounts similar to those used by Tang. This last medium was found to be particularly favorable for the growth of Opallna. Hew techniques for effecting separation of opallnlds from flagellates should be sought. The import ance of ridding the cultures of these latter associates cannot be overemphasized. On one occasion I inoculated two of Professor Tang’s cultures with a few flagellates from the rectum of Bufo vallloepa. Complete displacement of opalines by flagellates was observed within four days. Serial washing may remove most of the flagellates and t bacteria but many opallnlds are lost in the process. Tang has pointed out that inoculation with at least a hundred individuals is necessary for the establishment of a culture. CHAPTER VII ECOLOGY Habitat Konsuloff (1922) and Metcalf (1923) remarked that opallnlds are most frequently encountered In the upper end of the rectum near its juncture vlth the Ileum, Hunter (1955) observed that Opallna carollnenals In Rana plplens ooourred In greatest numbers toward the center of the rectum. In healthy well-nourished hosts, Zellerlella hirsute tend to concentrate at the periphery of the fecal mass between the middle and anterior end of the rectum. Several authors have reported finding opallnlds in the lower end of the small intestine. Konsuloff (1922) found Opallna ran arum in the small intestine of hibernating Rana temoorarla. Hunter (1955) observed that 0. carolln- ensls migrated from the rectum to the small intestine sometime after the death of Rana plplens. Wessenberg (1951) found that the opalines of Bufo boreas and Hvla regllla occur in greatest numbers in the small intestine just prior to metamorphosis. Marx (1963) reported opalines present in the same area in two starved Rana plplens and in a third individual with a malformed rectum. 119 120 I have found Z. hirsute, in the lleuai of its host “ “ i on several occasions, usually when the toad had been In oaptlvlty for some time or harbored extraordinarily large numbers of nematodes or flagellates* Marx proposed that opallnlds migrate to the small Intestine during hiberna tion and metamorphosis. Such migration may be a general reaction to unfavorable ohanges in the reotal milieu, oeourrlng not only during metamorphosis and hibernation but under conditions of high parasitemia by other groups and various other pathological states* Zellerielles in tadpoles are distributed in a manner similar to that seen in the adult host* In older tadpoles they are oonoentrated in the rectum* In small tadpoles with undifferentiated recta, zellerielles are more numerous in the distal intestine* Assoolates Hazard (I939) in a brief paper on ecology of opallnlds, observed that they are frequently "orowded out" by large numbers of the flagellate Trichomonas augusta and that a trematode, Dlplodiscus tewporatus, infrequently present, feeds on them* Bhatia and Gulatl (I927) sug gested that the greatest enemies of opallnlds were nematodes which ate them* Hunter (1955) found an Inverse ratio between Opallna carollnensls and either kind of worm* He did not believe that the worms fed on the 121 opallnlds but rather that they were killed by an aocumu lation of toxic waatea produoed by the worma or aa a reault of the depletion of nutrients. I have on only two oooaalona encountered trematodea In the reetun of anurana though I have found them Infre quently In the amall In tea tine. Nematode a have been abundant In my material, particularly in toads a long time In captivity. I agree with Hunter that an Inverae ratio ezlata between nematodea and opalinlda. I have never obaerved nematodea feeding on them however. Flagellates have Invariably been present In my specimens • As la the oaae with nematodea, flagellates usually occur in greatest numbers In hosts a long time In captivity. However, they may also be extremely abundant In newly taken anurans. An inverae ratio exlata between numbers of flagellates and zellerlelles similar to that existing between sellerlellea and nematodes. Other protozoans observed in association with Zellerle11a include the heterotrichous dilates Nvctotherus (frequent) and Balantldlum (rare). Sandon (1949) reported the same two dilates present In the large Intestine of eight species of fresh water flah harboring opallnlds. He found the resulting fauna remarkably similar to that of anurans, suggesting that there appeared to be no special conditions favoring the presence of opallnlds In these fish but rather that conditions were favorable to dilate inf**©tion in general* On ••▼•ml oocations enormous numbers of rotifers war* found in tho terminal portion of the intestine of tadpoles of B. woodhousil and B* oocaatua maintained under crowded conditions* In such instances, metamorphosis was delayed* I am uncertain whether this was simply due to orowding or to some interference by the rotifers with the nutritional or endocrine state of the tadpoles* Zeller- lella oocurred together with the rotifers but always in small numbers* There was no evidence that rotifers fed on them however* Among other unusual associates of Zellerlella (and other opalinid genera) are the hyperparasitio entamebas. First observed by Metcalf (1923) in zellerlelles from B. woodhousil and B. cognatua. but mistakenly Identified as "secondary" or "abnormal disintegrating" nuclei, their parasitic nature was recognized by Carlnl (1933) who plaoed them in a new genus, "Brumptina," Stabler (1933) correctly identified Metcalf's "secondary nuclei" and \ Carini's "Brumptlna” as Entamoeba, in a report confirmed by several later workers* Chen and Stabler (1935* 193&) and Stabler and Chen (193&) have presented the most thorough studies to date on the morphology, mode of trans mission and geographic distribution of entamebas hyper parasitio in opallnlds. These authors found them widely distributed and present in representatives of all opalinid genera* The taxonomic position of the entamebas has not been settled* Chen and Stabler (1936) found then very similar to Entamoeba ran arum, the common enterie maeba of anurans Whose life oyele has been described by Sanders (1931)* while Amaro (19&2) believes them to be E. paulista. another anuran smeba similar in appearance to B* ran arum* Hoare (19^3) observed that morphologically indistinguish able groups of Protozoa have historically been attributed to independent speoles on the grounds of biological differences between them* There is little structural difference between Entamoeba ranarum of frogs, E. histo lytica of man, E. lacertlcola of lizards, E* lnvadens of snakes, E. terrapinae of turtles and the saprozoic E. moshkovski* Biologically, E. histolytica differs from the others in its temperature requirements (Dobell, 1916) and Cairns (195>3) found it impossible to infect bullfrogs with E. lacertlcola or turtles with E. ranarum. The question of genetic relationships between these morphologically identical species of enterie amebas found in different hosts is interesting and important but not within the scope of this paper* Some evidence is available suggest ing that the entamebas of opallnlds may be biologically distinct from either E* ranarum or E. paulista* Carinl (19^3) has found entamebas present in zellerlelles in five species of bold snakes while Misra (1943) has observed them in Opallna from the lizard Varanus monitor. 12k Whether aore than one apeoles of Zelleriella may ocour simultaneously in a host’is a matter of conjecture. Metcalf (19^0) examined thirteen Leptodactylus ooellatus and found some to harbor Z. Uruguayans!s. some Z. brasili- ensls and a few Z. an tunes!. He remarked that, "In one Individual, Z. braslllensls and Z. antunesi were present • • • this Is one of the few Instances in which I have found in one individual host what seem to be two species of opallnlds*" Carini (1938) reported that Z. truncate from L* ooellatus ocourred rarely in Leptodactylus ooellatus, always In the presence of other sellerlelles, (an observation I regard as due to Carlni's failure to reoognize fission variants), I have not found what I consider more than a single species of Zellcriclla present at one time* Other genera of opallnlds, particularly Opallna. may occur in coamon infections with Zelleriella. In the United States multiple infeotlons of these two genera have been observed in B. woodhousil. B. cognatus. B. alvarlus. B. punotatus. B. valllceps. and Scaphlopus holbrookl. Other Horth American hosts reported to harbor Zelleriella. including one speeles of spadefoot toad and two Ranas have been found to harbor Opallna as well but not in joint infections. Opallna and Zelleriella occur together in several hosts In Mexloo and Central Amerloa. It has been my observation that when Opallna occurs 12$ simultaneously with Zallarlslla the latter Is prsssnt only In small numbsrs. Host Specificity Metoalf (1909) performed several transfaunatlon experiments with opallnlds from various speoles of European anurans. He found It possible to Infeot almost any larval host speoles with the opalinid from another. The ultimate fate of these Infeotlons in the transformed host was not observed. Metoalf believed that suoh unaooustomed Infec tions could occur in nature but would fall to become permanently established due to the inability of the "unnatural" opalinid to respond to the host stimuli in the pre-sexual and sexual periods. Opalinid infeotlons in such non-anuran vertebrates as salamanders, snakes and lisards were considered adventitious, resulting from predation by the host on frogs. Metoalf (1923) further suggested that, based on host distribution, speoifioity must have been less rigid in past times. He was aware that morphologically identical forms ooourred in a variety of hosts, sometimes in different families, but regarded these as physiologically specific. Subsequent investigators have disagreed with Metoalf*s conclusions. Wenrloh (1935) reported finding apparently identloal opalinlda in a snake from Cuba and a tree frog from Florida, holding this and other reports 126 of opalinlda from non-anuran vertebrates to ba evidence for tha abaanoa of boat specificity, Mohr, Geiger and Brahm (1961) found it possible, in a United number of experi ments, to Infeot free-swimming tadpolaa of several apaelaa with foreign opalinid oyata and ooeaalonally aatabliah flourlahlng InfeotIona In adulta by pipetting trophlo opalinlda Into tha rectum, It vaa their conclusion that auoh specificity aa exists between opalinlda and their hoata la due primarily to ecological rather than physio- logioal barriers. Ecological laolatlon of an opalinid In a particular host apeoles may be readily envisioned. All speolea of anurans In a given area do not oviposit simultaneously nor do the different tadpoles necessarily share oosason feeding habits, When these factors overlap, sharing of opallnlds may ooeur, Thus Zelleriella hlrauta la found in common