Morphological and Cytological Observations on Iwo Opalinid Endocommensals of Acanthixalus Spinosus (Amphibia, Anura)

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Morphological and cytological observations on two opalinid endocommensals of Acanthixalus spinosus (Amphibia, Anura)

Article in Canadian Journal of Zoology · February 2011 DOI: 10.1139/z96-171

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The user has requested enhancement of the downloaded file. 1573 Morphological and cytological observations on Iwo opalinid endocommensals of Acanthixalus spinosus (Amphibia, Anura)

Félix-Marie Affa'a, Jean-Pierre Mignot, and Jean-Louis Amiet

Abstract: The morphology and cytology of two new opalinid species were studied using silver impregnation and fixation, which preserves the microfibrils. Both species, commensal on Acanthixalus spinosus, are hast-specifie. Light microscopy showed the existence of a posterior secant system in Opalina proteus n.sp. and its absence in Cepedea couillardi n.sp. (in agreement with the differences presently recognised between the two genera). At the ultrastructural level, however, bath species present a posterior fibrillar zone that seems to be homologous with the secant system. This apparent contradiction may be explained by the fact that the secant system is visible under light microscopy only in O. proteus because its fibrillar zone is more developed than in C. couillardi. The life cycle of C. couillardi spans stages from the tadpole to the adult; in contrast, O. proteus completes its cycle before metamorphosis of the hast.

Résumé: Les auteurs ont étudié la morphologie et l'ultrastructure de deux nouvelles opalines par imprégnation à l'argent en microscopie optique et en microscopie électronique, après fixation par une technique réputée préserver les microfibrilles. Les deux espèces, commensales d'Acanthixalus spinosus, sont spécifiques de leur hôte. La microscopie optique a montré l'existence d'un système sécant postérieur chez Opalina proteus n.sp. et son absence chez Cepedea couillardi n.sp. (ce qui est conforme à la différence jusqu'ici reconnue entre les deux genres). Cependant, en microscopie électronique les deux espèces présentent une zone fibrillaire postérieure qui paraît correspondre à un système sécant. Cette contraction apparente peut être due au fait que le système sécant est visible en microscopie optique seulement chez Opalina parce que cette zone fibrillaire y est plus développée que chez Cepedea. Au cours de son cycle vital, Cepedea couillardi passe des têtards aux adultes alors qu'O. proteus effectue tout son cycle avant la métamorphose de la grenouille. 1

Introduction here. The general characteristics of these two genera, bath multinucleate, are presented in Table 1. Further details cali Acanthixalus spinosus (Buchholz et Peters 1875) is the sole be round in Affa'a (1992) and Affa'a and Lynn (1994). species in a genus whose morphology and anatomy set it apart frOIDother African tree frogs (Perret 1962). It repro- duces exclusively in tree cavities of sufficent size to hold Materials and methods water year-round (Perret 1962). A few other African anuran Frogs were captured in some 20 localities in the South Cameroon species exhibit such reproductive behaviour: Hyperolius Plateau, south of Yaoundé (11-12°E, 3 - 3.5° N), at altitudes from if acutirostris, H. mosaicus, Nectrophryne afra, and N. batesi 650 to 750 m. During the 5 years (1989-1994) when we specifi- (Amiet 1989). These breed in smalIer cavities that lose their cally looked for opalinids from Acanthixalus spinosus, more than a water in the dry season. 100 tree cavities were prospected and frogs were found in only 28. The unique systematic position and ecology of A. spinosus Ten ofthese 28 were visited many times a year to collect pneumonts calI for particular attention to be raid to ifs endocommensal and tadpoles at different stagesof developmentand at metamorphosis. Protozoa. Thus, one of us was able to describe several species ln total, 136 tadpoles and 59 pneumonts were collected, 1 of the of nyctotherans that are endemic to it (Affa'a 1979, 1980, new species being found in 74 (54%) tadpoles and the other species in 18 pneumonts (30.50%) and 14 tadpoles (10.29%). 1986, 1988). The abject of the present parer is to describe ifs The difficulty in finding hasts, the scarcity of one of their endozoic opalinids. Only two species, belonging to the genera opalinids, and the difficulty in redreating in the laboratory the partic- Cepedea and Opalina, were round, and they are described ular conditions oftree cavities colonized by A. spinosus or offinding a medium suitable for cultivating opalinids did not allow us to Received August 8, 1995. Accepted February 23, 1996. follow the developmental life cycle of the species described here. F.-M. Affa'a.1 Département de biologie, Université The presence of adults and tadpoles of A. spinosus in the tree cavi- d'Ottawa, 30 Marie Curie, Ottawa, ON KIN 6N5, Canada. ties year-round, however, allows us to propose that transmission J.-P. Mignot. Laboratoire de biologie des protistes, Unité de of Protozoa to very young tadpoles occurs when cysts contained in recherche associée n° 1944, Université Blaise-Pascal de the faeces of pneumonts and aider tadpoles are swallowed. Clermont-Ferrand II, 63177 Aubière Cédex, France. Preparation of opalinids by silver impregnation for light micros- J.-L. Amiet. 48, rue des Souchères, 26110, France. cary (Affa'a 1979, 1986, 1988, 1991, 1992) and special fixation procedures for ultrastructural studies of the fibrillar system (Mignot Author to whom ail correspondence should be addressed 1994; Mignot and Affa'a 1994a, 1994b, 1994c) has been previously (e-mail: [email protected]). described.

