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Ultrastructure of a Colourless Amoeboid Flagellate, Cercomonas sp.
Arch. Protistenkd. 131 (1986) : 2 39-247 VEB Gustav Fisch er Verlag J ena
Institute of Biology of Inland \Vaters , U SSH, A cademy of Sciences, Borok
Ultrastructure of a Colourless Amoeboid Flagellate, Cercomonas 8p. By A. P.
~IYLNIKOV
With 11 Figures
Summary Th e ultrastructure of a colourless amoeboid flagellate, Cercomonas ep, isolated from fresh water, is co ns ide re d . This minute organism p ossesses two heterodynamic fl ag ell a. When moving it actively crawls al ong the substratum, p ersistently forming pseudopodia of various shape and siz e. The v esi cular nucleus with a central nucl eolus is located anteriorly, close the two kinetosomes, The sys tem of kinetosome-derived mi erotubules forms a conical sh eat h a r ou n d the nucleus. The Golgi a pparat u s, the endoplasmic re t icu lu m , and mitochondria with t u b u la r cristae have usual str uc t u re. A paranuclear body surro u n de d by a unit m embrane is o bse r ve d in the ce lls. The cyto pl a sm is filled with food vacuoles a n d drops of s t orage su bstances. B eneath the body sur face , th ere a re st ing ing organell es a ffec t ing ba cteria. Peculiarities of t h e ul tra s tructure relate Cercom onas with the ch rysophyte al ga e a n d m yxomy cet es.
Introduction Presently, the protozoa whi ch com bine features of both Sarcod ina and Mastigophora are ext ensively studied. These include the gen era Pseudosp ora, Het eromita, Jlfastigamoeba, J.l f astigella, and Cercomonas. The taxonomic position of some of them ha s already been' cleared up, whil e for t he others it r emains quite problematic. The genus Cercomonas D UJARDIN (syn. Cercobodo KRASSILSCHIK) includes colourless flag ellates having two heterodynamic flagella and a single nucleus. They regularly form pseudopodia whi ch serve for locomotion and food ingestion. Unlike the a moeboflagellat es of the order Schizopyrenida , the represen tatives of Cercomonas do not sh ed their flagella during the amoeboid stage and do not form pseudopodia of the " limax" type. Detailed studies of these organisms hav e been carried out on both light microscope (KRASSILSOHIK 1886 ; WENYON 1910; H OLLANDE 1942) and electron m icros cope levels (MlGNOT and BUUGEROLLE 1975a; SCHUSTJm and POLLACK 1978; ANIKUSHIN 1978). Earlier, some authors (HOLLANDE 1942 ; ZHUKOV 1971) united Cercomonas with the representatives of the present order Kinetoplastida. However, the ab sen ce of the kinetoplast in Cercomonas and other pe culiarities distinctly separate them from the kinetoplastids. Mod ern investigations sh ow a wide distribution of these organisms in sewage treatment plants, as well as in seas, fresh waters, and soil (ZUUKOV 1971).
Material and Methods The organism used in this s t u d y was isola t ed from near-bot t om wa ters of the R ybinsk r eservoir a nd was clon ed . The re sults of stu d ies m ade wi th th e st ra in C-45 a r e presented b elow. The flagell a t e was cu lt u red in P etri di sh es u sin g the P RA= m edium with a d d ition o f b a ct eria A erobacter aero-
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MYLNIKOV
genes, amounting 10-100 million cells per ml. The population doubling time was 8-12 h at 20-22°C, and the number of individuals reached 0.1-0.5 million eellsjml on the 6-IOth day after inoculation. With aging of the culture, some of the flagellates formed cysts. When the cysts were transferred into a fresh medium, or when bacteria were added, excystment was observed. The cysts remained viable for a long time (up to a year). Data on the fine structure of the cysts are not included into this paper. Living organisms were studied using a Peraval microscope (Zeiss) with interference-contrast optics. For electron microscopic investigations, the flagellates were collected from the bottom of the dish, and the suspension was concentrated by centrifugation. The sedimented cells were collected and fixed with a mixture of 2 % OS04 and 0.6 % glutaraldehyde in 0.05 M cacodylate buffer for 15 min at 1 DC. After dehydration in a series of alcohols and water- free acetone, the cells were embedded into a mixture of Epon 812 and Araldite M. The sections were stained according to REYNOLDS and inspected with a JEM·I00 C microscope at 60 kV.
Results In young cultures, the flagellates actively crawl along the dish bottom, permanently forming pseudopodia of various shapes. When the number of individuals is great, some of them may swim irregularly for a short time. The length of the crawling individuals reaches 3.3-6.0 pm (pseudopodia excluded), the width being 1.8-2.8 pm. The cell shape is more or less oval. The flagellum directed forward (2-3 pm in length) performs rowing movements. The proximal part of this flagellum often passes through a cytoplasmic outgrowth, which can make independent movements without partici-
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Fig. 1. Structural scheme of Cercomonas sp, Designations: cv - contractile vacuole; er - endoplasmic reticulum; fe - flagellum emergence; fk - flagellar kinetosomes; fv - food vacuole; ga - Golgi apparatus; If - locomotory flagellum; m - mitochondrium; me - microtoxicyst; mb - microtubular band; n - nucleus; p - pseudopodium; pb - paranuclear body; rf - recurrent flagellum; sm - solitary microtubules; s s storage substances.
