I . Protozoal., 26( I), 1979.
p11. 56-(iP 0 1979 by the Souety of I'roio~ot~loqi\ti
Structure of Trichomonads as Revealed by Scanning Electron Microscopy*?
SYNOPSIS. A scanning electron microscope ( S E M ) study of H1,potrichoiiionav acosta (Moskowitz), Trichornonas uaginalis Donne. Pentatrirhornona\ honiini.t (Davaine). and T r i t r i c h o m o n m loetus (Riedmiiller) provided new information about the structure of the periflagellar cnnal: emergence of the flagella froin the cell body: structure of the undulating membrane; and position, shape. and size of the pelta. Of special interest were the spatial relationships of the attached part of the recurrent flagrllum a n d the accessory filariient in Hypotrichornonns and in the members of Trichomonadinae. i.e. Trichomonas and Pentatrirhomonas. Index Key Words: H ~ p i , t i i c i i o ~ i i ~ ~ nrrcosta: ci.\ ?'richo~nonusilaginalis: Pentatricho~non(i.rhorninis ; Tritrichornotzas f o e t u s ; periflzigcllar c;inal: flasella : undulLrting mrnibrane : pelta : scanninq rlectron microscopy.
UC:II
h ; i \ h t ~ y ilearned ; i l ) o i i t \ti'iictui-e of 'Irirhoiiion;ididn, f i n t I ) \ light iiiici-o\(~q>~ ' s e ( ~IZvf. ! I ) and then 1)y trancniission rlectron iiiicroccop\ ' T E J I isre Krfs. 13, ?:I' ' I . Only a fc.\\- rrpnrts, ho\ve\ er. Iiavc, Ixwi t h r t result o f ohsrrvations b y qc,aiinirig c.Iwtrori riiic.rosc.opy II SEA1 and. ;I< far :IS ii-c k n o r t ~ , tiioct of thein \vcre roric.erncd \ \ ith ? ' / i r h o i i t o n ~ . s~ n g i n a l i sDonn6 14, 1 !), 28 . Preuumah1~-bccaust. of tec~hriical difficultit-? the rcwilts obraincd \\ ith this oi-C;iiii\iii \\err not spectacular. T h v 1-c-ccrit 2 rrlativcl!- lo\\ -~ria~iiific~atitrn wanning clcrtronmicrographs o f ?'l-irholl7ito~JJi.\ f c o p s i d i 5 ir sc)\wal tvecks, the exact time depending upon the numbers o f trichomonads found in cultures, the flagellates were used for thr preparation of specimens for SEM. Upon thawing, the strains of the remaining 3 sprcirs werc culti\.ated in T Y M f trypticase-yeast-iiialtose) inediuni ( 5 ) with 5% (\. Iv \, inactivated nornial horse seniin. After 2 serial transfers a t 37 C in T Y M , a t 48-h intervals, t h r strains were passaged twice, ayain c\.ert- 2 days a t 37 C , in the same niediiim, from which the asiir \t-a\ omittcd. Cultures obtained froin tht, 2nd transfer in the lattcr nicdiuni \\ere used for SEM studies. SE.\I .Methods.-In all instances the culturcs ivere ccntrifuged i i t -.XO g for 1 5 niin. T h e resulting pellets wen' fixed in 2% '\. '1.; glutaraldehyde in 0.1 11cacodylate buffer, pH 7.4, with 1%
56
SEM
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(w/v) sucrose. After 4 to 6 h of fixation a t 4 C, the flagellates were washed twice with the cacodylate buffer, then postfixed in 1% (w/v) OsO, in the same buffer for 1.5 h. Thereafter, the specimens were rinsed in several changes of the cacodylate buffer with sucrose. A Unipore ( 1 p m ) Polycarbonate membrane (BioRad Laboratories, Richmond CA) was placed in a Swinny syringe filter head (Millipore). T h e washed cells were then aspirated into a 10-ml syringe. T o this syringe was attached the Swinny filter with the Unipore membrane and the protozoa-containing suspension was expelled from the syringe. The flagellates adhered to the membrane which was then transferred to a special metal holder. This assembly was used during dehydration through a series of graded ethanols and acetone. Next, the material was subjected to the critical-point drying method using the acetone/ CO, system. Subsequently, the cells were coated with platinum and examined in a JEOL SEM-35 scanning electron microscope. RESULTS T o facilitate discussion, the 4 genera, represented by as many yxcies, except for Tritrichomonas foetus the type species, will be discussed separately. Since these species have been described in considerable detail on the basis of light microscopy (10, 16, 18, 20, 26, 30-34) and T E M (2, 11, 12, 24, 27, 29), we shall concern ourselves only with those morphologic feature? the understanding of which was improved with the aid of SEM.