Cano J. Zoo\. 74: 1573-1584 (1996). Printed in Canada / Imprimé au Canada 1574 Cano J. Zool. V

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Figs. 1-5. Cepedeacouillardi n.sp., ammoniacalsilver nitrate impregnationsafter Fernandez-Galiano(1976).Fig. 1. Mature individual as found in pneumonts. Figs. 2 and 3. Sigmoid or arcuate immatures from young tadpoles. Fig. 4. Anterior part of a mature individual. Scale bars = 50 Jtm. Fig. 5. Anterior zone of a mature individual seen from the right lateral side, showing the semicircular falcu1ar zone. Scale bar = 20 Jtm.

Table 1. Morphological differences between Opalina and Cepedea.

Opalina Cepedea

Body shape Wide, leaflike Elongated, narrow, round, or flattened Caudal secant system and (or) kinety Present Absent coalescence zone at posterior pole Ratio of length of falcular zone to >40% <25% cell perimeter

Results Fig. 6. Outline drawings of the various forms of Cepedea couillardi from Protargol-impregnated individuals. Endozoic organisms and host specificity The commensal fauna of the lower intestine of Acanthixalus spinosus varies with the age of the hast and, to a lesser extent, with microhabitat and locality. Opalinids have been found associated with diverse Metazoa such as nematodes (espe- cia11yoxyurids), which occur in adults as we11as in tadpoles. Rotifers were found only in tadpoles froID4 of the tree cavities sampled. Protozoa are far more abundant, as bath species and individuals. Six species of nyctotherids have been described (Affa'a 1979, 1980, 1986, 1988), and a further species has yet to be described. One unspecified Trichodina species has also been found in tadpoles froID two sites. Nyctotheroides augeri is transmitted frOIDtadpoles to adults in the course of its life cycle, the others being restricted to the larval stage of the hast. Proteromonads, diplomonads, and (or) oxymonads and trichomonads are found in bath tadpoles and adults. ln the latter, they coexist with one undetermined Balantidium species and three nyctotherids. Both opalinid species described here differ from a11those observed during a study of the endocommensal Protozoa of 100 anuran hast species froID different location, primarily Cameroon. They also differ froIDthose described by Metcalf (1923, 1940) froIDthe Afrotropical region. ln particular, we have searched for these new opalinid species in Hyperolius acutirostris, a frog that breeds in the saille tree cavities as A. spinosus, known to be a hast ofthese opalinids. Examina- tion of20 tadpolesand 15adults of H. acutirostrisrevealedthe presence of one opalinid, Cepedea affinis, and two nyctho- terids, one specimen of Albaretia terroni and two of Albaretia maxima. It thus seems that our two new opalinids are oioxenous, that is, they are restricted to a single hast (Euzet and Combes 1980), as are the numerous nyctotherid species 1 ribbonlike (Figs. 1 and 6) with a wide and latera11yflattened commensal on A. spinosus (Affa'a and Amiet 1990, 1994). anterior extremity (Figs. 4 and 5) and a minute caudal tir. The numerous nuclei are difficult to stain by conventional Cepedea couillardi n.sp. techniques. The falcular zone is subapical and slightly dis- Figs. 1-15 placed to the right side. It often takes the form of a half-circle Morphological observations with moderatelytapered extremities. Kinetiesoriginate directly Mature trophonts of this opalinid, which are very large frOIDthis zone. They mn straight on the right side and curve (Table 2), have so far been found only in adult frogs and around to lie more or Jess diagona11yon the left surface. young pneumonts (Affa'a et Amiet 1994) in 30% (18/59) of Immature forfis, found exclusively in tadpoles (14/136, the A. spinosus population sampled. They are elongated and or 12.35%), are sma11erand variable in shape. The sma11est, 1576 Cano J. Zool. Vol. 74, 1991 Affa'a et al. 1577