A Colo urless Amoeboid F lagellate
241
F ig . 2. L ongitudin a l sectio n of t he fl a gell a t e. Th e recu rrent fl agellum (r f) is seen passin g in a n inva gi nation, t h e a n t er ior en d of t h e nucl eu s (n) is po inted, t he wa ll of t h e con t ract ile v acuole (cv) is sin u ou s, the mi cr ot ubu les b and (mb) p ass es n ear a k inet oso m e , the st orage subs t a nc es (ss) arc located n ear the para n u clea r b ody (pb ). X 30, 000 .
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A Colo u rles s Amoebo id Fla gell a t e
243
pation of the fla gellum. The seco nd fla gellum is direct ed ba ckwa rds and equa ls t he bod y len gth ; ther efore, it is usu all y incon spicuous. After fixa t ion with mer cury bichlor ide or osmiu m vap ours, the second flagellum separates in some individual s fr om the cell body along it s entire len gth except the proximal end . When obs er ving the live orga n ism from ben eath , the rec ur re nt fla gellum is seen to p erform undulating movemen t s. This is prob ably indi cati ve of t he a ct ive ro le of t he p ost erior flagellum in locomotion. At the di st al end t he flagella becom e thinner. No ma sti gonem es wer e found on dried prepara ti on s. The ves icula r nucleus (1.2-1.4 pm in di am eter ) has a cent ral nucleolu s a nd is more or less egg -shape d . A nu III bel' of refr actory gra n ules occur in the cy t oplas m. The pseudopodia are formed along the entire p erimeter of the bod y , bu t more frequ en tl y at t he rear end. The food vac uo les a re few. No cy toproct has been found. The single cont rac t ile va cu ole is located on the side or t he middle of the body a nd changes its position cons tantly . T he dir ection of the cell m ovem ent often cha nges. After stain ing with iron haem atoxyline, a rod-like st r ucture (0.4- 0.6 pm ) is visibl e behind the nucleus. No kinetopl ast wa s observed. The cys ts, having a diameter of 2.0-2.5 pm, show a central nu cleu s a nd are surrounded by a t hin wall. The elect ron microscopic d ata are summarized on the di agram (Fig. 1). The general p osition of the organelles is shown in Figs. 2 and ~. The nucleus is situated ne ar t he two kinetosomes of the flagella. Its shape is usually egg-like, the pointed end being direct ed forward. The nucleolu s (0.5-0.6 pm in di a met er ) lies in the cente r of t he nucleu s. Lumps of chro matin occ ur in the karyoplasm . The nuclear en velope d ispla ys consp icuous p or es in so me pl aces. The kinetosom e of the recurrent flagellum gives rise t o a bundle of mi crotubules, These s play ou t for ming a cone whi ch envelops t he n ucle us as the mi crotubules pass back wa rds (Fig. 3). So metimes t he bundle of m icrot ub ules d epresses t he surface of the nucleu s. The k inetosom es are ar ranged at rigth or, mor e ofte n, at obtuse angles t o eac h other (F ig. 5). N o ribbon s, strand s, bridges or other connections are found between t he m, alt ho ug h osmiophilic p at ch es are some t imes obse rved between the ba ses of t he two kinet osom es, The two flagellar axonemes and both kinetosom es are of the usu al struct ure. No coiled st ruct ure, as it occ urs in t he representa ti ves of Chrysop hy ta (H rnnn n n 1976 ), ha s been fou nd in the axone me . A ba nd of 3-4 microt u bules, wh ich pass und er t he cell membrane to t he rear end of t he ho dy, emerges fr om one of t he kinetosom es. Se ve ral solitary microtubules occur in t he cy to plas m. As see n on t he sec tions, t he rec ur rent fla gellum is fixed t o t he surfa ce of t he cell bod y in it s p ro ximal part (Fig. 4). Th e Golgi apparatus and t he cont ract ile vacu ole lie usu ally n ear the ventral side of the body . The wall of the con t r act ile vacuole shows in vagin at ions. The v esicl es of t he Golgi apparatus are consp icuously curved (Figs. 3 a nd 10). The oval mito-
Fig. :1 . Anter ior p art of th e body. So litary m icro t ub u les (sm) em erge f rom t h e fl agell ar kinet oso m e, th e Golgi a p parat us (ga ) is cu rved, t he mi to ch ond ria (m) h ave tubular cr istae. X 40 ,000 . Fig. 4. Fixation of t he recurrent fl a gellum (in cro ss sect ion ) to t h e b od y s ur fac e. X 60 ,000. F ig. 5. Orientation of t he fl a gell a r ki netosomes in rela t ion t o each other. So litary mi cro tubules (s m ) a re seen . X 45,0 00 . Figs. 6, i. Lon gitudinal a n d cr oss sec t ions of mi crot oxi ey s t s (m e). Fig. 6, 10*
X
150,000; F ig. i ,
X
100,000.
244
A. P.
]\fVLN IK OV
-
b
~---mc
.'
9
Fig. 8. The p aranuclear b ody (pb) n ear t he nucleus (n). X 66 ,000. Fig. 9. A mier otoxiey st (m c) in ject s it s con ten t s into a bacterium (b).
X
100,000.
F ig. 10. Micr ot ox icy st s (m e) are be in g formed in vesicles produced by the Go igi apparatus (ga), X 70,000. F ig . 11. A food va cuole (f v ) con t a in s a b acterium (b ) which was affected by a microtoxicys t (me). T h e con te n ts of the bacterium appear lysed. Another fo od vacuole (a rrow) con tains a n intact bacterium. X 50 ,000.