Hypotrichomonas acosta The diameter of the single recurrent and 3 anterior flagella (Figy. 1, 3, 4 ) is as noted previously in living organisms examined by phase-contrast and Nomarski interference contrast optics (Honigberg, unpublished data). As seen in a polar view of the organism, all 4 flagella emerge from the periflagellar canal (Fig. 4 ) . The recurrmt flagellum can be differentiated from the anterior locomotor organelles, which emerge very close to one another, by its direction within and outside the canal (Fig. 4 ) . The wall of the periflagellar canal is reinforced by the pelta seen in scanning electronmicrographs as a cytoplasmic ridge around the canal (Fig. 4 ) . The feeblc undulating membrane appears to consist of the recurrent flagellum and a narrow strip, probably the poorly developed accessory filament (Fig. 2 ) reported by Mattern et al. (Figs. 27-30 in Ref. 24). The posterior end of the undulating
57
membrane is clearly evident in Fig. 2, in which it is indicated by an arrow. The posterior axostylar “projection” of medium diameter is seen in many specimens (Fig. 1 ) . T h e surface of the organism is quite smooth (Figs. 1-4).
Trichomonas vaginalis The diameters of the anterior and recurrent flagella (Figs. 6, 7, 9, 10) correspond to those seen in living organisms ( 10) . The anterior flagella emerge from the periflagellar canal very close to one another (Figs. 5, 7-9). The recurrent flagellum emerges, independently of the anterior flagella, from the dorsal part of the canal (Figs. 7, 8). Within the canal, the former locomotor organelle appears to be separated from the latter flagella by a kind of partition (Fig. 8 ) . The wall of the periflagellar canal is reinforced by a well developed pelta whose outline is seen at the anterior end of the cell (Fig. 8). A comparison of members of a population reveals variability in length of the undulating membrane (Figs. 5-10). The spatial relationships between the recurrent flagellum and the accessory filament in the outer margin of the undulating membrane are illustrated in Figs. 7 and 8. The accessory filament originates very close and ventral to the recurrent flagellum. I t courses posteriad and to the right of, paralleling the flagellum and, in many areas, folding over onto its dorsal surface (Fig. 7 ) . T h e slender and, in some organisms, quite long terminal segment of the axostyle “projects” from the posterior surface of the cell (Figs. 6, 7, 9, 10). I n all urogenital trichomonads of man seen in the course of this study, the cell surface appeared to be rather smooth (Figs. 5-10). The small pits evident in the surface of some of the organisms (Fig. 8) may be openings of pinocytotic canals. Pseudopodia are present in some individuals (Fig. 10).