Figs. 7 and 8. Cortex of Cepedea couillardi. Tangential sections of the cortex at various levels, showing the superficial cytoskeletal network. Fig. 7. 21000 x. Scale bar = 1 pm. Fig. 8.42000 x. Scale bar = 0.5 /lm. Edv, endocytotic vesicles; Exv, exocytotic vesicles; F, fibrillar bundles. Ks, kinetosomes of somatic flagella. Fig. 9. Ectoplasm and endoplasm of Cepedea couillardi at low magnification. 11 000 X. Scale bar = l /lm. D, dictyosome; Edv, endocytotic vesicle; F, fibrillar bundles; G, glycogen; M, mitochondrion; Re, endoplasmic reticulum.

Figs. 10 and 11. Sections at various levels of the transition zone between somatic kineties and the fa1cular zone. 20 000 x and 12000 x. Scale bar = 1 /lm. F, microfilament bundles; KF, fa1cular kinetosomes; Ks, kinetosomes of somatic flagella; MF, fa1cular matrix. 1578 Cano J. Zool. Vol. 74, 1!

Figs. 12 and 13. Detail of the falcular zone in Cepedea couillardi. 40000 x. Scale bar = 0.5 /lm. Des, interkinetosomal desmoses; F, microfilament bundles; KF, base of falcular flagella; Ks, somatic flagella; MF, falcular matrix; Mt, microtubular ribbons. Affa'a et al. 1579

Figs. 14 and 15. Whole-cell sections showing the superficial (Fig. 14) and deep (Fig. 15) zones. Scale bar = 1 J.tm. D, dictyosome; Edv, endocytotic vesicles; G, glycogen; M, mitochondrion; N, nucleus; EN, nuclear envelope; RE, endoplasmic reticulum. - 1580 Cano J. Zool. Vol. 74, 1996

Table 2. Morphometric data for the various forms of Cepedea couillardi n.sp. LC WC LZF CFO CFG PC RAP

Adult trophonts (30 specimens) Mean 1322 145 161 85 91 2157 7.53 Median 1357 146 148 89 95 2195 6.69 Variance 21764 802 2396 262 317 51735 5.57 50 147.5 28.3 48.9 16.2 17.8 227.4 2.36 Intermediate trophonts (25 specimens) Mean 154 47 302 15.9 Median 158 - 50 - - 308 15.3 Variance 440 173.9 1812 13.1 50 20.99 13.19 42.56 3.62 Young trophonts (10 specimens) Mean 246 48 44 24 23 437 10.9 Median 240 50 41 24 24 416 10.1 Variance 1954 62.4 156.2 48.32 41.6 8868 13.6 50 44.20 7.90 12.49 6.95 6.45 94.17 3.69

Note: LC, celllength; WC, cell width; LFZ, length of the falcular zone; CFD, number of kineties on the right side; CFG, number of kineties on the left side; PC, cell perimeter; RAP, LFZ/PC ratio as a percentage. Lengths and widths are glven III micrometres.