A Colourless Amoeboid Flagellate
245'
chondria (0.35-0.40 {tm) contain tubular cristae which are located along the periphery of the organelle, its central part being occupied by an osmiophilic structure. Usually 5-10 mitochondria are seen in a single section; they are evenly distributed in the cytoplasm. Almost in every section a structure is seen behind the nucleus, called by MIGNOT and BRUGEROLLE (1975a) the paranuclear body. Serial sections have revealed that this oblong structure is surrounded by a unit membrane. A dark matrix and probably some tubular elements can be discerned inside it (Fig. 2). Sometimes the dark matrix alternates with light areas (Fig. 8). In some cases, a contact is observed between the nuclear envelope and the paranuclear body. Drops of storage substances occur near the paranuclear body. The cell is surrounded by a layer of fibrous glycocalix, 50-75 nm thick, which is seen not in all sections. The flagellate feeds on bacteria caught by the pseudopodia. Besides the several food vacuoles and the endoplasmic reticulum, some spherical stinging organelles (130-150 nm) were detected in the cytoplasm. Here they are called microtoxicysts. Their maturation takes place in the vesicles pinching off the Golgi apparatus (Fig. 10). Mature microtoxicysts lie close to the body surface. These organelles consist of two cylinders placed one into another and surrounded by a heterogenous matrix and a membrane (Figs. 6 and 7). When discharging the outer cylinder turns inside out as a glove finger and pushes the inner cylinder out. The contents of the stinging organelle becomes extruded. The cylinders are oriented perpendicularly to the cell surface. The discharge of a microtoxicyst appears to be caused by a contact with a bacterium, with a flagellar axoneme, or with the flagellate's body; it proba bly occurs also during fixation (Figs. 9 and 10). The extruded contents of the microtoxicyst apparently lyse the bacterium. Usually 2 to 7 microtoxicysts occur in a section of a flagellate; they are situated more or less evenly along the perimeter of the cell body.
Discussion An analysis of the above results shows that the general organization of the cells strain C-45 is similar to that of the genus Cercomonas according to the descriptions made using both light and electron microscope. A number of authors published detailed studies of morphology, feeding, locomotion, and cyst formation in Cercomonas (KRASSILSCHIK 1886; WENYON 1910; Hovxssn 1937; HOLLANDE 1942). All the species of this genus have an amoeboid body, two permanent heterodynamic flagella, and usually an egg-shaped anterior nucleus. The flagellates are thought to move with the help of movements of the directed forward locomotory flagellum. However even KRASSILSCHIK (1886) noted undulating movements of the recurrent flagellum, which was recently confirmed by MIGNOT and BRUGEROLLE (1975a) as well as by the present work. In all species of Cercomonas investigated by now, fine structural studies have shown that the recurrent flagellum is fixed in its proximal part to the cell body. When comparing the strain C-45 with other species of Cercomonas (MIGNOT and BRUGEROLLE 1975a; SCHUSTER and POLLACK 1978), one can see the similarity of their principal cell structures and of the general pattern of organization. The differences concern mostly the body size and the presence of microtoxicysts in the strain C-45. The following characteristic features are shared by Cercomonas and the strain here studied: absence of the kinetoplast and of the paraxial rod in the flagella, tubular cristae in the mitochondria, absence of the system of subpellicular microtubuless ability to form pseudopodia, and presence of the paranuclear body. All these feature, indicate a significant difference between Cercomonas and the kinetoplastids. In
246
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MYLNIKOV
contrast to the strains studied earlier (MWNOT and BRUGEROLLE 1975a; SCHUSTER and POLLACK 1978), the kinetosomes of the strain C-45 lack a fibrous band fixing them together. In all the three strains, the microtubules proceed backwards as a cone enveloping the nucleus, and a band of 3-4 microtubules emerges the kinetosome. The paranuclear body, which lies behind the nucleus, very much resembles the microbody known in many flagellates (DODGE 1973). In the cytoplasm, there are granules of storage substances which impart a granular appearance to the cytoplasm. The glycocalyx of the strain C-45 is similar to that in other species of Cercomonas. In the organism presently studied we found organelles, the stinging nature of which is established rather firmly. Trichocystlike structures have also been found in the strains studied before-.As supposed by the authors, they serve for production of mucilage during the sliding of the flagellates along the substratum; their discharge was however not observed. Similar organelles occur in many biflagellated colourless flagellates and some other protozoa. They were found in Colponema loxodes (MWNOT and BRUGEROLLE 1975b), Gyromitus limax (SWALE and BELCHER 1975), Heteromita globosa (MACDONALD et al. 1977), Pseudospora sp, (SWALE 1969), and other species. The term microtoxicyst is quite applicable to the stinging organelles of our organism (HAUSMANN 1978), because this minute organelle injects into a bacterium and lyses it. The taxonomic position of the strain C-45 and of other Cercomonas species remains uncertain. The permanent presence of two unequal flagella, the formation of pseudopodia, the structure of mitochondria, the type of the microtubular system, and the presence of trichocyst-like structure closely relate all known species of Cercomonas to Heteromita and Pseudospora; the latter genus has also microbodies. However, they all differ essentially from the typical amoebo-flagellates of the order Schizopyrenida (genera Tetramitus and N aeqleria: SCHUSTER 1963; OUTKA and KLUSS 1967), which have a different system of microtubules, a rhizoplast, peculiar mitochondria, and kinetosomes arranged parallel to each other. The genus Cercomonas may probably be related with the Chrysophyta, where a tendency is found to form pseudopodia and to lose chloroplasts, e.g., in Chrysamoeba radians (HIBBERD 1971). However, the Chrysophyta are characterized by mastigonemes on their flagella, by coiled structures in their axonemes, and by some other features (HIBBERD 1976) not found in Cercomonas. The organisms here considered possess a certain similarity with flagellate and amoeboid stages of development in Mycetozoa. Thus, they have two flagella without mastigonemes, filose pseudopodia, mitochondria with tubular cristae, a microbody, a cone of microtubules enveloping the nucleus (OLIVE 1975). An especially great similarity is found between Cercomonas and the protostelids (HUNG and OLIVE 1973). It is quite possible that Cercomonas and the Mycetozoa originated from a common ancestry.