In Bufo woodhousil, B. cognatus and B. alvarlua which ooour together in canals and drainage ditohea around Yuma, with reproductive oyclea at least potentially overlapping. The Opallna found in the same three hosts may also be a single species, though I have not studied these critically. In a paper given before the Western Sooiety of Naturalists at Stockton, California In the winter of 1963 I presented a similar case for sharing of Opallna, Two disjunct popula tions of the Yosemite toad, Bufo canorus. were found to harbor distinctly different opalines. One was similar 127 to Opallna vlrguloldea of Hyla regllla vhlla tha other closely resembled the Opallna (unnamed) of R*»a musoosa. It la significant that In the first ease B. oanorus shared a oommon habitat with H. regllla while In the seoond it occurred with R. musoosa. One of the weaknesses of transfaunatlon experiments to date is that Infeotlons with foreign opallnlds have not generally been followed subsequent to metamorphosis. In order to test Metcalf's hypothesis that an "unnatural1 1 opalinid would fall to beoome permanently established due to an inability to respond to host stimuli, I infected tadpoles of a host not previously reported as harboring opallnlds, (but in fact occasionally sharing Opallna with its neighbors), Bufo oanorus. with Z. hirsute from B. woodhousil. Tadpoles were collected from seepage ponds at the southern end of T. J. Lake, Inyo County, California, at an elevation of nearly 11,000 feet. Many individuals were nearing metamorphosis, others had rear limbs only or were limbless. They were divided into two groups on the basis of whether limbs were evident or not, exposed to Zelleriella infection cysts for a period of five hours, then removed. Limbed tadpoles were plaeed in a small amount of water in tilted flat-bottomed pans, permitting exit from the water as necessary. Limbless tadpoles were treated as in earlier infection experiments, and removed to flat-bottomed pans as metamorphosis approached. 128 Juveniles, maintained on damp paper toweling in Jars, were fed Drosophila or Tubifex at two day Intervals* Daily examination of both groups showed a high ineidenoe of initial infection* Fifteen of 18 juveniles exposed to eysts before limb formation showed vigorous infeotlons ten days after metamorphosisv as did 5 of 7 examined twenty-one days after metamorphosis* Zellerlelles in older individuals were more numerous and larger than in newly transformed individuals* Ten of 13 transformed juveniles exposed to eysts after formation of limbs were similarly infected twenty-one days after metamorphosis* These results indicate that conditions in the host did not act to restrict the development of an "unnatural" zellerielle at least during the tadpole, metamorphic and immediate post-met amorphic stages. Whether the infections would have been sustained through subsequent development to maturity was not determined. To assume that any opalinid may infect any anuran would, on the basis of present evidence, be unwarranted* Physiological differences have been shown to exist between different opallnlds, as for example Kedrowsky’s (1931) demonstration that various speoles of Opallna stain differently with toluldlne blue and the reports by Suckhanova (1962c), and Poljansky (cited by Levitt, Meryman and Prosser, 1963) that Opaline from a stream frog (Rana temporarla) has a lower heat tolerance than Opallna 129 from a lake frog (Rana rldlbunda). Whether such physio logical differences may ba ralatad to hoat spaolflo faatora la, at tha praaant time, unknown. Parhapa more garmane to tha problam ara tha observations of Hatoalf (19^0) and Hegnar (1931) that eartaln froga with over- wintaring larvae may ba Inaapabla of auatalnlng opalinid infaetlona through metamorphosis. Matoalf (1923) auggaatad that anurana without free-living larvae would llkaly ba unlnfaetad with opali nid a* This aaaumption appeara to ba largaly trua. Aacaphus trual harbora an opalinid whlla tha ralatad Lolopelma. which laeka an aquatlo larval atago, ia barren. Metcalf found no opalinlda in twelve apeolmena of South American plplda, whose young develop directly or in pockets on tha back of tha female. Since these hosts ware museum speci mens tha possibility that any opalinlda praaant may have decomposed cannot ba ruled out. An unusual report of an opalinid in a host without a free-living larvaa comas from Mlara (1960). Ha found vigorous infeotlons of Opallna in several specimens of tha desert monitor, Varanua grlaeua. a speciet which, according to Mlara, has little chance to coma aoroaa froga in nature. It was Misra* oontantion that, oontrary to Metoalf*a (1940) opinion, opalinid infections in monitor llzarda (four reported hoat speoles) are not adventitious or temporary. He did not comment on how the life cyole might go to 130 completion in V* grlseua however* Car ini (191*3) reviewing tha reoord of tallaria11aa in South Anarioan bold snakes, was unaura of vhathar thay vara natural or faoultatlve infections* A final oanaideration of faetora influencing infaction involvea tha partioular anuran larval type dealt with. Tadpolaa have, in raaponaa to tha aeleetive preaaure of tha habitat, undergone adaptive radiation in feeding node and nouth nod if 1 oat Iona, without partioular regard to phylogenetic lines* Included in the adaptive typea are oarnivoroua tadpolaa with enlarged beaks which feed on invartabrataa aa well aa larval vertebrataa. In this category are aavaral Old World ranids, and tha tropical American leptodaetylid Ceratophrva. (Hobla, 1933)* Other anuran larvae have dorsally-directed moutha and feed on aurfaoe plankton, for example the Aaian Mlcrohvla hevmonal (Noble, 1933) and Hyla aetakl from Coata Rica (Starrett, I960), It would aeam unlikely that either of thaaa tad pole types would acquire opalinid cysts in tha usual manner, that is, by feeding on tha faoea of the adult, Anurans with carnivorous tadpolaa might ba axpaotad to share opallnlds with associated species whose larvae are preyed upon by the former* Anurans whose tadpoles feed on aurfaoe plankton may not harbor opallnlds at all* Studies on this aspect of opalinid eoology are totally • lacking at the present time* Geographic Distribution Zellerlellee have been reported from or are known to oeeur in 78 apeelee of anuran, 67 of which belong to a single suborder, Proooela* All but 7 anuran hosts are from tha Saw World. Son-anuran hosts include a gymnophi- onan, several speoles of bold snakes and 2 eatfishes* A regional oheok-list of dasorlbed and undesoribed teller- lelles Is lnoluded in this paper as Appendix I* In the introduction to this paper it was pointed out that the distribution of opallnlds among tha anurans has led to theories on host-phylogeny and migration routes* Developed and extended by Metcalf (1923, 19if0 and several intervening papers^ these notions have, despite early and severe criticism, retained considerable popu larity, particularly with parasitologists* Briefly, Metcalf held that opallnlds and anurans evolved in parallel and that from the distribution of opalinid genera and subgenera whose phylogeny was presumed known, could be adduced the phylogeny and probable migration routes of anuran families and, in some cases, genera* This view necessitated the assumption of specificity between opalinid and host at least at the family level. Judging from its persistence in the literature, the most acceptable evidence used by Metoalf to support his theories was the restricted distribution of seller- ielles. Metcalf held that Zelleriella was exclusively confined to tropical and semi-tropical America and Australia* Evidence to the contrary was either dismissed* ignored, or both. For example, Bezzenberger *a (190l|.) report of Zelleriella macronucleata from an aelatlc Bufo was regarded by Metcalf (1923) as ". • • so strange that one must question the Asiatic origin of the host • • « one wonders if there might not have been any oonfusion of labels or if the host in question might have become infected from some South American anuran with which it may have been kept and which it may have eaten." Metcalf (19l|.0) noted, in a footnote in small type, Nie's (1935) report of Zelleriella orlentails in Microhyla omata from Nanking. Sandon's (1938) discovery of zellerlelles in two South African Hanas was ignored altogether in the same work, though included in the bibliography. Since the publication of the second half of Met- * calf's monograph several additional reports of Old World zellerlelles have been published. Sandon (19^9) found Zelleriella in a Sllurold fish from the upper Nile River in Africa, TJttangi (1951» 19&1) has described three species from Indian frogs, and Liu (1957) has reported finding Z. orlen tails in Mlorohyla omata from Taiwan. During the same period, 10 species of Zelleriella have been described from the New World and 3 unnamed forms reported from new American hosts. Eighteen hosts not previously known to harbor zellerlelles are reported for the first time in this paper (Appendix I). Metoalf noted that, in its range, Zelleriella \ was frequently encountered in leptodaotylld frogs, a group he oonsidered exclusively neotropical and Australian, nerer occurring in the Old World. Reluctant to attribute the coincidence in distribution of leptodaetyllds and zellerlelles to simultaneous convergent evolution, Metcalf oonoluded that they had evolved together in Pategonia, some leptodaetyllds later migrating to Australia over a land bridge between southern continents similar, as Duxm (1925) pointed out, to the corridor erected by earlier soogeog- rsphere to permit marsupials access to South America and Australia. Metcalf’s conclusions are still considered attractive by certain contemporary workers. Manter (1963) expressed wholehearted approval of Metoalf*s notions in his reoent paper on parallel evolution of trematodes and their hosts. Noble and Noble (i960) aeoept Metcalf’s work quite uncritically in their recent text on the biology of animal parasites as apparently does Kudo (195&) in his standard text on the Protosoa. Duxm (1925, 191+1)* Mathew (1929* unpublished MS) and Noble (1925* 1931) criticized Metcalf's theories on the grounds that his anuran phylo- genies were poorly drawn and his interpretations of paleogeography not in aoeord with geologic evidenoe. Present day biogeographer a, including Simpson (19^0)> Romer (19^5) snd Darlington (1957) reject a South 13k Auer loan-Australian corridor aa essential to explain the well-known similarities in flora and fauna. These authors support the view given bj Moble that anurans probably reached the southern eontinents by invasion from the north* As Dunn remarked* Metcalf's observation simply supports herpetologloal evidence that the leptodaetyllds are a natural group and in no respect shows how they reached their present ranges* A particularly conservative opinion on the history of anuran dispersal has been recently offered by the paleobatrachlologlst Max Hecht (1963)* who holds that* "Until a more complete fossil record becomes available, zoogeography of the frogs will remain in the realm of mythology*" Mohr (194lc) criticized Metcalf's theories on protozoologioal grounds. He pointed out that most of Metcalf's materials were in poor condition* implying that distinctive features assigned to lower taxa were as likely derived from the manner in which they had been killed and preserved as from their parents* MoreoverA the low order of morphological distinction between species rendered opallnlds unfit as indicators at this level* Mohr also argued that Metcalf's sampling was inadequate numerically and his hosts poorly soattered geographically* eoologi- cally and taxonomioally* Metcalf's theories were developed from the distri bution of opallnlds among l£3 anuran species. Today* tha list has baen extended to over 270 apeolas In l j . 