from very young tadpoles (2/136, or 1.5%), are laterally lies of the endoplasmicreticulum, dictyosomes, and associated bellI or even sigmoid, with a more tapered posterior end vesicles (Figs. 7 and 8). (Figs. 2, 3, and 6). ln aIder tadpoles, with no legs or only Tangentialsectionstaken at variousdepths (Figs. 10and Il) rear legs (9/136, or 6.6% ), they are club-shaped. ln tadpoles through the junction zone between the somatic kineties and undergoing metamorphosis (more than IWOlegs, with the tail the faIx show a network of microfilament bundles arising in still present; 3/136, or 2.2%) the trend is towards longer the falcular zone. They are associated on one band with the and more flattened forms intermediate between the club- falcular kineties (Fig. 12) and on the other with the somatic shaped young trophonts and the elongated, ribbonlike mature kineties. ln addition, it cao be seen in Fig. 12 that the micro- trophonts (fig. 6). tubular ribbonsprecede the corticalfoldings. As in aIl opalinids As expected, these observations of the guI contents of reported in Ibis series (Mignot 1994;Mignotand Affa'a 1994a, several hasts at aIl developmentalstages entail a certain degree 1994b, 1994c), the somalie kineties of C. couillardi show, of variation, yet al114 tadpoles ofvarious ages that harboured in their proximal zone, interkinetaldesmoses oriented towards C. couillardi contained laterally curved, sigmoid, or club- the falcular matrix (Fig. 13). shaped and DeveTribbonlike forms. Metamorphosingtadpoles, with a tail and more than IWOlegs, includeintermediate forms. Endoplasmic zone Fully mature hasts contain only ribbonlike forms. ln Ibis denser region are found nuclei, granular endoplasmic Cepedeacouillardin.sp. populationstherefore show impor- retieulum cisternae, and abundant mitochondria with tubular tant modifications in individual morphology related to the cristae. Some microfibril bundles run through it (Fig. 9). developmental stage of their hast. This type of temporal The central part of the cell often shows a lighter zone probably evolution of morphology bas DeveTbeen reported in opalinids rieh in glycogen (Fig. 15). Some authors have interpreted it until now, and might, then, be considered a character exclu- as a central vacuole. sive to C. couillardi. As in other multinucleate opalinids, nuclei of C. couillardi It is still possible, however, that ailier similar cases remain lack the fibrillar layer that overlies the aliter nuclear enve- unknown, because it is exceptional to follow opalinid popula- love of protoopalinids. The chromatin appears homogeneous, tions from the tadpole stage to the pneumont in the Saillehast with an eccentric nucleolus (Fig. 14). species (Wessenberg 1961), particularly in the African fauna. Ultrastructural observations Affinities 1 This new opalinid is close in size to Cepedea daloalensis (Tuzet Ectoplasmic zone and Zuber-Vogeli 1954) commensal in Hemisus marmoratus, This aliter zone is highly vacuolized, with membrane folds a burrowing amphibian. Cepedea daloalensis is more anteriorly stabilized by microtubular ribbons. These surface folds alter- rounded with a uniform posterior taper, while C. couillardi Datewith somatic kineties. Tangential sections taken in the is ribbonlike with a wide, laterally flattened anterior end in cortex area (Figs. 7 and 8) reveal a superficial cytoskeletal mature individuals. Cepedea couillardi is also different from network of longitudinal and transverse bundles of miero- the known opalinid species in ils individual morphological fibrils. Longitudinal microtubules parallel somatic kineties, modifications related to ifs host's metamorphosis. while transverse bundles surround rows of endocytotic and Although, ultrastructurally, C. couillardi shares many char- exocytotie vesicles. Also in the ectoplasm cao be seen cavi- acIers with other opalinids, it shows some unique particulari- 1582 Cano J. Zool. Vol. 74,1996

Figs. 17-22. Opalinaproteusimpregnatedwith ammoniacalsilver after Fernandez-Galiano(Figs. 17, 18,20, and 21) and with Protargol (Figs. 19 and 22). Fig. 17. Mature trophont. Fig. 18. Posterior pole of a mature trophont, showing the secant system (arrow). Figs. 19 and 20. Young trophonts. Fig. 21. Anterior part of a mature trophont seen from right side, showing the marginal falcular zone (arrow). Scale bars = 50 /Lm. Fig. 22. Precystic individual. Scale bar = 2.5 /Lm.

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Table 3. Morphometric data for the various forms of Opalina proteus n.sp. LC WC LZF CFD CFG PC RAP

Adult trophonts (30 specimens) Mean 465 420 629 252 254 1376 45.8 Median 466 416 634 219 238 1423 45.3 Variance 5931 14995 19235 8431 7367 86541 16.6 SD 77.01 122.4 138.7 91.82 85.83 294.2 4.08 lntermediate trophonts (25 specimens) Mean 215 90 119 42 47 498 24 Median 216 88 116 39 43 486 24 Variance 427 183 316.9 64.55 130.9 2585 6.67 SD 20.66 13.53 17.80 8.03 Il.44 50.85 2.58 Young trophonts (10 specimens) Mean 139 61 87 320 26.7 Median 141 59 91 - 325 26.1 Variance 814.8 276.7 632.4 5222 19.3 SD 28.55 16.63 25.15 72.27 4.39