Literature ANIKUSHIN, N. F. (1978): On structural heterogeneity of colourless freeliving flagellates of the suborder Bodonina Hollande. In: Biology of Inland Waters, Inform. Bull. No. 38: 10-12 (in Russian). DODGE, J. D. (1973): The fine structure of algal cell. London and New York. HAUSMANN, K. (1978): Observations on the extrusive organelles in Protists. Int. Rev. Cytol. 52: 197-276. HIBBERD, D. J. (1971): Observations on the cytology and ultrastructure of Chrysamoeba radians KLEBS (Chrysophyceae). Brit. Phycol. J. 6: 207-224. _ (1976): The ultrastructure and taxonomy of Chrysophyceae and Prymnesiophyceae (Haplophyceae): a survey with some new observations on the ultrastructure of the Chrysophyceae. Bot. J. Linnean Soc. 72: 55-80.
A Colourless Amoebo id F lagell ate
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HOI,(,ANDE, A. (1942) : Etude cy t olo g iq ue d e quelques F'lagelles Iib res, Arch . Zool. E x p er. Gen. 83 : 1- 268. H OVASSE, R. (1937 ): Cont.ribut.ion a I'e t u de de I' ap p a re il d e Golg i de s Flagelles Iibres : L ' ex ist ence d 'un cor ps parabasal ch ez Cercomon as lonqi cauda D UJ . Arch . Zoo l. Exper. Gen. 79: 43- 4 6. H UNo, C. ' Y., and OLIVE, L. S. (197 3) : U ltra structure of th e a moebo id ce lls of P rotostelli u m zona tu m (Mycetozoa) . J. Protozool. 20: 252 -263. KRA SSILs CHIK, 1. ( 1886) : Material s t o th e n atural history a n d taxonomy of flagellates. About Cerco bodo laciniaeqerens nov . gen. et no v . sp . Zap . Novorossyi sk. obsch . estestvoispyt. 2: 2 11- 245 (in R u ssia n ). MACDoNALD , C. 1\1., DARBYSHIRE, J . F ., a nd OGDEN, C. C. (1977) : Th e m orphology of a com m o n so il fl agellate, H etero mi ta glob osd STEIl' (Mast.igophorea : Protozoa). Bull. B r it. Mu s, (Natur. Hist.) Zool. 31 : 255- 264. :l\IrGKOT, J. F. , and B RUOEROLLE, G. (1975 a) : Etude u ltrastru ctura le d e Cer com onas D UJARDIK ( = Cerc obodo KRASSILSCHICK), Protiste f'lag elle. P rot is t ol og ica 11 : 547 -554. - - ( 1975 b) : Etude ul t r a st ruct ur al e du flagel le phagotrophe Colpon ema loxodes STEIN. Protistologi ca 11 : 429-4 44 . O LIVE, L . S. ( 1975) : T h e Mycetoz oan. N ew Y or k. O UTKA, D . E ., a n d KL USS, B. C. ( 1967): The a me b a -t o- f1ag ellate transformat io n in Tetramit u s rostratus , J. Cell. BioI. 35: 323 -:146. Scn cs 'n sn , F . (1963): An electron m icroscope study of t h e amoebofl agellate , Ntteqleria grub eri (S CHARDIKOER). 1. Th e amoeboid and fl agellat e stages. J. Protozool. 10 : 297 -313. S CHUSTER, F . L. , and POLLACK, A . ( 1978): Ultrastr u ct ure of Cercomonas sp., a free-living ameboid fl agellat e . Arch . Pro tist en k d. 120 : 206 -2 12. SWAI,E, E . M. F . (19 69) : The structure of a species of the amoeboflageUate Pseudospora CIENK. Arch . Mierobiol. 67 : 7 1-90. - a nd BELCHER, J . H. ( 1975) : Gyrom ituslimax nov. sp . - Free-living colo u rless a moebo -flagellate. Arch. Prot.is tenkd. 117 : 20- 26. ' YENYON, C. J\1. ( 19 10) : Some observation on flagellates of th e genus Cercom onas , Quart. J. Mi crosc. Sci. 55 : 24 1-260. Z IIUKOV, B. F . (197 1) : K ey to colou r less fr ee-living fl agellates of th e su b or de r B odon ina H oll ande. In : B iol ogy and productivit y of fr eshwater organisms, pp. 241- 284. Leningrad (in Russian) . Author 's address : A. P . J\IYLKIKOV, Laboratory of biology of lower organisms, I ns t it u t e of Biology of I n la n d Waters, USSR Academy of Scien ces , 15274 2 Borok (Y a ro slav l D istrict ), USSR .