8 genera. Tablaa 3 “id If summarise tha distribution of opalinid ganara among anuran families and suborders. Aa nay ba lnfarrad froai tha tablaa, many of tha inadequacies in sampling noted by Mohr ramain. Among tha mora obvious deficiencies ara tha daarth of raaords from Afrioa, Australia and much of Aala and tha unequal scatter among oertaln taxonomic catagoriaa. For example, tha opalinlda of Old World miorohyllda, a group vail represented in tropical Asia and Africa, ara virtually unknown as ara tha opalinlda of South Amerioan miorohyllda and 3 exclusively tropical American families, Dendrobatldaa, Atelopodidaa, and Centrolenidae. Sampling from North American hoata would seem reasonably adequate. Opallnlds have been reported from 51 of tha 66 or so species of anuran of tha United States and Canada, Including repreaentatlvea of all genera. Regionally in South and Central America a rather large number of raoords has bean published. Opalinlda from froga in tha neighborhoods of Rio da Janeiro, and sXo Paulo Brazil, Montevideo, Uruguay, and La Plata, Argentina have baen sampled in some depth. Several opalinlda from Costa Rioa and Nloaragua have been described by Metoalf from preserved hosts. In terms of host categories, the greatest number of records come from the advanced suborders Diplasioooela and Procoela (21+3 host species). Opallnlds are known TABLE 3 SUMMARY DISTRIBUTION OF OPALINID GENERA AMONG ANURAN FAMILIES1 South North America^ Am erica Europe Asia Africa Australia^ O P Z O P Z O P Z O P Z O P Z OP z Liopelmatidae (1-1) (1-3) 1 Discoglossidae (3-5) (2-3) 3 3 2 Pipidae (1-5) (3-11) 2 Rhinophrynidae (1-1) Pelobatidae (1-6) (2-5) (6-41) (3-4) 2 3 2 2 2 1 Microhylidae (16-42) (2-2) (22-126) (17-60) (2-3) 111 51 1. Numbers in parentheses reflect genera and species of anura in each region according to Gorham (1963). 2. Includes Central America and Mexico. 3. Includes New Zealand. (CONTINUED) Leptodactylidae Bufonidae Hylidae Centrolenidae Atelopodidae Dendrobatidae Ran id a e South North Am erica America Europe Asia Africa Australia O P Z O P Z O P Z O P Z O P Z O P Z (38-495) (3-5) (2-6) (15-66) 5 3 24 1 5 2 (1-63) (1-14) (1-3) (7-65) (7-60) 2 1 18 12 5 1 14 3 1 2 3 (29-390) (3-20) (1-1) (2-55) (1-31) 17 7 11 1 1 ^ 4 (3-29) (1-27) 2 (3-58) 1 5 (1-13) (1-18) (1-6) (16-216) (20-213) 3 3 12 2 4 1 27 9 5 8 2 Rhacophoridae (2-95) 5 1 1 (16-340) 7 1 TABLE 4 SUMMARY DISTRIBUTION OF OPALINID GENERA AMONG ANURAN SUBORDERS1 South North America Am erica Europe Asia Africa Austr AMPHICOELA Opalina 0 0 2 genera & P'opalina 1 0 4 species Zeller r la 0 0 OPISTHOCOELA Opalina 0 3 0 0 9 genera & P'opalina 0 3 2 2 25 species Zeller'la 0 0 0 0 ANOMOCOELA Opalina 2 0 2 0 11 genera & P'opalina 3 2 1 0 56 species Zeller'la 2 0 0 0 MESOCOELA1 Opalina 0 1 5 ♦ 0 0 47 genera & P'opalina 1 1 0 0 0 229 species Zeller'la 0 0 1 0 0 PROCOELA Opalina 25 23 2 15 2 0 104 genera & P'opalina 4 0 0 3 4 9 1386 species Zeller'la 56 5 0 1 0 2 DIPLASIOCOELA Opalina 3 12 4 32 12 52 genera & P'opalina 0 0 1 10 9 892 species Zeller'la 3 2 0 1 2 1. Term of Professor Jay Savage in lit, includes the single family Microhylidae 139 from 10 of 26 spaolea comprising tbs two primitive sub orders Amphlcoela end Oplsthocoela* Several authors, Including Dunn (192$, I9I 4 .I)» Noble (192$, 1931), Wenrieh (1935), Mohr (19l|lc, 19$9) «nd Mohr, Oelger and Braba (1961) have further ohallenged Metoalf*s theories on grounds that opallnlds laek host specificity and are, in part, where they are today as a result of transmission of Infection cysts between hosts of similar habitat and habits irrespective of genetic rela tionship, This conclusion is supported by the oolleotlon record and a Halted number of field and experimental observations* In the absence of a thorough understanding of factors influencing infection, the probable consequences of straggling on the distribution of opallnlds can only be a matter for speculation* Metoalf (1923, 19M)) supported his opalinid-anuran distribution theories by contending that oertaln anuran groups were: (1) resistant to infection by oertain genera (leptodaetyllds to Opallna. hylida to Zelleriella): (2) particularly susceptible to infection by a genus (Bufo to Zelleriella): or (3) that certain opalln genera were prone to Infect any host ("Cepedea adopts anything it meets")• These generalizations are unfounded* They were not tested by Metoalf nor have they been tested by later workers* Experimental investigation of whether, in a lifO particular host category, one sort of opalinid fares better than another would perhaps bo useful in helping explain present distributional patterns, Inconclusive evidence gathered In the oourse of my study suggests that, In the 3 Arisonan Bufoa, Opallna may be better adapted than Zellerlella, The ratio of occurrence of Opallna to Zellerlella In adult hosts examined was approximately 3 to one In 68 speolmens of B. woodhousll and 37 specimens of B. cognatus, 2 to one In 20 specimens of B. alvarlua. In mixed Qpallna-Zellerlella infections, (9 B. woodhousll. ^ £• eoanatus. 5 > B. alvarlua) zellerlelles were greatly subordinate to opalines In numbers. CHAPTER VIII TAXONOMY Relationship to Other Protoaoa Based on their uniform holotriehoua oillature and laok of a mouth, opallnids were originally grouped vith the astoaatous dilates. Toward the end of the last century, several investigators observed that in certain respeots opallnldf differed from typioal dilates. The absence of a maoronnoleus was first remarked by Parker and Haswell (1897). Leger and Dubosoq (190lf) and Neresheimer (1907) recommended removing opallnids from the dilates and placing them with the flagellates on the basis of similarities in life cycle. Hartog (1906) made a similar suggestion based on the nuclear condition. Cepede (1910) separated opallnids from the group with which they had long been associated, the astomatous dilates, in his monographio work on the latter. Metoalf (1918a, 1918b, 1923) placed the opallnids firmly in the dilate lineage as subclass Protoelliata, the choice of name indicative of his view that opallnids were primitive representatives of the Cillata. This arrangement necessitated a second sub- class, Euclllata, for the remainder of the group. l l fl 1 1* 2 Basle to Metcalf's position were two assumptions: first* that opallnld nuolei contained both "macro- and mloroohromosomes*" representing a transition stage In the evolution of dimorphic nuclei typloal of higher dilates; second* that fission was of a sort intermediate between that of dilates and flagellates* being sometimes longi tudinal* sometimes transverse* With few exceptions* contemporary workers followed Metcalf's classification* Dissidents fell Into two cate gories; those who wished to retain the opallnids within the dilate line but disagreed with one or another of Metcalf's assumptions* and those who favored their removal from the Ciliata altogether* Inoluded In the pro-oillate group was Konsuloff (1930* 1931) *ho held to astome affinities* He believed that true macronuolel were present In the form of disc- shaped bodies in the cytoplasm (the "endospherules" of Metoalf* mitochondria of Hunter* and probably the mlto- ohondrla of Noirot-Timothee). Tonniges (1927) had previously homologlzed these structures with the dispersed macronuolel of the gymnostome dilate Dllcptua glgas* Chen (1932a, 1932b) convincingly demonstrated that Metoalf's "amphinucleus" was the product of misinterpreta tion* Calkins (1933) In opposition to those who wished to assign flagellate affinities to the opallnids observed that gametic fertilization Is not wholly unique to 11*3 opallnids and flagellates but occurs In modified form in trus dilates, Mohr (1941a) oompared the fibrillar system of opallnids and ths astome Anoplophrva and found them similar, holding this to be strong evldenoe for oillate affinities* He took the position that opallnids were neither primitive dilates nor direotly in the astome line but rather represented a group removed from the main stream of oillate evolution. Oatenby and King (1925) and Kofold and Dodds (1928) favored removal of the opallnids from the Giliata and their inclusion vith the flagellates on the basis of presumed anatomical similarities. Their observations have not been and probably cannot be verified. The strongest pro-flagellate arguments have been developed during the past quarter century in Europe. Chatton and Braohon (193&) found similarities in fission in Opallna and hypermastlgote flagellates and homologlzed the anterior-most "generative kinetles" in the former vith centrloles in the latter* This finding, together with the reported occurrence of syngamy and the unquestionable presenoe of monomorphic nuolei in opallnids has resulted in their removal from the oillate line and their inclusion smong flagellates by a majority of reoent students of protozoan taxonomy, among them Lvoff and Valentlnl (1948). Ulrich (1950), Grasse (1952), Faure-Fremiet (1950* 1953)* Blooca (1956)and Corliss (195&)* ilA Corliss (1955) Wessenberg (1961) have reviewed ths evidence for affinities with elliates and flagellates* Corliss commended the position of Grasse who treated opallnids as a superorder within the mastlgophora, while Wessenberg considered the opallnids sufficiently distinct from either group to warrant placement in a separate olass. Chiefly on Wessenberg's reaffirmation of transverse fis sion in Ooallna. Corliss and Balamuth (1963) have recently removed the opallnids from a position subordinate to the flagellates and created for them a new subclass within the Sarcomastlgophora, but at a level equivalent to true flagellates* Corliss accepted the view that opallnids divide only longitudinally and that daughters are symmetrlgenic, as put forth and elaborated by the French schools of Chatton, Faure-Fremiet, Lwoff, and Grasse* Wessenberg holds that in Opallna transverse fission also occults, producing homothetigenic daughters* Zellerlella invariably divides longitudinally but, except for the fact that the fission plane passes between ciliary rows, there appears little else in oommon between fission in Zellerlella and flagellates* Fission in hypermastigote flagellates, to which fission in opallnids is most frequently compared, involves a series of phenomena quite lacking in Zellerlella and other opallnids. In hypermastlgotes (Cleveland, 1938, * 4 5 1949) tb. olll*ry band* appaar to b* darlTad from a pair of centrloles. At fission, ons osntrlols and half the ciliary bends go to each daughter, whereupon the eentrlole dlTldes and new bands are produced.