Note: LC, celliength; WC, cell width; LFZ, length of the falcular zone; CFD, number of kineties on the right side; CFG, number of kineties on the left side; PC, cell perimeter; RAP, LFZ/PC ratio as a percentage. Lengths and widths are given in micrometres.

ties: (i) a relative paucity of endoplasmic fibers compared with Fig. 16. Outline of the various forms of Opalina proteus. Protoopalina pseudonutti and Cepedea sudafricana (Mignot Sca1ebar = 100 /lm. and Affa'a 1994b, 1994c), with more abondant cortical fibers; (ii) a clear axial zone, which could be mistaken for a sucker disk under the light microscope. ln our opinion, these specifie characteristics justify the creation of a new species that we paille Cepedea couillardi in honour of Pierre Couillard, Professor Emeritus, Univer- sité de Montréal, on the occasion of bis retirement.

Opalina proteus n.sp. 1 Figs. 16-22 Morphological observations Althoughmore common than C. couillardi, this multinucleate opalinid bas so faTbeen found only in A. spinosus tadpoles (74/136, or 54%). It effectively disappears when the hast metamorphoses.No representativeofthis specieswas observed in the 59 adult hasts we examined. Younger individuals are generally longer than wide, while the smallest are oblate. They grow into more elongated forms with the posterior pole obliquely eut dorsoventrally (Figs. 6, 19, and 20). Mature trophonts have a wide, leaflike flattening. They often show a conspicuous caudal tir. The smaller oblate forms are found only in younger tadpoles. Table 3 lists measurements for the various categories. ln mature trophonts, the fa1cular zone extends along the margin of the anterior half of the cell (Figs. 17 and 21). Kineties arise direcdy froID the fa1cular zone and run uni- formly towards the posterior pole. Those on the right face are straight, while those on the opposite side appear curved. As they near the posterior pole they give rise to a long dorsoventral secant system (Fig. 18). A precystic individual 1985; Mignot 1994). While cortical structures are charac- (Fig. 22) hints at a reduction in size and number of nuc1ei. teristic of this group, the cortical fibrillar system appears more developed than its endoplasmic counterpart. The right Ultrastructural observations and left faces appear to be separated by a thick bondie of Opalina proteus shares many ultrastructural features with fibers that exclude the kinetosomes frOIDthe posterior pole O. ranarum (Noirot-Timothée 1959; Mergner 1985;Patterson of the cell. This would correspond to the posterior secant 1582 Cano J. Zool. Vol. 74, 1996

Figs. 17-22. Opalina proteus impregnated with ammoniacal silver after Fernandez-Galiano (Figs. 17, 18, 20, and 21) and with Protargol (Figs. 19 and 22). Fig. 17. Mature trophont. Fig. 18. Posterior pole of a mature trophont, showing the secant system (arrow). Figs. 19 and 20. Young trophonts. Fig. 21. Anterior part of a mature trophont seen from right side, showing the marginal fa1cular zone (arrow). Scale bars = 50 /lm. Fig. 22. Precystic individual. Sca1ebar = 2.5 /lm.