Institute of Biology of Inland \Vaters , U SSH, A cademy of Sciences, Borok
Ultrastructure of a Colourless Amoeboid Flagellate, Cercomonas 8p. By A. P.
~IYLNIKOV
With 11 Figures
Summary Th e ultrastructure of a colourless amoeboid flagellate, Cercomonas ep, isolated from fresh water, is co ns ide re d . This minute organism p ossesses two heterodynamic fl ag ell a. When moving it actively crawls al ong the substratum, p ersistently forming pseudopodia of various shape and siz e. The v esi cular nucleus with a central nucl eolus is located anteriorly, close the two kinetosomes, The sys tem of kinetosome-derived mi erotubules forms a conical sh eat h a r ou n d the nucleus. The Golgi a pparat u s, the endoplasmic re t icu lu m , and mitochondria with t u b u la r cristae have usual str uc t u re. A paranuclear body surro u n de d by a unit m embrane is o bse r ve d in the ce lls. The cyto pl a sm is filled with food vacuoles a n d drops of s t orage su bstances. B eneath the body sur face , th ere a re st ing ing organell es a ffec t ing ba cteria. Peculiarities of t h e ul tra s tructure relate Cercom onas with the ch rysophyte al ga e a n d m yxomy cet es.
Introduction Presently, the protozoa whi ch com bine features of both Sarcod ina and Mastigophora are ext ensively studied. These include the gen era Pseudosp ora, Het eromita, Jlfastigamoeba, J.l f astigella, and Cercomonas. The taxonomic position of some of them ha s already been' cleared up, whil e for t he others it r emains quite problematic. The genus Cercomonas D UJARDIN (syn. Cercobodo KRASSILSCHIK) includes colourless flag ellates having two heterodynamic flagella and a single nucleus. They regularly form pseudopodia whi ch serve for locomotion and food ingestion. Unlike the a moeboflagellat es of the order Schizopyrenida , the represen tatives of Cercomonas do not sh ed their flagella during the amoeboid stage and do not form pseudopodia of the " limax" type. Detailed studies of these organisms hav e been carried out on both light microscope (KRASSILSOHIK 1886 ; WENYON 1910; H OLLANDE 1942) and electron m icros cope levels (MlGNOT and BUUGEROLLE 1975a; SCHUSTJm and POLLACK 1978; ANIKUSHIN 1978). Earlier, some authors (HOLLANDE 1942 ; ZHUKOV 1971) united Cercomonas with the representatives of the present order Kinetoplastida. However, the ab sen ce of the kinetoplast in Cercomonas and other pe culiarities distinctly separate them from the kinetoplastids. Mod ern investigations sh ow a wide distribution of these organisms in sewage treatment plants, as well as in seas, fresh waters, and soil (ZUUKOV 1971).
Material and Methods The organism used in this s t u d y was isola t ed from near-bot t om wa ters of the R ybinsk r eservoir a nd was clon ed . The re sults of stu d ies m ade wi th th e st ra in C-45 a r e presented b elow. The flagell a t e was cu lt u red in P etri di sh es u sin g the P RA= m edium with a d d ition o f b a ct eria A erobacter aero-
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MYLNIKOV
genes, amounting 10-100 million cells per ml. The population doubling time was 8-12 h at 20-22°C, and the number of individuals reached 0.1-0.5 million eellsjml on the 6-IOth day after inoculation. With aging of the culture, some of the flagellates formed cysts. When the cysts were transferred into a fresh medium, or when bacteria were added, excystment was observed. The cysts remained viable for a long time (up to a year). Data on the fine structure of the cysts are not included into this paper. Living organisms were studied using a Peraval microscope (Zeiss) with interference-contrast optics. For electron microscopic investigations, the flagellates were collected from the bottom of the dish, and the suspension was concentrated by centrifugation. The sedimented cells were collected and fixed with a mixture of 2 % OS04 and 0.6 % glutaraldehyde in 0.05 M cacodylate buffer for 15 min at 1 DC. After dehydration in a series of alcohols and water- free acetone, the cells were embedded into a mixture of Epon 812 and Araldite M. The sections were stained according to REYNOLDS and inspected with a JEM·I00 C microscope at 60 kV.
Results In young cultures, the flagellates actively crawl along the dish bottom, permanently forming pseudopodia of various shapes. When the number of individuals is great, some of them may swim irregularly for a short time. The length of the crawling individuals reaches 3.3-6.0 pm (pseudopodia excluded), the width being 1.8-2.8 pm. The cell shape is more or less oval. The flagellum directed forward (2-3 pm in length) performs rowing movements. The proximal part of this flagellum often passes through a cytoplasmic outgrowth, which can make independent movements without partici-
f e -1'-+-I--7Vi~
go ./"l>--"l"-\- Pb r.;-~ ~~""--r 55
er
Fig. 1. Structural scheme of Cercomonas sp, Designations: cv - contractile vacuole; er - endoplasmic reticulum; fe - flagellum emergence; fk - flagellar kinetosomes; fv - food vacuole; ga - Golgi apparatus; If - locomotory flagellum; m - mitochondrium; me - microtoxicyst; mb - microtubular band; n - nucleus; p - pseudopodium; pb - paranuclear body; rf - recurrent flagellum; sm - solitary microtubules; s s storage substances.