^ Centrloles of hyper- mastlgotes play an aotive role In the formation of the so-ealled achromatic figure. Astral rays arise from the distal ends of each and form a central spindle which is pulled apart as the daughters separate. Centrloles are lacking in Zellerlella and other opallnids. During fission, the falx is transected. New primary ciliary meridians in eaoh daughter are apparently formed at one end of the falx, seoondary rows at various Intermediate positions along ita length. Daughters are dissimilar in shape and may be dissimilar in size. In terms of readily identifiable features of fixed orientation, for example the apical end of the falx, dividing zeller- lelles exhibit one for one correspondence in parts, not mirror-image symmetry. There is no real evidence that faloular basal granules are specialized morphologically or that they give rise to somatic basal granules by simple division. Wessenberg's observation that after transverse fission in Opallna a new falx is organized in the poste rior daughter from basal granules of transected somatic oillary rows suggests that individual basal granules are more nearly equlpotent than has been heretofore suggested. The faloular field in Opallna has no real 1 1* 6 equivalent among the flagellate or Clllophora, though it is somewhat reminiscent of tha so-called "dorsal brush" described by Tannreuther (1925) in tho rhabdophorino gymnostome Prorodon. Tho anterior furrow of opalinids resembles the anterior suture of astome dilates in the genus Anoplophrva as figured by MaoKlnnon and Hawes (1961) and by Ptxytorae (1954K Wessenberg found detorslon in ciliary rows to be similar In the ontogenies of Opallna and apostome dilates. In Opallna. but not in apostomes, detorsion was followed by slnlstral or antl-clookwise torsion. Ciliary rows in Zellerlella do not spiral about the body and for this reason may not be oompared precisely with those of Opallna. Based upon the angular relationship of olllary rows in trophlo sellerielles they would spiral olookwlse as in Opallna trophozoites if permitted to continue beyond the margins. In small pre-oystlo and recently exoysted zellerielles ciliary rows are more nearly meridional than in trophozoites. No evidence for slnlstral torsion was found at any stage of the life cycle in Zellerlella. Unlike the situation found in apostomes and other dilates which undergo periodic diminution in size, the ciliary rows in Zellerlella are not shortened by successive trans verse divisions but by the repeated production of ever shorter rows during successive unequal longitudinal fissions. Infraolllature morphogenesis In Zellerlella seems to have littla In oommon with tha similar a rent In elliataa, and to ba quite unlike that In hypermastIgote flagellates. Pltalka (1963) remarked that, "Electron microscopy doea not yet add fuel to arguments concerning opallnld affinities." Thla would a earn to ba largely true, However, tha quaatlon raised by Corllaa (1955) *■ to whether a typloal oillate klnetodesma la present in opallnids has been raeantly answered by electron microsoopists. In addition, comparisons may ba made of tha fine structure of thla element in opallnlda, dilates and hypermaatlgotea. The klnetodesmata In Opallna and Zellerlella differs some what from that in typical elliatea, and bears even less resemblance to the klnetodeamal homologs described thus far from hypermastigotes. In Opallna and Zellerlella each ciliary baaal granule transmits two fibrils anteriorly which join and (nearly ?) reach the next anterior basal granule. The overlapping fibrils linking several basal granules together, characteristic of clliates, are lacking. Sleigh (1962) believes, basing his conclusions on electron miorographs of Pitelka and Nolrot-Timothee, that the fibrils In opallnids pass forward on the left side of the ciliary rows rather than on the right as In dilates (the so-called rule of desmodexy of Chatton and Imoff, 1935)* l l j . 8 Fibrils associated with basal granules in hyper- mast igote flagellates hare been desorlbed for Trlohonwnha and Hblonastlgotoldes by Gibbons and Grlmatene (i960) and for Lpphonas by Beans, King,Tahnlslan and Devine (i960). In Trlohonvmpha and Lophonas basal granules within a row are Joined to neighboring basal granules in the sane and adjacent rows by one or several extremely fine filaments. In Holonastlgotoldes a thick fibrous band runs parallel each ciliary row, on the right side, connecting all basal granules in a row together, Corliss has suggested that the position of the opallnids night be clarified if It were known whether the mechanics of ciliary notion differed fundanentally between dilates and flagellates, pointing out that locomotion in opallnids appeared very much like that in typical dilates. Sleigh (1962) in a recent monograph on the biology of cilia and flagella states that in the hypermastigote Trlchonynroha the flagella are more or less fixed in their orientation and can produoe only forward movement, changes in direction being effected by the "steering” action of the front end of the body while in Opallna (and dilates) change in direction is brought about by ciliary reversal, that is, change in the direotlon of the ciliary beat. In terms of the movement of an Individual oilium, Sleigh found greater similarities between opallnids and flagel lates than between the former and dilates. The wave-llke Iif9 metaehronal beat In Opallna was considered by Sleigh to be more highly evolved than In Trlchonvmpha. though primi tive in terms of metaehronal patterns found In true dilates. I have not studied ciliary aotlon in Zellerlella directly. Superficially there do not seem to be any fundamental dlfferenoes In this oharaeter between Zeller lella and Opallna* Sleigh’s observations do little toward olarlfylng relationships between opallnids, flagellates and eiliates beyond suggesting that the meehanlcs of elliary action In opallnids Is more specialised than In highly evolved representatives of the flagellates, less specialised than in primitive members of the Cillophora. Those characters considered by the majority of contemporary workers as linking opalinids with flagellates Include particularly; gametic reproduction, monomorphie nuolei, and symmetrigenio fission. Biocca (1956) and Chelasin and Poljansky (1963) regard the first two characters as of such fundamental phylogenetio significance for the Protosoa that they group together In a single subphylum (Homokaryota) all repre sentatives that have monomorphie nuclei and which reproduce by syngamy. Included within this taxon are the Mastigo- phora, Saroodina and Sporosoa of Calkins (1933). The Cillophora are Included by Blooca and Cheissin and Poljansky in a second subphylum, Heterokaryota. 150 Faure-Freniet (1950) and Corlisa (1956) hold that equal weight should he accorded homo the tl genic fission as a oharaeter to be used In separating dilates from other Protozoa* The occurrence of syngamy maong true dilates seems doubtful* Ihe report of fusion of isogametes during the life oyole of the oillate, Glaucoma frontata. by Calkins and Bowling (1929) has never been oonflrmed* Wessenberg*a observation that syngamy occurs in suetorIans and peri- trichs Is not in accord with the views of specialists on these two groups, including Davis (19l|7), Finley (1952) and Grell (1953) who hold that apparent gametes are micro- and macroconjugants* Elaboration of the question of the phylogenetic significance of syngamy in Protozoa is not within the limits set for this paper. It is appropriate to point out, however, that in assessing relationships between higher and higher categories, it is usually necessary progressively to replace objective evidence with subjective opinion* The possibility that closer relationships may exist between certain members of the oonsiunity of Protozoa reproducing by syngamy and others that reproduce by conjugation cannot be ruled out* To judge, for example, that the occurrence of conjugation (regarded by Sonneborn, 1957, as modified syngamy) is a more valid measure of evolutionary diversity than the absenoe of 151 centrloles la largaly subjective. dilates reproduce by conjugation; opallnlda and flagollatea by syngamy; clllataa and opallnlda laok eentrlolea while In flagollatea tha eentriole plays a major role In tha mitotle sequence. Tha deelaion aa to tha weight to be aecorded these charaetera in aaalgnlng affinities la arbitrary. Monomorphie nuclei, known to ba praaant in at laaat one true oillate, Stophanoppgon. appear to be other- wiae restricted to those Protozoa included by Blooca in the aubphylum Homokaryota. Cheissin and Poljansky (1963) offered in support of their view that flagellates and sporozoans share a common heritage the observation that in both, diploidy is restricted to the zygote, all other developmental forms being haploid. The chromosome comple ment of moat Protozoa is unknown. Available evidence indicates that, in the species studied, flagellate tropho zoites are typically haploid (Kudo, 195^) while in trophlo dilates the mioronuoleua is diploid (Sonneborn, 1957) as waa also the nucleus in the several opallnids studied by Chen. How widespread these phenomena are among the various protozoan groups and whether the chromosome comple ment adheres to phylogenetio boundaries remains to be discovered. Cheissin and Poljansky appear to regard haploldy, in trophic sporozoans and flagellates at least, as tazonoulcally significant. My studies add little to what has already been 152 written on nnolear and reproductive homologies between opallnlda and othar protosoan gronpa. However, flaalon In opallnlda baa baan hare ahown to ba of a unlqtte type with out equivalence among either tha clllataa of flagellates. Attempts to assign flagellate affinities to opallnlda on tha baala of similarities In flaalon Mode and infraellia- tura homology are oontrary to tha evidence. In eartaln othar particulars (ciliary kinetics, flna atructure of tha pellicle, intraoytoplasmio fibrils and vesloles) opallnids also diffar from representatives of other groups in which these characters have been studied, Opallnlda resemble dilates