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system observed with the light microscope. AlI these fibers microfibrils. Even ifthey resemble microf1lamentsin diameter arise froIDthe falcular zone. (6-7 nID), their dynamism sets them apart. The endoplasmcontainsfewer fibrillar structures but shows Finally, we must note that in ils development, C. couillardi numerous clear areas probably rich in glycogen. Mitochon- adopts the now classical pattern among numerous commensal dria have tubular cristae. protists of the anuran digestive system (Metcalf 1909; Hegner As in the other multinucleate opalinids, the aliter nuclear 1923; Wichterman 1936; McArthur 1955;Wessenberg 1961; membrane lacks the fibrous layer found in protoopalinids and Affa'a and Amiet 1985). Its life cycle spans stages (Mignot and Affa'a 1994a, 1994b). The nucleoplasmis homo- frOIDthe tadpole to the adult. ln contrast, the life cycle of geneous, with an eccentric nucleolus. O. proteus is completed before metamorphosis of the hast. Affinities It must be noted that many individuals in our O. proteus Acknowledgements populations show a posterior end with one side rounded and Support for this work was provided by the Laboratoire de the other straight, as in Opalina duquesnei Delvinquier et al. Zoologie de la Faculté des Sciences de l'Université de 1991). This species was newly described froID only four Yaoundé1;the Laboratoire de Biologie des Protistes de l'Uni- specimens of Chiromantis xerampelina. This makes compar- versité Blaise Pascal de Clermont-Ferrand II; and the labora- ison with our material, consisting of numerous organisms tories of Dr. D.A. Hickey and Dr. D. Brown, Department froIDa greater number of hasts, rather difficult. There is no of Biology, University of Ottawa. Professor P. Couillard question that we are dealing with different species, consider- translated the manuscript froID French to English. We are ing the differences and the variability in shape and size. The very grateful for the help provided. ultrastructural characteristics of O. proteus confirm ils position within the genus Opalina, with a definite kinship with References O. ranarum. Its status as a new species is supported by, among other features, ils greater variabilityin shape, ils dorso- Affa'a, F.-M. 1979. Nyctositum amieti n.gen., n.sp., Cilié endo- ventrally truncated posterior pole, and the great maximal commensaldu têtardd'Acanthixalusspinosus(AmphibienAnoure). size of mature trophonts. We have chosen to name it Opalina Protistologica, 15: 333- 336. proteus to bring attention to ils changing shape. Affa'a, F.-M. 1980. Nyctotheroides nouveaux ou peu connus du Cameroun (première série). Ann. Fac. Sci. Yaoundé, 27: 47 -67. Discussion Affa'a, F.-M. 1986. Description de nouveaux Sicuophoridae (Ciliés Hétérotriches) endocommensaux d'Anoures camerounais. Arch. ln describing these two hast-specifie opalinids froIDAcan- Protistenkd.132: 201-211. thixalus spinulosus we have raid particular attention to the Affa'a, F.-M. 1988. Nyctotheroides nouveaux ou peu connus du relationship between the morphological characteristics of Cameroun (deuxièmesérie). Ann. Fac. Sci. Yaoundé,3(5): 5-50. populations and life-cycle status of the hast, as recommended Affa'a, F.-M. 1991. Observations morphologiques sur deux nycto- by Sandon (1976). Peculiarities of the infraciliature have also thères (ciliés hétérotriches) commensaux de batraciens anoures du Québec. CanoJ. ZooI. 69: 2765 - 2770. proved to be of value as a taxonomic criterion. For Sandon, Affa'a, F.-M. 1992. À propos des critères de séparation des genres variations in infraciliature are of minor importance, as they d'Opalines: ProtoopalinaMetcalf, 1918, CepedeaMetcalf, 1920 mostly result froIDconstraints imposed by cell size and shape. et Opalina Purkinje et Valentin, 1835. Arch. Protistenkd. 141: This is not yet proven. However, our comparative study of 304-314. the two new species has confirmed the existence of a posterior Affa'a, F.-M., and Amiet, J.-L. 1994. Progrès récents dans la secant system in Opalina as well as ils absence in Cepedea. connaissancedes Nyctothères (Protozoaires, CiliésHétérotriches) At the ultrastructural level, the posterior secant line in associés aux Anoures. Alytes, 12(2): 75-92. O. proteus might be homologous to the kinetosome-freeposte- Affa'a, F.-M., and Amiet, J.-L. 1990. Les modes d'association avec rior fibrous zone described by Mignot (1994) in O. ranarum. les espèces-hôtes chez les nyctothères d'Amphibiens du Sud- Unfortunately, such a structure has also been observed in Cameroun. Rev. EcoI. Terre Vie, 45: 345-355. Affa'a, F.-M., and Amiet, J.-L. 1985. Quelques observations sur C. sudafricana. It is possible that the secant system is visible l'évolution de la faune d'Hétérotriches endocommensaux chez under light microscopy only in Opalina because ils fibrillar Bufo regularis et B. maculatus. Protistologica, 21: 273 -278. zone is more developed than in Cepedea. Affa'a, F.-M., and Lynn, D.H. 1994. A review ofthe classification UltrastructuralIy, C. couillardi apparently lacks the endo- and distribution of five opalinids from Africa and North America. plasmic fibrillar system shawn to be present in C. sudafricana, CanoJ. ZooI. 72: 665-674. 1 P. pseudonutti (Mignot and Affa'a 1994a, 1994b), and Amiet, J.-L. 1989. Quelques aspects de la biologie des Amphibiens Cepedea dimidiata (Fernandez-Galiano 1947). However, this Anoures du Cameroun. Ann. BioI. 28(2): 73-136. may not be the case. Indeed, we cali consider the endoplasmic Delvinquier, B.J.L., Markus, M.B., and Passemore, N.I. 1991. Opalinidae in African Anura. 1. Genus Opalina. Syst. ParasitoI. fibrillar system of C. sudafricana, P. pseudonutti, and several 19: 119-146. other African protoopalinids to be a dynamic system; it is not Euzet, L., and Combes, C. 1980. Les problèmes de l'espèce chez les developed to the saille extent in all individuals, and is even animaux parasites. ln Les problèmes de l'espèce dans le règne lacking in Saille trophonts, as shawn under the light micro- animal. Tome III. Soc. ZooI. Fr. Mem. n°40. pp. 239-285. scope (personal observation). How does this structure evolve Fernandez-Galiano, D. 1947. Observaciones citologicas sobre las during the life cycle? Does it arise and later disorganize? For opalinas. Trab. Inst. Cienc. Nat. "José de Acosta," Ser. BioI. 1: a definitive answer we need an in vitro study of the opalinid 353-422. life cycle. We also need to know the chemical nature of the Fernandez-Galiano,D. 1976.Silverimpregnationof ciliatedProtozoa: 1584 Cano J. Zool. Vol. 74, 1996