A Colo urless Amoeboid F lagellate
241
F ig . 2. L ongitudin a l sectio n of t he fl a gell a t e. Th e recu rrent fl agellum (r f) is seen passin g in a n inva gi nation, t h e a n t er ior en d of t h e nucl eu s (n) is po inted, t he wa ll of t h e con t ract ile v acuole (cv) is sin u ou s, the mi cr ot ubu les b and (mb) p ass es n ear a k inet oso m e , the st orage subs t a nc es (ss) arc located n ear the para n u clea r b ody (pb ). X 30, 000 .
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A Colo u rles s Amoebo id Fla gell a t e
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pation of the fla gellum. The seco nd fla gellum is direct ed ba ckwa rds and equa ls t he bod y len gth ; ther efore, it is usu all y incon spicuous. After fixa t ion with mer cury bichlor ide or osmiu m vap ours, the second flagellum separates in some individual s fr om the cell body along it s entire len gth except the proximal end . When obs er ving the live orga n ism from ben eath , the rec ur re nt fla gellum is seen to p erform undulating movemen t s. This is prob ably indi cati ve of t he a ct ive ro le of t he p ost erior flagellum in locomotion. At the di st al end t he flagella becom e thinner. No ma sti gonem es wer e found on dried prepara ti on s. The ves icula r nucleus (1.2-1.4 pm in di am eter ) has a cent ral nucleolu s a nd is more or less egg -shape d . A nu III bel' of refr actory gra n ules occur in the cy t oplas m. The pseudopodia are formed along the entire p erimeter of the bod y , bu t more frequ en tl y at t he rear end. The food vac uo les a re few. No cy toproct has been found. The single cont rac t ile va cu ole is located on the side or t he middle of the body a nd changes its position cons tantly . T he dir ection of the cell m ovem ent often cha nges. After stain ing with iron haem atoxyline, a rod-like st r ucture (0.4- 0.6 pm ) is visibl e behind the nucleus. No kinetopl ast wa s observed. The cys ts, having a diameter of 2.0-2.5 pm, show a central nu cleu s a nd are surrounded by a t hin wall. The elect ron microscopic d ata are summarized on the di agram (Fig. 1). The general p osition of the organelles is shown in Figs. 2 and ~. The nucleus is situated ne ar t he two kinetosomes of the flagella. Its shape is usually egg-like, the pointed end being direct ed forward. The nucleolu s (0.5-0.6 pm in di a met er ) lies in the cente r of t he nucleu s. Lumps of chro matin occ ur in the karyoplasm . The nuclear en velope d ispla ys consp icuous p or es in so me pl aces. The kinetosom e of the recurrent flagellum gives rise t o a bundle of mi crotubules, These s play ou t for ming a cone whi ch envelops t he n ucle us as the mi crotubules pass back wa rds (Fig. 3). So metimes t he bundle of m icrot ub ules d epresses t he surface of the nucleu s. The k inetosom es are ar ranged at rigth or, mor e ofte n, at obtuse angles t o eac h other (F ig. 5). N o ribbon s, strand s, bridges or other connections are found between t he m, alt ho ug h osmiophilic p at ch es are some t imes obse rved between the ba ses of t he two kinet osom es, The two flagellar axonemes and both kinetosom es are of the usu al struct ure. No coiled st ruct ure, as it occ urs in t he representa ti ves of Chrysop hy ta (H rnnn n n 1976 ), ha s been fou nd in the axone me . A ba nd of 3-4 microt u bules, wh ich pass und er t he cell membrane to t he rear end of t he ho dy, emerges fr om one of t he kinetosom es. Se ve ral solitary microtubules occur in t he cy to plas m. As see n on t he sec tions, t he rec ur rent fla gellum is fixed t o t he surfa ce of t he cell bod y in it s p ro ximal part (Fig. 4). Th e Golgi apparatus and t he cont ract ile vacu ole lie usu ally n ear the ventral side of the body . The wall of the con t r act ile vacuole shows in vagin at ions. The v esicl es of t he Golgi apparatus are consp icuously curved (Figs. 3 a nd 10). The oval mito-
Fig. :1 . Anter ior p art of th e body. So litary m icro t ub u les (sm) em erge f rom t h e fl agell ar kinet oso m e, th e Golgi a p parat us (ga ) is cu rved, t he mi to ch ond ria (m) h ave tubular cr istae. X 40 ,000 . Fig. 4. Fixation of t he recurrent fl a gellum (in cro ss sect ion ) to t h e b od y s ur fac e. X 60 ,000. F ig. 5. Orientation of t he fl a gell a r ki netosomes in rela t ion t o each other. So litary mi cro tubules (s m ) a re seen . X 45,0 00 . Figs. 6, i. Lon gitudinal a n d cr oss sec t ions of mi crot oxi ey s t s (m e). Fig. 6, 10*
X
150,000; F ig. i ,
X
100,000.
244
A. P.
]\fVLN IK OV
-
b
~---mc
.'
9
Fig. 8. The p aranuclear b ody (pb) n ear t he nucleus (n). X 66 ,000. Fig. 9. A mier otoxiey st (m c) in ject s it s con ten t s into a bacterium (b).
X
100,000.
F ig. 10. Micr ot ox icy st s (m e) are be in g formed in vesicles produced by the Go igi apparatus (ga), X 70,000. F ig . 11. A food va cuole (f v ) con t a in s a b acterium (b ) which was affected by a microtoxicys t (me). T h e con te n ts of the bacterium appear lysed. Another fo od vacuole (a rrow) con tains a n intact bacterium. X 50 ,000.