superficially in the disposition of cilia and basal granules and in having an anterior marginal furrow in the pellicle. The most distinctive dilate feature of opallnids, however, is their lack of centrloles, Monomorphio nuclei are common to both opallnids and flagellates. Differences may be noted between tha two groups, however, in mitotic behavior (without kinetlo oenters in opallnids, with kinetic oenters in flagellates) and possibly in the chromosome complement of trophic forma (diploid in opallnids, haploid in flagellates), Wessenberg*s opinion that ancestral opallnids formed a transitional group between flagellates and dilates seems unjustified. The high degree of 153 specialisation and differentiation seen in opalinida suggests, rather, that they have undergone a long evolu tionary history apart from the main-line of development of other protoaoan groups* Clear-out evidenoe for affinities is laoklng. a I find it diffieult to aeoept the inolusion of opalinids among either the sareomastigophoran or eillo- phoran lineages. Removal of the opalinida from their ourrent plaoe among the Sareomastlgophora would enhance the character of the latter group as a natural assemblage. The same may be said for their separation from the Cilio- phora. For the present, it seems more reasonable to oonsider the opalinids as a group lnoertae sedls. separate from but provisionally equivalent to, the subphyla Sareomastlgophora and Cillophora* Relationships within the Opalinldae Metcalf's classification for the family Opalinldae, first published in 1918* revised in 1923 and reaffirmed in I9I 4 .O is as follows: Opalinldae Protoopalinae (blnuoleate opalinids) Protoopalina (eylindrioal in cross section) Zellerlella (flattened in cross section) Opallnlnae Cepedea (cylindrical In cross scetion) Opallna (flattened In cross seotlon) Opallnae angustae (oblanoeolate in outline) Opallna latae (rounded in outline) Carini (1938b), following Metcalf's arrangement of the opalines, divided the sellerielles into Zellerlellae angustae (narrow forms) and Zellerlellae latae (broad forms). Based on the lack of a discemable boundary between Opallna and Cepedea Mohr (1959) plaoed the latter in synonomy with Opallna. Metcalf considered Protoopallna the most arehalo member of the family on the basis of similarity in form to less modified flagellates. The Opalininae were believed to have arisen from Protoopallna through supresslon of fission of cytoplasm in the presence of continued nuolear division, Cepedea being the more primitive based on its cylindrical form. Zellerlella and Opallna were derived from Protoopallna and Cepedea respectively by flattening of the body. Within the opaline lineage, broad forms were considered anoestral to narrow Opallnae angustae. Metoalf supported his phylogenetic scheme with evidenoe adduced from the life cycle. He contended that all genera began development in the tadpole in the condi tion of the more primitive protoopalines and passed through successively higher stages until the definitive 155 form was attained. This view has been aooepted by Konsuloff (1922), Overbeek da Meyer (I929) and Wasaanbarg (1961). That variation in ontoganatio pattern ooours Is Indicated by tha studies of Hara (I938) who observed reversal In the broad to narrow sequence In Opallna ranarun and Mohr (1933) who noted that larval stages of a truncate Opallna in Rana boylll were flattened rather than cylin drical. I found no evidence for a protoopaline larval stage In tha ontogeny of Zellerlella hirsute. Snail uninucleate pre- and post-cystic sellerlallas resemble, In broad outline, some of the more robust protoopallna s. In oross section, however, they are flattened. By the time blnuolearlty is aohieved, the typical zellerlella body character has bean established. Metcalf's ideas on rela tionships between major opallnld groups may be oorreot but he goes beyond his evidence. Mohr ( ) has written that due to the generally poor oondition of most of Metcalf's material the validity of certain characteristics regarded as pivotal In the evolution of the family is questionable. A phylogeny based on the morphology of presumed transitional series seems still bast regarded skeptically. Many authors have remarked the correspondence between geographic distribution and the presumed place of related organisms on the evolutionary scale. On the strength of What wo know at present, the best evidence for relationships within the Opalinidae cones from their distribution* All opalinid genera are cosmopolitan, with the possible exoeptlon of Opallna whloh has not been reported froai Australia* The distribution of Protoopallna Is discontinuous; it is nowhere dominant but is perhaps the most common opalinid genus found in primitive frogs (Lelopelmatldae, Disooglossldae, Flpidae) and intermediate or advanced frogs of highly specialised habits, (Rhino- derma. Scaphlopus. Oastrophrvne). Zellerlella is poorly represented in the Old World and North America but is encountered frequently in advanced frogs (Frocoela) of tropioal America while Opallna appears dominant among higher frogs (Proooela, Diplasloooela) in the Old World tropics, Eurasia and North America* The frequenoy with which Zellerlella has been reported and the proportionally few records of Opallna from South America suggest possible isolation of Zeller lella there lint 11 more recent times* There is, moreover, in the known distribution of sellerlelles and opalines in the New World, some indication of faunal interchange between North and South Amerioa* Opallna and Zellerlella are known from 39 host speolea in the United States* All have been reported as hosts for Opallna. 8 as hosts for Zellerlella. No anuran in the United States is known to harbor Zellerlella exclusively* In Mexico, Central and 157 South America Zellerlella alone has boon reported from $0 host species, Opallna alona fro* 17. Twelve tropical American host a have baan found to harbor both genera. Tha known gaographie distribution of Opallna and Zallarlalla In tha Haw World la given in Tabla 5* Tha comparatively large nunbar of hosts known to shara both ganara In tha southwestern United States, Mexico and Central Amarloa Is reminiscent of ecotonal patterns observed In overlapping ranges of free-living faunal assemblages (Odum, 1959)* The discontinuous distribution of Protoopallna and 2ellerlella and the apparent dominance of Opallna suggests that older opalinid groups have possibly been replaced or displaced by more recant descendants. Protoopallna and Zallarlalla may have been dominant at different times, the latter replacing the former over much of Its range to be In turn replaced In tha Old World, and later North America, by Opallna. These ourrently dlsoernable trends may, with more thorough sampling, be proven mistaken. Until valid transi tional forms within and between opalinid genera are identified, the likelihood of parallel evolution within the family can only be a matter of conjecture. At the present time, Metcalf's contention that Zallarlalla Is monophyletie may ba neither proven nor disproven. Tha corollary that Zellerlella evolved In Fategonla appears 158 « TA BLE 5 DISTRIBUTION O F OP A LINA AND ZELLERIELLA IN NEW WORLD ANURANS Q palina Z e lle r ie lla Both T otal H osts only only G enera R eported R eported R eported R eported U nited S ta tes 39 30 0 9 M ex ico , C entral 44 8 25 11 A m erica South A m erica 35 9 25 1 159 to have boon constructed primarily to support his zoogeo- graphioal hypotheses and Is Inoonslstont vith tho eolloetion record. Mohr'a (I938) argument that, basod on Its sporadic oeeurronoo in tho Old World, Zollorlolls is probably polyphylotio, dorlvod from cosmopolitan proto- opallnos at various times and plaeos may bo oorroot but, on morphologloal grounds, oannot bo substantiated. I find it more difficult to believe that tho selleriolles of tho Nov World, South Afrloa, tho Sudan, Bombay, Australia and Nanking are tho products of several independent evolutions than that tho genus was onoo widely distributed and now persists only in reliot groups ezoept in one (troploal America) and possibly two (Australia) barrier-limited regions. Taxonomy of Zellerlella Inapplicability of Non-Morphological Characters Breeding Incompatibility has been used extensively as a measure of divergence between closely related, sexually reproducing organisms. Even assuming sexual reproduction by all opalinid groups, the real and theo retical difficulties Involved in testing for intraspeolfle fertility or cross-specific sterility are so great as to make such studies impracticable, if not Impossible. Experimental transf&unation of symbionts to unusual l6o hosts has bssn used with sons success for the separation of biologically distinct but Morphologically similar spades* InfeetIon experiments with opallnids to date indloate that physiological bonds restricting a speoles to a particular host are either absent or of a wery low order. Morphologioal Species Characters in Opalinids Metcalf (1923) set forth basic morphological criteria to be used for the delineation of opalinid species. These criteria were reaffirmed with soant modification in 19^0. Virtually all characters were established after study of opallnids obtained from preserved museum specimens of anurans. Nineteen of the 24 species and subspecies of Zelleriella named by Metcalf came from such hosts* Metcalf’s speoles have been recognised and his criteria aocepted by the majority of opalinid taxonomists to the present* Inoluded in this number are Fantham (1923-1931), Fantham and Robertson (1927, 1928), Tuxet and Zuber-Vogell (1954) «&d Bolsson (1959a, 1959b, 1963) in Africa; Bhatia and Oulati (1927)# Mello (1931, 1932), TTttangi (I9I 4 . 