procedureyieldinggood resultswiththepyridinatedsilvercarbonate Endocytofibrillar complex in Protoopalina pseudonutti Sandon method. Trans. Am. Microsc. Soc. 95: 557-560. 1976. Eur. 1. Protistol. 30: 324-332. Hegner, R.W. 1923. Observations and experiments on Euglenoidina Mignot, 1.-P., and Affa'a, F.-M. 1994c. Patterning in opalinids. III: in the digestivetract of frog and toad tadpoles. Biol. Bull. (Woods The cytoskeletonof Cepedeasudafricana(Fantham, 1923)Affa'a Hole, Mass.), 45: 162-180. and Lynn (1994). Anintermediate type between Opalina ranarum McArthur, H.W. 1955. Observations on the enteric Protozoa of andProtoopalinapseudonutti. Arch. Protistenkd.145: 241-249. Rana pipiens during larval development and metamorphosis. Noirot-Timothée, C. 1959. Recherches sur l'ultrastructure d' Opalina Iowa Acad. Sei. 62: 640-651. ranarum. Ann. Sei. Nat. Zool. Biol. Anim. (Sér. 12),1: 265-281. Mergner, V. 1985. Opaliniden im Licht- und Elektronenmikroskop. Patterson, D.J. 1985. The fine structure of Opalina ranarum (family Mikroskosmos, 74: 233-239. Opalinidae): opalinid phylogenyand classification.Protistologica, Metcalf, M.M. 1909. Opalina, its anatomy and reproduction with a 21: 413-428. description of infection experiments, and a chronological review Perret, J.-L. 1962. La biologie d'Acanthixalus spinosus (Amphibia of the literature. Arch. Protistenkd. 13: 195-375. Salientia). Rech. Etudes Cameroon 1,16: 822-828. Metcalf, M.M. 1923. The opalinid ciliate infusorians. V.S. Nad. Sandon, H. 1976. The species problem in the opalinids (Protozoa, Mus. Bull. No. 120. Opalinata), with special reference to Protoopalina. Trans. Am. Metcalf, M.M. 1940.Further studiesontheopalinidciliateinfusorians Microsc. Soc. 95: 357-366. and their hasts. Froc. V.S. Nad. Mus. 87: 465-634. Tuzet, O., and Zuber-Vogeli, M. 1954. Recherches sur les opalines Mignot, J.-P. 1994. Patterning in opalinids. 1. Implications ofnew et les ciliés parasites des batraciens recoltés à Daloa (A.O.F.). morphological and ultrastructural findings on the genesis of Bull. Inst. Fr. Afr. Noire, 16: 822-828. kineties. Eur. J. Protistol. 30: 196-210. Wessenberg, H.S. 1961. Studies on the cycle and morphogenesis of Mignot, J.-P., and Affa'a, F.-M. 1994a. Étude structurale et ultra- Opalina. Vniv. Calif. Publ. Zool. 61: 315-369. structurale de Protoopalina drachi Tuzet et Knoepffler, 1968. Wichterman, R. 1936. Division and conjugation in Nyctotherus Arch. Protistenkd. 144: 173-183. cordiformis (Ehr.) Stein (Protozoa, Ciliata) with special reference Mignot, J.-P., and Affa'a, F.-M. 1994b. Patterning in opalinids. II. to the nuclear phenomena. J. Morphol. 60: 563-611.

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