A Colourless Amoeboid Flagellate
245'
chondria (0.35-0.40 {tm) contain tubular cristae which are located along the periphery of the organelle, its central part being occupied by an osmiophilic structure. Usually 5-10 mitochondria are seen in a single section; they are evenly distributed in the cytoplasm. Almost in every section a structure is seen behind the nucleus, called by MIGNOT and BRUGEROLLE (1975a) the paranuclear body. Serial sections have revealed that this oblong structure is surrounded by a unit membrane. A dark matrix and probably some tubular elements can be discerned inside it (Fig. 2). Sometimes the dark matrix alternates with light areas (Fig. 8). In some cases, a contact is observed between the nuclear envelope and the paranuclear body. Drops of storage substances occur near the paranuclear body. The cell is surrounded by a layer of fibrous glycocalix, 50-75 nm thick, which is seen not in all sections. The flagellate feeds on bacteria caught by the pseudopodia. Besides the several food vacuoles and the endoplasmic reticulum, some spherical stinging organelles (130-150 nm) were detected in the cytoplasm. Here they are called microtoxicysts. Their maturation takes place in the vesicles pinching off the Golgi apparatus (Fig. 10). Mature microtoxicysts lie close to the body surface. These organelles consist of two cylinders placed one into another and surrounded by a heterogenous matrix and a membrane (Figs. 6 and 7). When discharging the outer cylinder turns inside out as a glove finger and pushes the inner cylinder out. The contents of the stinging organelle becomes extruded. The cylinders are oriented perpendicularly to the cell surface. The discharge of a microtoxicyst appears to be caused by a contact with a bacterium, with a flagellar axoneme, or with the flagellate's body; it proba bly occurs also during fixation (Figs. 9 and 10). The extruded contents of the microtoxicyst apparently lyse the bacterium. Usually 2 to 7 microtoxicysts occur in a section of a flagellate; they are situated more or less evenly along the perimeter of the cell body.
Discussion An analysis of the above results shows that the general organization of the cells strain C-45 is similar to that of the genus Cercomonas according to the descriptions made using both light and electron microscope. A number of authors published detailed studies of morphology, feeding, locomotion, and cyst formation in Cercomonas (KRASSILSCHIK 1886; WENYON 1910; Hovxssn 1937; HOLLANDE 1942). All the species of this genus have an amoeboid body, two permanent heterodynamic flagella, and usually an egg-shaped anterior nucleus. The flagellates are thought to move with the help of movements of the directed forward locomotory flagellum. However even KRASSILSCHIK (1886) noted undulating movements of the recurrent flagellum, which was recently confirmed by MIGNOT and BRUGEROLLE (1975a) as well as by the present work. In all species of Cercomonas investigated by now, fine structural studies have shown that the recurrent flagellum is fixed in its proximal part to the cell body. When comparing the strain C-45 with other species of Cercomonas (MIGNOT and BRUGEROLLE 1975a; SCHUSTER and POLLACK 1978), one can see the similarity of their principal cell structures and of the general pattern of organization. The differences concern mostly the body size and the presence of microtoxicysts in the strain C-45. The following characteristic features are shared by Cercomonas and the strain here studied: absence of the kinetoplast and of the paraxial rod in the flagella, tubular cristae in the mitochondria, absence of the system of subpellicular microtubuless ability to form pseudopodia, and presence of the paranuclear body. All these feature, indicate a significant difference between Cercomonas and the kinetoplastids. In
246
A. P.
MYLNIKOV
contrast to the strains studied earlier (MWNOT and BRUGEROLLE 1975a; SCHUSTER and POLLACK 1978), the kinetosomes of the strain C-45 lack a fibrous band fixing them together. In all the three strains, the microtubules proceed backwards as a cone enveloping the nucleus, and a band of 3-4 microtubules emerges the kinetosome. The paranuclear body, which lies behind the nucleus, very much resembles the microbody known in many flagellates (DODGE 1973). In the cytoplasm, there are granules of storage substances which impart a granular appearance to the cytoplasm. The glycocalyx of the strain C-45 is similar to that in other species of Cercomonas. In the organism presently studied we found organelles, the stinging nature of which is established rather firmly. Trichocystlike structures have also been found in the strains studied before-.As supposed by the authors, they serve for production of mucilage during the sliding of the flagellates along the substratum; their discharge was however not observed. Similar organelles occur in many biflagellated colourless flagellates and some other protozoa. They were found in Colponema loxodes (MWNOT and BRUGEROLLE 1975b), Gyromitus limax (SWALE and BELCHER 1975), Heteromita globosa (MACDONALD et al. 1977), Pseudospora sp, (SWALE 1969), and other species. The term microtoxicyst is quite applicable to the stinging organelles of our organism (HAUSMANN 1978), because this minute organelle injects into a bacterium and lyses it. The taxonomic position of the strain C-45 and of other Cercomonas species remains uncertain. The permanent presence of two unequal flagella, the formation of pseudopodia, the structure of mitochondria, the type of the microtubular system, and the presence of trichocyst-like structure closely relate all known species of Cercomonas to Heteromita and Pseudospora; the latter genus has also microbodies. However, they all differ essentially from the typical amoebo-flagellates of the order Schizopyrenida (genera Tetramitus and N aeqleria: SCHUSTER 1963; OUTKA and KLUSS 1967), which have a different system of microtubules, a rhizoplast, peculiar mitochondria, and kinetosomes arranged parallel to each other. The genus Cercomonas may probably be related with the Chrysophyta, where a tendency is found to form pseudopodia and to lose chloroplasts, e.g., in Chrysamoeba radians (HIBBERD 1971). However, the Chrysophyta are characterized by mastigonemes on their flagella, by coiled structures in their axonemes, and by some other features (HIBBERD 1976) not found in Cercomonas. The organisms here considered possess a certain similarity with flagellate and amoeboid stages of development in Mycetozoa. Thus, they have two flagella without mastigonemes, filose pseudopodia, mitochondria with tubular cristae, a microbody, a cone of microtubules enveloping the nucleus (OLIVE 1975). An especially great similarity is found between Cercomonas and the protostelids (HUNG and OLIVE 1973). It is quite possible that Cercomonas and the Mycetozoa originated from a common ancestry.