8-I961) and Khan (1962) in India; Nle (1932, 1935), Lu (1945), Liu (1957) and Bolsson (1957) in other parts of Asia; Carini (1933-1943)# Beltran (194l) Otamendi (1945) in Latin America* Others have suggested new taxonomic characters or criticized those considered dependable by Metcalf and his follower*• Here (1938) considered the engle between cillery rows end the long body ezis of diegnoatle value for •peeies of Opel Inc. Mohr (19M>) proposed that the asym metry of eillery rows on either surfaoe could be used In making coeiperlsons within the family end Mohr (19lfla) demonstrated thet pellleuler furrowing, regerded by Meteelf end others es e speoifle trelt, oould be experi mentally Induced by placing opallnids in hypertonic media. Chen (1932a, 1932b) showed thet, contrary to Metcalf, opalinid nuclei do not rest In Intermediate mitotic states end Chen (19M*) used the number of nuoleoli, nuolsolar- ohromosome character, end degree of eetoplesmlc alveolatlon In separating Zellerlella species from Bufo Talllceps. Otamendl (19^5) computed length to width ratios for body and nuclei In several Argentinian zellerielles but failed to Indloate where on the body the measurements were taken. Modern taxonomy emphasises study of intraspeclfle or population variation as the proper approach to under standing evolutionary relationships among speoles. Manwell (1956), writing on intrasppolflo variation In parasitic protozoans, points out that earlier taxonomists were aware of such variation, but held more rigidly to the concept of living things as divisible Into fixed species than do many taxonomists today. Failure to identify normal speoles variants as members of a oomon genetic community, added to the belief In a relatively strict host-parasite specificity, appears to have resulted in tha assignment of spaaifle status to many opalinids merely on tha basis of their occurrence in a nav host species. Dlffieulty in reconciling an opalinid from a naw host vith dasariptions alraady published has doubtless aided in tha perpetuation of tha praetioa and resulted in the prolifera tion of numbers of questionable speoles. An example of arbitrary assignment of specific status to selleriellas from different hosts in the absence of any real morpholog ical distinction is found in Metcalf»s (1923) treatment of selleriellas in the United States, to be considered in the concluding section of this chapter. Hot all opalinid taxonomists hare held to the ▼ievs on host specificity put forth by Metcalf. Workers in South Amerloa appear to have frequently gone to the other extreme and named many species from the same host. Most remarkable of these is Leptodaotylua ooellatua from whioh no fever than 10 species of Zellerlella hare been described. Observations by the authors of these several species are informative. For example, Carini (1938) distinguished the trumpet shaped Z. cornucopia from the similarly formed Z. oorniola solely on sise differences, vhlle the vedge shaped Z. truncate was found alvays in combination vith "another species" oval in shape. Otamendi (I9I 4 . 5), obviously unavare of Chen's vork, found the most distinctive trait of Z. cuneata from Leptodaotylua 163 ooollatua to bo nuelol vith four "naeroohronosones" vhleh "rested In anaphase*" Metcalf's (194°) observation on living sollerlollea from L* ooollatua and throo othar South Anar la an anurana la particularly revealing* Ho vrote, "In thoao four hoata • • • thoro aro Zelleriell as that it la dlffioult to distinguish, for aftor study of many infactIona one roalisoa that they aaan to grade into one another*" Morphologioal char ae tor a in Z* hirauta nay ba eategorlzed aa: (1) phyaiologically variable; (2) onto- gonatieally variable; and (3) ontogenetically oonatant* Theea have baen oonaidarod in appropriate aeetlona of this paper and are summarised be loo: Phyalologically Variable Charactera (a) General Appearance of tho Cvtoplaan: Shape and alee of vacuoles, "endospherules," and other incluaion bodlea appear to depend on nutritional or other physiological states obtaining at the tine of fixation* (b) Thickness: Aa la the case vith most, if not all opalinids, Z. hirauta lacks an excretory system. Swelling and shrinkage nay occur over a vide range in response to tho environmental osmotic pres sure. Cytologleal fixation preserves theso variant characters. (o) lumber of Muolcolls Variation In apparent naoloolar number nay con# about by fusion of adjacent hypertrophic nucleoli car, possibly, by regression In slse of a nucleolus to the point of near disappear ance • (d) Huolcolua-Chromoaomc Association: Evidence froai other studies suggests that In general, the nature of a particular nucleolus- chronosone association depends on the physiological state of the oell. Whether this is true or not for Zellerlella. the technloal difficulties attendant upon demonstration of this oharaeter are such as to render it of United value for the vorking taxonomist. Ontogenetically Variable Characters (a) Fora and Slse of Body Initially variable due to the oourse of the fission plane, these characters are subse quently variable depending on fission fre quency, this latter apparently related both to intrinsic and extrinsic (host-induced) factors. (b) Position of the Kuclel The distance between quo1*1 varies apart fro* A size, balng apparently dependent on tba fission interval* Tba distance from the right nuoleus to tba adjacent margin, measured along the line of nuclear mid-points inoraasea irregularly with site* (c) Nuclear Site Nuclear diameter varies with body else but not proportionally* Small zellerlelles bays proportionally larger nuclei than large individuals. (d) Ciliary Row Number General correspondence exists between size and number of olliary rows* Ontogenetically Constant Characters (a) Ciliary Length Length of ollia in all zellerlelles studied was about 10 to 11 microns regardless of the size of the individual or the location of the cilia on the body* Ciliary density is always greatest along the falx, increasingly sparse posterlad* (b) Relative Proportions of Ectoplasm and Endoplaam Ectoplasm!c width varies little (one to one and a half miorons) between larga and small sellerielles. Consistent diffaraneas in this character vara not observed among sallarlallaa from numerous boat species, apart from the three Arisonan Bufos. (e) Angular Relationship between Falx and Ciliary Rows In Z. hlrauta the angle at whleh the recti linear ciliary rows intersect the falx remains constant during ontogeny. Near the apex this angle is approximately 90 degrees* It increases gradually toward the left end of the falx to about I30 degrees in large tropho zoites. (d) Course of Reotlllnear Ciliary Rows and Right Marginal Profile Proceeding posterlad, rectilinear ciliary rows *n hirsute curve slightly. The profile ohar&oteristio is determined by the oourse taken by these rows since at fission the division the fission plane passes between and parallel to them. (e) Relationship of Axis of Nuclear Centers to the Falx The parallel relationship between the axis of nuclear mid-points and the anterior margin 167 observed in Z. hirsute appears to ba wide spread among member a of tha genua. The aaae relationship oocurred In zellerlelles from 17 different hoat species atudiad by me. Thla eharaoteriatlo la illustrated In moat of tha drawings of sallarlallaa in tha literature* (f) Chromosome Humber Chan found 12 ehromoaome pairs in tha several speoiaa of zellerlelle found in B. valllcepa. A similar number appaara present in Z. hirsute. Identification of individual chromoaoaiea for purposes of counting requires careful attention to staining procedures, a high quality optical system Including apoohromatlo objectives and achromatic condenser, and favorable material in which division stages with large nuclei are well represented. Conclusions Hunter (1955) remarked that, for Opaline, the usual characters such as size, shape of body, and the appearance of the cortical region of ectoplasm will not adequately separata the opalines into their respective apeoiflo groups. Zellerlelle similarly displays few characters of obvious use to the taxonomist. Those here regarded as particularly promising Include the ratio of 168 breadth of body to nuolear diameter and details of tha infraciliature, aspsolally the number and disposition of tha olllary rows* Broad outline and size are charaotars to be used with oaution* Intraspeoifio variation may oecur to same degree* Ontogenetlo variation may be marked. Ontogenetic variation in nuolear diameter and olllary row number la not strlotly proportional to body size* Identi fication of normal variability ranges is possible, but only if infections from numerous hosts of various ages and physiological states are oompared* Some characters, notably ciliary length and the parallel relationship between nuclei and anterior margin have been found oonstant in zellerlelles from many host species* Further study may show these to be generic traits* Historical concepts that speoies of Zellerleila. or probably any opalinid, may be adequately described by reference to the features of one or a few "modal individ uals" should be discarded* Zellerlella in the United States Zellerlelles have been reported from, or are known to ooour in, 9 speoies of anurans in the United States. Four species, one subspecies and 2 probable speoies have been described by Metcalf (1923), all from different hosts. An additional 4 speoies were named by Chen (19^-B) 1 6 9 froa a single host* Two now Borth American hoot reoorda are reported for the first tine in this paper. Naned speoies and subspeoies, undesignated foras, hoots and authors are sunaarised below: Zelleriella oouehil Metoalf 1923, froa Soaphiopus oouohii. Z. soaphiopodos Metoalf 1923, froa Soaphiopus holbrooki. Z. hirauta Metoalf 1923, froa Bufo cognatua. Z. ranaxena Metoalf 1923, froa Rana aurora. Z. intermedia ouneata Metoalf I923, froa Bufo ▼allloops. J5* elllptlea Chen 19lf8, from B. Tallloepa. Z. loulslanensls Chen 19^8, from Bufo Tallloepa. Z. ▼alllceps Chen 19^-8, from Bufo vallleeps. Z. pfltsnerl Chen 19^-8, froa Bufo Tallloepa. Z. (of Bufo woodhousii) Metoalf 1923, from Bufo woodhousll. Z. (of Bufo punctatua) Metoalf 1923, from Bufo punotatua. hirsute Metoalf 1923, from Bufo alTarlus (this paper, page 9). Z. hirauta Metoalf 1923, from Bufo woodhousii (this paper, page 9). Z. speoies froa Rana ausoosa (this paper, page 170). 170 Chen's taxonoalo treatment of the sellerielles froa Bufo Tallloepa has been eonaldered elsewhere In thla paper* Metoalf'a work on Bfclted Statea selleriellea la here preaented ohiefly to point up the Inadequaolea in taxonoalo methodology for the Opallnldae* The numbers of host individuals froa whleh apeolea were deaerlbed by Metoalf (I923) are the following: Z. oouohii* two aoant infections $ Z* aoaphiopodos* one infeotion; Z* interaedla cuneata. two Infeotiona; Z. ranaxena. one infeotion; Z. hirauta* two infeotiona. With the exoeption of Z* ranaxena all apeoies were deaoribed froa TT. S. National Muaeua apeolaena* Regarding the host of Z. ranaxena Metoalf wrote* "Of numerous hosts of this apeolea (Rana aurora) purchased in San Franoiseo and said to hare been eolleoted in the vioinlty* one contained Z. ranaxena and two others had Opallna draytonll*" Subsequent exaaination of 83 specimens of Rana aurora by Metoalf (1923)» Mohr (unpublished)* Lehman (i960) and me* has failed to confirm the presence of Zelleriella in this host. In Tlew of the questionable origins of the frog identified by Metoalf as R* aurora and in the absence of substantiat ing reports of Zelleriella in this host* It seems probable that it was either incorrectly identified or carried an accidental infection* Zelleriella is known to occur in natural infeotiona in Rana anacoaa. whose range overlaps that of R* aurora in California. 171 tfata used by Metoalf la distinguishing speoies and subspecies of zellerlelles froa Unitad Stataa anurans ara given In Tabla 6* Maaaurananta vara raportad for alngla "average" individuals only* Metcalf's llluatratlons of Z. hirauta ara ahovn In Plata XIV, Figure* 3I 4 . to 39* Matoalf further distinguished thaaa zallariallaa In tha following manner: Z. couohli— small fora with amall nuclei, may ba wedge-shaped or rounded; Z. aoaphlopodoa— raaaablaa Z. eouohll but ha a relatively aueh larger nuclei; Z. ranaxena— a large thin fora, poatarior and alwaya rounded; Z* hirauta— vary danaa 0illation, usually narrow at anterior end, nuclei wall back in body; Z. intermedia ouneata— nuolel of intermediate aixe, not connected by a thread* It is obvious that moat of Metoalf' a measurements have little meaning. The proportional difference in size between nuolel of Z* oouohii and Z* scaohlooodos would possibly be significant if the zelleriellea had been protosoologloally fixed and if the differences were constant for individuals of various sizes* Opallnid nuolel, improperly fixed, may become pyonotio or shrunken* Cilia on op&linida from preserved hosts are ordinarily matted* In this condition neither density nor length may be satisfactorily determined* I have found the cilia of Z. hirauta no aore dense than those of other zellerlelles* In view of the normal range of variation, there is little 172 TABLE 6 CHARACTERS USED BY M ETC A LF (1923) FOR THE SEPARATION O F SPECIES AND SUBSPECIES OF ZELLERIELLA FROM FIVE UNITED STATES ANURANS z. z. z. z. z. cou ch ii sca p h io - ran a- h irsu ta in te r - podos xena m ed ia Length (p) 160 155 167 U 3 160 W idth fyi) 87 90 91 60 74 T h ick n ess (p ) 16 13 - - 22 N u clear D ia m eter (p) 14 21 17 11 14 C ilia r y Line in terv a l (;a) A n terior 1. 6 2 .6 3 .0 - - 1 .7 P o ste r io r 3. 3 3. 8 4. 5 - - 2 .3 M ach roch rosom e num ber 6 .8 - - - - — 4 Length and Width of E n d os- p h eru les (ji) 2 . 5 x 2 -- -- — 2.2 x 1.3 PLATE XIV Figures 3^ to 38* Trophozoites of Zelleriella hirsute from Metoalf, 1923. Smaller spheres in Figures 36, 37 and 38 are hyperparasitic entamebas, mistakenly identified by Metoalf as "abnormal nuclei*" Figures to 37 are X lf6o, Figure 38 X 1,000* Figure 39. Cysts of Zelleriella hirauta from Metoalf, 1923, X 1,000* 17k PLATE XIV in Metoalf»s writtan descriptions or illustrations to distinguish thess species from Zelleriella hirsute* as redeseribed in this paper* APPENDIXES REGIONAL CHECK LIST OF ZELLER IELLES AND HOSTS A LIST OF PAPERS CONTAINING ORIGINAL OBSERVA TIONS ON OPALINIDS SINCE I93O AND NOT SUMMARIZED BY METCALF (I9I 4 .O) APPENDIX I REGIONAL CHECK LIST OF ZELLER IELLSS AND HOSTS1 *Indioatea described from preserved material. I* NEOTROPICAL ZBLLERIBLLES2 ♦Zallarlalla antllllanala Metoalf i Jamaloa 8b Bermuda, Matoalf ( 1923) Coat. Rloa, Ruls & Alfaro (1959) Zallarlalla antuneel Peaaoa Brasil (Sao Paulc), Paaaoa (1934) Uruguay (Montevideo) and Brasil (Rio da Janeiro), Metoalf (1940) Zallarlalla antunael quadrata Brasil (Rio da Janeiro), Metoalf (1940) 1Spaelaa names hare not been brought Into conform ity with modarn uaaga in all eases. Generic names ara according to Gorham (1963). ^Includes Mexico, Central and South America. 177 Bufo marlnua Bufo marlnua Bufo marlnua Bufo arenarum Bufo cruolfer Bufo dorblgnyl Leptodactylua ocellatua Bufo crucifer Zelleriella artlgaal Untl Brasil, Unti (1935) ♦Zallarlalla atelopyxona Metoalf Cotta Rica, Matoalf (1923) ♦Zallarlalla atelopyxona atolsnorl Panama, Metoalf (1923) ♦Zallarlalla blnghaml Metoalf Peru, Metoalf (1923) Zelleriella bolpevae Carlnl Braall (Sao Paulo), Carlnl (1933«) Brasil (Sao Paulo), Carlnl < 1910) Brasil (3»o Paulo), Carlnl (19^3) Zelleriella bollrarl Beltran Mexloo (Guerrero), Beltran (19^1a) ♦Zelleriella braalllenola (Pinto) Brasil (Rio de Janeiro), Metoalf (19l(.0) Brasil (Rio de Janeiro), Metoalf (19I 4 .O) Argentina (La Plata), Otamend1 (19l*5) Argentina (La Plata), Otamendl (1945) ♦Zelleriella bufoxena Metoalf Nicaragua, Metoalf (1923) 178 A Bufo marlnua Atalopua yarlua Atelopua atelsnerl SleutherodaotTlua plngkaml Onhla mere— 11 Lelmadronhls poecllogyrua Lelmadrophlo almadenola Bufo namoreua Crop aodactrlua gaudlckaudll LeptodaotTlua ooellatua Leptodaotylua ooellatua Bufo arenarum Bufo haenatitlcua Cost*. Rica* Ruiz & Alfaro (1959) Zallarlalla oarlull Otanendl Argentina (La Plata)* Otaaandi (19^5) Zallarlalla eornlola Carlnl Brazil (Sao Paulo)* Carlnl (1938a) Zallarlalla cornucopia Carlnl Brazil (Sao Paulo)* Carlnl (1933 e) ♦Zallarlalla ouaoonla Metcalf Paru* Matcalf (I923) Zallarlalla cuneata Otamendl Argentina (Quilmes), Otanendl ( 191^ 5) ♦Zallarlalla darwinll Metcalf Chile, Metcalf (I923) ♦Zelleriella dendrobatldla Metcalf Hlcaragua & Costa Rioa* Metoalf (1923) Zallarlalla dubla Metcalf Brazil* Metcalf falcata Car ini Brazil (Sao Paulo)* Carlnl 0-933*) 179 Bufo haanatltlcua Bufo aranarun Leptodactylua ooellatua Leptodaotylua ooellatua Eleutherodactylua footal Leptodactylua ooellatua Rhlnoderma darwinll Dendrobatea tlnctoriua Dendrobatea typographua Eupemphlx nana Leptodactylua ooellatua Eupemphlx ovale 160 Zallarlalla follacea Carlnl Brazil (Sao Paulo)* Carlnl (1936b) ♦Zallarlalla hylaxena Matoalf Paraguay, Matoalf (I923) ♦Zallarlalla hypopacheoa Matoalf Ouatanala* Matoalf (I923) ♦Zallarlalla Intermedia Matoalf Maxloo (Guanajuato), Metcalf (1923) Zallarlalla Jaegerl Carlnl Brazil, Carlnl (1933d) ♦Zallarlalla laptodaptyll Matoalf Mexico (Tehuantepec), Metoalf (1923) Guatemala, Metcalf (1923) Zallarlalla laptodelrae Beltran Maxloo (Purlflcaolon), Beltran (19Ub) ♦Zallarlalla aagna Metoalf Vanazuela, Metoalf (1923) Brazil (Sao Paulo), Carlnl (1938b) Paraguay, Schouten (I9I 4 .I) Zelleriella menendezl Schouten Paraguay, Schouten (1934) Leptodactylua ooellatua Hyla pulehella Hypopachua yarloloaua Bufo lnteraodlua Llophla Jaagarl Leptodactylua albllabrla Leptodactylua oallgl- noaua Leptodactylua graollla Leptodactylua nlorotla Leptodeira napulata Leptodactylua typhonlua Leptodactylua ooellatua Leptodactylua ooellatua Leptodactylua callgi- noaua 181 ♦Zellarlalla nlorooarya Matoalf Puerto Rico, Matoalf (1923) ♦Zallarlalla oplsthooarya Matoalf Bufo lanur Hlearagua & Costa Rica, Matoalf Bufo conlfarua (1923) ------------ Panama, Matoalf (1923) Maxloo (Guanajuato), Matoalf (1923) Zallarlalla ovonuoleata Matoalf Brasil, Metoalf (I9M)) Zallarlalla ovonucleata bufonls Maxloo (Tehuantepec), Metoalf (19M>) ♦Zallarlalla paludlcola Metoalf Chile (Toloahuano), Metoalf (1923) Vanesuela, Metcalf (I923) ♦Zelleriella Patagoniansla Metoalf Bufo typhonlus Bufo nonxlas Leptodactylua penta- daetylus Bufo aternoatlgnatua Pleurodena blbronll Pleurodena brachyops Argentina (Straits of Magellan) Pleurodena bufonla Metcalf (1923) Zelleriella plelcola Da Cunha & Penldo Brasil (Paraguay River). Plmelodus elarlaa Da Cunha & Penldo (192o) Zallarlalla slphonopsl Carlnl Brasil (Rio de Janeiro), Carlnl Slphonops annulatus ♦Zelleriella telnatobll Metcalf Ecuador & Peru, Metoalf (1923) Telnatoblus jelskll Zelleriella truncate Carlnl Brasil (Sao Paulo), Carlnl (1938b) Zollorlolla Uruguayans!a Matoalf Uruguay (Montevideo) k Brasil (Rio do Janeiro), Matoalf (191^ 0) Zollorlolla Uruguayans! s quadrata Montevideo k Rio da Janeiro, Matoalf (19lf0) Brasil (Rio de Janairo), Matoalf (I9I 1O) ♦Zallarlalla venezuelae Matoalf Vanazuala, Matoalf (1923) ♦Zallarlalla (atalonodoa) Costa Rloa, Matoalf (1923) ♦Zallarlalla (boulangarl) Colombia, Matoalf (1923) ♦Zallarlalla (engyatosiopala) Panama, Matoalf (1923) Maxloo (Tehuantepec), Matoalf (1923) ♦Zallarlalla (trlnltatla) Vanasuala, Metoalf (1923) Brasil, Matoalf (19^0) ♦Zallarlalla (of Bufo naltocephalus) Cuba, Matoalf (1923) 182 Laptodaotylus ooallatus Laptodaotylus typhonlus Bufo arenarun Laptodaotylus ooallatus Bufo eruelfar Bufo dorblgnyl Hyla vanuloaa Atalopus yarlus Prostharapsla boulangarl Eupemphlx stentor Eupemphlx pustuloaus Phyllobatas trlnltatla Elosla latarlstrlgata Bufo peltocephalua 183 ♦Zollerlolla (of Bofo s£inulo«us) Brasil & Peru, Metoalf (1923) Bufo aplnuloeue ♦Zelleriella (of Bleutherodaetylua Miliaria) Brasil, Metoalf (191*0) Eleutherodaetylua m ifera — *— ♦Zelleriella (of Hyla aeptentrlonalla) Bahanaa, Metoalf (1923) Zelleriella ap • Brasil, Metoalf (19l*0) Zelleriella ap. Brasil, Carlnl (19^3) Zelleriella ap. Brasil, Carlnl (19^.0) Zelleriella ap. Cuba, Wenrioh (1935) Zelleriella ap. Mexloo (Coyoacan), Mohr (19l(.ld) Zelleriella ap. Mexloo (Masatlan), Mohr (unpublished) Zelleriella ap. Mexico (Coyoacan), Mohr (unpub 1 ladied) Zelleriella ap. Hyla aeptentrlonalla Eloala laterlatrlgata Chlorosona aeatirua Ceratophrya aiaerlcana Tropldophla nelanura Hyla exlmla Dlaglena apatulata Bufo compactllla Chile (Valpariao), Mohr (unpublished) Bufo apinuloaua 1 8 1 * . Zelleriella ap. Chll* (Valparlao), Mohr (unpub11shod) Zelleriella ap* Chlla (Valpariao), Mohr (unpubllahad) Zallarlalla ap* Maxloo (Maaatlan), Brookaa (unpubllahad) Zallarlalla ap* Coata Rloa (Tras Rloa), Brookaa (unpubllahad) Zallarlalla ap* Coata Rloa (Liberia), Brookaa (unpubllahad) Zallarlalla ap* Coata Rieaf Brookaa (unpubllahad) Zallarlalla ap* Coata Rica (Las Crnaa), Brooke a (unpub11ahad) Zallarlalla ap. Costa Rloa (San Joae), Brookaa (unpubllahad) Zallarlalla ap* Coata Rloa (La Lola). Brookaa (unpubllahad) Zallarlalla ap* Coata Rloa (Barranoa), Brooke a (unpubl1shed) Pleurodena blbronl Calyptooephalua gaTl Bufo naaatlananala Rana warachawltaohll Rana plplona .Rana palnlpaa Bufo leutkanll Bufo oooolfar Leptodactylua penta- daotylua La Ta ptodaotylua oris macull- 185 Zallarlalla ap. Coata Rloa (Tres Rloa), Brookaa (unpubllahad) Zallarlalla ap. Coata Rloa (La Lola), Brookaa (unpubllahad) Zallarlalla ap. Coata Rloa (La Lola), Brookes (unpubllahad) Elautharodaotrlua flelsehmanni Hyla alaaoohroa Dendrobatea auratua II. NORTH AMERICAN ZELLERIELLE3 ♦Zallarlalla oouchll Matoalf Halotaa, Texas, Matoalf (1923) Zallarlalla elllptlea Chan New Orleans, La. Chan, (I9I 4 . 8) ♦Zallarlalla hirauta Matoalf Port Mojave & Phoenix, Arizona Matoalf (I923) Yuma, Arizona Brookes (unpub11 shad) Zallarlalla loulalanensla Chan New Orleans, La. Chan (19^8) Zallarlalla pfltznerl Chan Soaphiopus oouchll Bufo vallloeps Bufo eognatua Bufo oognatua Bufo woodhouall Bufo alvarlus Bufo vallloeps New Orleans, La. Chen (I9I 4 . 