Literature ANIKUSHIN, N. F. (1978): On structural heterogeneity of colourless freeliving flagellates of the suborder Bodonina Hollande. In: Biology of Inland Waters, Inform. Bull. No. 38: 10-12 (in Russian). DODGE, J. D. (1973): The fine structure of algal cell. London and New York. HAUSMANN, K. (1978): Observations on the extrusive organelles in Protists. Int. Rev. Cytol. 52: 197-276. HIBBERD, D. J. (1971): Observations on the cytology and ultrastructure of Chrysamoeba radians KLEBS (Chrysophyceae). Brit. Phycol. J. 6: 207-224. _ (1976): The ultrastructure and taxonomy of Chrysophyceae and Prymnesiophyceae (Haplophyceae): a survey with some new observations on the ultrastructure of the Chrysophyceae. Bot. J. Linnean Soc. 72: 55-80.
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HOI,(,ANDE, A. (1942) : Etude cy t olo g iq ue d e quelques F'lagelles Iib res, Arch . Zool. E x p er. Gen. 83 : 1- 268. H OVASSE, R. (1937 ): Cont.ribut.ion a I'e t u de de I' ap p a re il d e Golg i de s Flagelles Iibres : L ' ex ist ence d 'un cor ps parabasal ch ez Cercomon as lonqi cauda D UJ . Arch . Zoo l. Exper. Gen. 79: 43- 4 6. H UNo, C. ' Y., and OLIVE, L. S. (197 3) : U ltra structure of th e a moebo id ce lls of P rotostelli u m zona tu m (Mycetozoa) . J. Protozool. 20: 252 -263. KRA SSILs CHIK, 1. ( 1886) : Material s t o th e n atural history a n d taxonomy of flagellates. About Cerco bodo laciniaeqerens nov . gen. et no v . sp . Zap . Novorossyi sk. obsch . estestvoispyt. 2: 2 11- 245 (in R u ssia n ). MACDoNALD , C. 1\1., DARBYSHIRE, J . F ., a nd OGDEN, C. C. (1977) : Th e m orphology of a com m o n so il fl agellate, H etero mi ta glob osd STEIl' (Mast.igophorea : Protozoa). Bull. B r it. Mu s, (Natur. Hist.) Zool. 31 : 255- 264. :l\IrGKOT, J. F. , and B RUOEROLLE, G. (1975 a) : Etude u ltrastru ctura le d e Cer com onas D UJARDIK ( = Cerc obodo KRASSILSCHICK), Protiste f'lag elle. P rot is t ol og ica 11 : 547 -554. - - ( 1975 b) : Etude ul t r a st ruct ur al e du flagel le phagotrophe Colpon ema loxodes STEIN. Protistologi ca 11 : 429-4 44 . O LIVE, L . S. ( 1975) : T h e Mycetoz oan. N ew Y or k. O UTKA, D . E ., a n d KL USS, B. C. ( 1967): The a me b a -t o- f1ag ellate transformat io n in Tetramit u s rostratus , J. Cell. BioI. 35: 323 -:146. Scn cs 'n sn , F . (1963): An electron m icroscope study of t h e amoebofl agellate , Ntteqleria grub eri (S CHARDIKOER). 1. Th e amoeboid and fl agellat e stages. J. Protozool. 10 : 297 -313. S CHUSTER, F . L. , and POLLACK, A . ( 1978): Ultrastr u ct ure of Cercomonas sp., a free-living ameboid fl agellat e . Arch . Pro tist en k d. 120 : 206 -2 12. SWAI,E, E . M. F . (19 69) : The structure of a species of the amoeboflageUate Pseudospora CIENK. Arch . Mierobiol. 67 : 7 1-90. - a nd BELCHER, J . H. ( 1975) : Gyrom ituslimax nov. sp . - Free-living colo u rless a moebo -flagellate. Arch. Prot.is tenkd. 117 : 20- 26. ' YENYON, C. J\1. ( 19 10) : Some observation on flagellates of th e genus Cercom onas , Quart. J. Mi crosc. Sci. 55 : 24 1-260. Z IIUKOV, B. F . (197 1) : K ey to colou r less fr ee-living fl agellates of th e su b or de r B odon ina H oll ande. In : B iol ogy and productivit y of fr eshwater organisms, pp. 241- 284. Leningrad (in Russian) . Author 's address : A. P . J\IYLKIKOV, Laboratory of biology of lower organisms, I ns t it u t e of Biology of I n la n d Waters, USSR Academy of Scien ces , 15274 2 Borok (Y a ro slav l D istrict ), USSR .