8) Bufo vallloaps 1 8 6 Zollorlolla ranaxena Metoalf San Pranolseo. Calif., Matoalf (1923) ♦Zallarlalla aoaphlopodoa Matoalf Raleigh, Mo* Carolina, Matoalf (1923) Zallarlalla Tallloepa Chan Haw Orleans, La., Chan (1948) Zallarlalla (of Bufo punctatua) Death Valley, Calif., Matoalf (1923) Zallarlalla (of Bufo woodhouaei) Provo, Utah, Matoalf (1923) Zallarlalla ap* Florida, Waarioh (1935) Zallarlalla ap* High Sierra, Calif*, Brookaa (unpubllahad) Rana aurora Soaphlopua holbrookl Bufo vallloapa Bufo punctatua Bufo woodhouaei Hyla clnerea Rana muaooaa III. ASIAN ZELLERIELLES Zallarlalla frollanol Uttangl Dharwar, India, Uttangl (1951) Zallarlalla indlea Uttangl Dharwar, India, Uttangl (i960) Phllautua ap. Mlorohyla ornata Zallarlalla aacronucleata (Bezzenberger) "Asia" Bezzenberger (190^) Bufo nolanoatlctus 187 Zallarlalla alcroirrlae Uttangl Dharwar, India, Mlorohyla ornata Uttangl (1951) Zallarlalla orientalla Via Banking, China, Via (1935) Mlorohyla ornata Taiwan, Liu (1957) Mlorohyla ornata IV. AFRICAN ZELLERIKLLES Zallarlalla (Afrloana A) South Africa, Sandon (1938) Rana dalalandl r. Zallarlalla (Afrloana B) South Africa, Sandon (193®) Rana fuaclgula Zallarlalla ap. Khartoum, Sudan, Sandon (19^9) Cltharlnua latua V. AUSTRALIAN ZELLERIELLES Zallarlalla blnuclaata (Raff) Auatralia, Raff (1911) Llanodynaatea doraalla Auatralla, Raff (1912) Llanodynaataa taaaanl- anala NOTE: Zallariallaa occur In Hawaii, Fiji and Tinian (Marlanaa) in tha imported toad Bufo aarinua. (Collection raeorda of Brookaa and Mohr, unpubllahad). APPENDIX II A LIST OP PAPERS CONTAINING ORIGINAL OBSERVATIONS ON OPALDTIDS SINCE 1930 AND NOT SUMMARIZED BY METCALP (I9I 4 .O) Amaro, A. 1962. Observaooes am zellrelellas hiperpara- sitadas por antamebas (Protozoa, Mastlgophora). Atlas. Soo. Biol* Rio da Janeiro 6:21-25. Bal, C. and P. V. RanJIni. 1955* 0n tha occurrence of Opallna soalprif orals in tha rectum of Rana tlgerlna. i»roc. Indian Sol. Congress lj.2: 281. Beltran, E. 19^0* Maynard M. Matoalf, su obra oiantifica y el conoolmlento da los protoeiliados. Rev. Soc. Mexieana Hist. Nat. 1:265-278. . 19lfl». Opalinidos parasltoa on anfibios mexi- oanos. Rot. Soo. Maxioana Hist. Nat. 2:127-136. 19i|.lb. Zallarlalla leptodierae sp. n. parasite da Esptodelra maculata. Rev. 5oc. Mexicans Hist. Nat. 2:267-272. Bhatia, B. L. 1936* Protozoa: Ciliophora, Jn Sawall, Tha Fauna of British India, including Ceylon and Burma, Taylor and Francis, London. 1^93 P> Blaniarz, J. 1950. Influence of vertebrate gonadotropic hormones upon tha reproductive oyele of certain protozoa in frogs. Nature 165:650-651* Biocca, E. 1956* Alcune eonslderazlonl sulla slstematioa del protocol a sulla utiliata dl ereare una nuova class© dl protocol. Rev. Brasil. Malarlol. 8:91-102. 188 189 Boisson, C. 1957* Opalines et ollles pereeltea de quel- quea batraoiens de la region de Saigon. Ann. Sol. Vat. Biol. Aniatale 19:£73-585. 1959** Cil^ea et opalines hotea du rectum de Rana occipitalis Gunther. Bull. Inat. Franeaia Afrique Noire 21:1-13. . 1959b. Quatre nouvelles opalines d»A.O.F. Bull. Inat. Franeaia Afrique Voire 21:14-20. . 1963* Deux nouvellea opalines de l'ouest Africain. Proc. Internatl. Congr. Zool. 16:123, (abstract). Blanokart, S. 1957. Die Oberflaohenstrukyuren von Paramecium ap und Opal in a ran arum. Z. Vlss. Mikroscop. fc>3: £76-287. Brace, E. C., •til. 1953* The frog, Hyla aurea, as a source of animal parasites. Tuatara 5:l2-Sl. Bradley, V. R. 1937* The incidence of protozoan parasites of frogs of the OkoboJI region. Anat. Rqc., Suppl., 70:128 (abstract). Brandt, B. B. 1936. Parasites of certain North Carolina Sallentla* Ecol. Monographs 6:491-532. Bretschneider, L. H. 1950. Electronenmikroskopisohe Unterauohung einiger Zillaten. Mikroskople 5:2^7-269. Brookes, J. A. and J. L. Mohr. 1963. Bile pigments in opaiinids. J. Protozool. 10:138-140. Brumpt, E. and G. Lavier. 1936. Sur 1?hyperparaaitism d’opalines par des amibes. Ann. Parasitol. 14:349-359* Calkins, G. V. 1933* Parasitic Cillata, p. 397-J4 .OO. In G. V. Calkins, Biology of the Protozoa. Lea and Feblger, Philadelphia, lf.64 P* Carini, A. 194°• Contribui$ao ao estudo dos nlctoteros dos batraqulos do brasll. Arquiv. Biol. 24:11*12. . 1942* Sobre uma Zelleriella do ceoum do SlPhonopa annulatus« Arquiv. biol. 26:76-77* . 1943* Novas observaooes, em batraqulos e ofidios, de Zelleriella hlperparasitadas por entamebas. Biol. 27:64-6'87 1 9 0 Cehovle, 0# 1956. Reoherehes experlmentales sur la correlation hormonal e antra la oycle salaonnler da la grenouille at oelul da sea paraaltas. Compt. Rand. Soo. Biol. 242:2176. Chardas, D. 1955* Protoxoaires andoparaaltaa ba batra- olana. Rev. Vervletolse Hist. Nat. 12:39-44. Chebofarev, R. S. 1957* On tha paraaitoeoanoala of froga in Kiev Province (In Russian); IX Confaranoa on Paraaltlo Problems, 270-271• Cheissin, E. M. and 6. I. Pol Jansky. 1963. On tha taxonomic aystom of Protosoa. Aota Protoaoologloa 1*328-352. Chen, T. T. 1944** Chromosome studies In opallnld clllata infusorians. Year Book Am. Philos. Soe. 123:27. . 1944b. Staining nuolei and chromosomes in proioxoa. Stain Technol. 19*83-90. 1948. Chromosomes in Opallnidae (Protosoa, cillata) with apaoial raferenoe to their behavior, morphology, individuality, dlploidy, haploldv, and association with nucleoli. J. Morphol. 83:261-358. Corliss, J. 0. 1955a. On the systematic position of the enigmatic opallnld infusorians. Anat. Record. 122:434* . 1955b. The opallnld infusorians: flagellates or elliates? J. Protoxool. 2:107-114* . 195ba. Evolution and aystematies of the oillated protosoa I. Systematic. Zool. 5*68-91. 1956b. Evolution and systematica of the oillated Protosoa II. Systematic Zool. 5J121-l40. 196l. Tha Ciliated Protosoa: Characterisation, classification and guide to the literature. Pergamon Press. New York. 310 p. . and W. Balamuth. 1963. Consideration of the opallnlds as a new superclass in the subphylum Saroomastigophora (Abstr.) J. Protoxool. 10 (Suppl.): 26. Cosgrove, W. B. 1947* Fibrillar structures in Opallna obtrlgonoldea Metcalf. J. Parasitol. 33*351-3577™"™" 191 Deride* Z. and L. Geltier. 19^7* Dia ehromosomen der Cillaten. Chromosoma 3:110-136. Dillon* L. g. 1963* A reclassification of tha major groups of organisms basad upon comparative cytology. Systematic Zool. 12:71-62. Divide* Z. 1950. Hromoemi Cilijata: Eucill lata I Opalinl- daa. Rad. Jugoslarenske Akad. Znatost 280:199-217. Dubinins* M. N. 1950. Eoologloal studies on the paraaitlo fauna of Rana rldlbunda (In Russian); Mag. Parasltol. Moscow 121^5-3^57“ Dunn* E. R. 1941* Comment on "Further studies on the opallnld olliate Infusorians and their hosts" by M. M. Metcalf. Am. Mat. 75:^99-503. Dutta, G. P. 195>8. Hlstochemloal studies of Protosoa I. Studies on the lipids In Opaline rananas and Opallna scalprlformls. Research bull. Punjab tonir. lljjT^T-ToS. el Mofty* M. and J. D. Smyth. i960. Endoorlne control of sexual reproduction in Opallna ranarum parasitic In Rana temporarla. Nature* 16b:559* . 1961|-. Endocrine control of encyst- ation in Opaline ranarum parasitic in Rana temporarla. Exp. ParasifolT" 152T55-T99• ---------- ------- ------ Fantham, H. B. 1931* Some paraaitlo Protosoa found in South Africa. South African J. Sci. l l j.5323-333* Faure-Fremiet, E. 1950. Morphologic compares et systema- tique des ollles. Bull. Soc. Zool. France 75:109-122. _______ • 1953* Morphology of Protozoa. Ann. Rev. Microbiol. 751-18. Fernandez-Galiano* D. 19^7. Observaclones citological sobre las opalines. Trab. Inst. Cienc. Nat. Biol. 1:3^9-1 ^ 22. Fukui* T. and Y. Hara. 1936. On Ccpedea pulchra laponica Metcalf* parasitic in Rana rugose (In Japanese); Proc. Japanese Soc. ParaaltolT, 9* 192 Grasse, P. P. 1952* Classe des zooflagellea Zooflagellata on Zoomastlglna. p. 57l|.-578: Supra-or dr • des opallna a (Opallnlna n.n.) p. 983-lOOlj. In P. P. Oraaaa' (editor) Tralta da Zoologla. Vol. 1, Taes. 1. Maaaon at Cie, Parla. Grail, K. a. 1950* Der Kerndualismua dar Cillaten und Suktorien. Dia Naturwlsaenschafter 15:3^7:356. Grewal, M. S. 1960. Morphology of Opallna pall sp. nor. Grawal 1960, fro* tha gut of Varanua grlaeua. Raaaarch Bull. Punjab Univ. 11:167-1727 ----- Hara, Y. 193^* Studies on tha general Morphology and tha neuronotor system of Protoopallna axonucleata lata Matoalf, parasite of ftana nlgromaotuLata (In~"7apaneae). Botan. and Zool: Theoreticalana Applied, Tokyo 2:2015-2022. . 1936. Studies on Cepedea dimidiate orientalla Metcalf, paraaitlo in Rana nlgromaculaia (In Japanese): Botan. and Zool: Theoretical ana Applied, Tokyo. Ip537-5U*. . 1938* 011 the life history of Opallna ranarum and the relationship among the genera of Opal ini 8* Rhythmic bio- eleotrle phenomena in the single-celled organism Opallna ranarum; (In Russian)* Blofizika 3 I 4 .O9. Lavier, G* 1936. Protoopallna duboscqul n* sp* opaline parasite d’un potssonmarln* Ann* Parasitol* 14:272- 277. Lehman, D. L* 1960* Some parasites of central California amphibians* J* Parasitol* lj.6:10. Leweranz, H. J* 1957* Versuche on Opallna ranarum uber das Elndrlnger saurer Farbstoffe* Wiss. £• Univ. Rostock, Math. Nat. 5:345-356. Liu, S* Y* 1957* On the opallnld Infusorians paraaitlo in the Formosan anurans* Quart* J. Taiwan Museum 11:131 -145. Lu, K* 1945* some parasitic dilates from frogs of Pehpei* Sinensia 16:65-72* Lucas, M* S. 1930. Results obtained from applying the Feulgen reaction to Protozoa* Proc* Soo* Exp. Biol* and Med. 27:258-260. Lwoff, A. and S. Valentin!• 194^* Culture du flagelle opalinide Cepedea dlmldlata. Ann. Inst* Pasteur 75:1-7. Manwell, R* 1961* Opalinldae, p 14-3-447. In R. D. Manwell* Introduction to Protozoology* ^t* Martin's Press, New York. Markov, G. S. and M. L* Rogoza. 1955* Annual differences in the paraaitlo fauna of the frog (Rana temporarla): (In Russian). Zool. Z. Moscow 34:1263-1209. 195 Marx, L. 19^3. Opallnld* In the snail Intestine of frogs* Wasnann J* Biol* 21:199“20l|.. MoConnaohie, E. V* i960. Experiments on the enoystatlon Opaline In Rana temporarla. Parasitology 50(171-181. MacKinnon, D* L. and R* S. J, Hawes* 1961* Inoertae sedls: Opalinata* p 11^9-155• la d* **. Rae^innon and P 7 X J* Hawes. Protozoa* Clarendon Press, Oxford* Mello, I* P. de* 1932* Contribution a 1*etude des infusolres parasites des anoures du Malabar* Reo. 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Zool. 29:352-380. 1 T h is d isser ta tio n has been 65-7223 m ic r o film e d ex a ctly as receiv ed BROOKES, John A rthur, 1930- STUDIES ON THE BIOLOGY OF ZELLERIELLA (PROTOZOA, OPALINIDAE). U n iv ersity of Southern C aliforn ia, P h .D ., 1965 B io lo g y — G en etics University Microfilms, Inc., Ann Arbor, Michigan PLEASE NOTE: Pages throughout tend to "curl" Filmed in the best possible way UNIVERSITY MICROFILMS, INC.

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Asset Metadata

Creator Brookes, John Arthur, 1930- (author)

Core Title Studies on the biology of zelleriella (protozoa, Opalinidae)

School Graduate School

Degree Doctor of Philosophy

Degree Program Biology

Degree Conferral Date 1965-01

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, genetics,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Mohr, John Luther (committee chair), Bils, Robert F. (committee member), Chew, Robert M. (committee member), Fertig, Norman R. (committee member), Stager, Kenneth E. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-152398

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Legacy Identifier 6507223.pdf

Dmrecord 152398

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Rights Brookes, John Arthur

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