Pentatrichomonas hominis Except for its possession of an indepmdent flagcllum (the prime generic characteristic) (Figs. 12-14), a somewhat better developed and typically longer undulating nicmbranc (Figs. 11, 12, 15), a free posterior flagellum, and a heavier axostyle, P. hominis, as seen with SEM, is generally similar to Trichomonas vagina& (cf. Figs. 5-10 and 11-14). Pentatrichomonas is unique in the possession of the independent flagellum which leaves the cell ventrally and at some distance
+ All figures are scanning electronmicrographs of trichomonads. Figs. 1-4.[Hypotrichomonas acosta strain 6.1 1. Right and somewhat ventral view. The anterior flagella (a.fl.) emerge from the cell in a bundle-like arrangement. A part of the undulating membrane is evident, as are also the free posterior flagellum (p.fl.) and the terminal segment of the axostyle (ax.) “projecting” beyond the posterior surface of the organism. X 6,270. 2. Posterior part of the dorsal surfarc of an organism seen at a relatively high magnification. I n the feeble and rather short undulating membrane one can resolve the recurrent flagellum (r.fl.) and the poorly-developed accessory filament (ac.f.), coursing ventrally and somewhat to the right of the flagellum. The posterior end of the undulating membrane, coinciding with that of the accessory filament, is marked by an arrow, which indicates also the beginning of the free posterior flagellum. This latter flagellum tends, however, to adhere to the cell surface for some distance beyond the end of the undulating membrane (cf. Fig. 1). x 9,130. 3. Dorsal view. Note the bundle-like arrangement of the proximal parts of the anterior flagella, which appear to be at the end stage of the effective stroke (see Ref. 15). The recurrent flagellum is seen on the dorsal cell surface to the right of the anterior flagella. X 9,280. 4. Top view. The outline of the well developed pelta (pe.) is seen on the wall of the periflagellar canal (P.c.) from which emerge the 3 closely apposed anterior flagella (a.fl.) and, at about a right angle to them, the recurrent flagellum (r.fl.) . x 11,000. Figs. 5-10.Trichomonas vaginalis strain JH34A, dorsaI or nearly dorsal views. In all figures, note the anterior flagella (a.fl. in Figs. 8, 9 ) which emerge from the penflagellar canal closely apposed to one another. The recurrent flagellum (r.fl. in Fig. 8) emerges from the periflagellar canal (P.c. in Fig. 8 ) posterior and dorsal to the anterior locomotor organelles (Figs. 5 - 9 ) , the area of its emergence being evident most clearly in Figs. 7 and 8. The area of emergence of the recurrent flagellum appears to be separated by a narrow strip of cytoplasm from the major part of the periflagellar canal from which emerge the 4 anterior flagella (Figs. 7, 8 ) . The wall of the periflagellar canal is reinforced by the pelta, the outline of which is seen best in Fig. 8. The outer margin of the undulating membrane, which varies in length among members of a population (cf. Figs. 5, 7, 8 ) , consists of the recurrent flagellum (r.fl. in Fig. 8 ) and the accessory filament (ac.f. in Figs. 7, 8) ; details of the relationships of the 2 structures are shown in Figs. 7 and 8. Note the pseudopod (ps.) extending from the surface of the flagellate (Fig, 10). Fig. 5 , x 3,000; Fig. 6, x 4,720; Fig. 7, X 9,600; Fig. 8, X 12,600; Fig. 9, x 4,640; Fig. 10, X 5,800.
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59
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from the periflagellar canal (Fig. 1 4 ; . Its arca of emergence is 180” anterior to the ventral margin of the prlta and a t from the rec‘urrrnt flagellum (Fig. 1 4 ) . In some specimens, one can s w the apparrntly some\\ hat pointed endings of the anterior flagella (Fig. 1 3 ) . T h e rrxcurrrnt flagclluni leaves thc cell posterior and dorsal to the 4 typical anterior flagella, and there is a strip of cytoplasm scparating the arcas of cnicrgencc of the former and the latter locomotor organellcs (Figs. 11, 1 4 ) . I t seems that the recurrent flagellum actually emerges outside the pcriflagcllar canal (Figs. 11, 14;. T h e outline of the c r t w r n t h p e d pelta is evident around thc ventral part of the c m a l (Fig. 1 1 , to the observer‘s left i. T h r accessory filament emergc.s from the ccll ventral to the recurrent flagcllum Figs. 11, 1-1-j. ?’he filament courses parallel and to the right of this flagellum, in man)- areas folding over onto its dorsal surface (Figs. 11, 15;. Nvar the posterior end of the organism, the recurrent flagellum, without the accessoIy filament, e s t m d s for some distance as the frec posterior flapcllum (Fig. 15 1. A rrlativclp heavy terminal scpment of the axost!-le “projects“ h y o n d the posterior body surface (Fig. 1 2 ;. T h e external surface of thc trichonionad \\-as smooth in the preponderant majority of specimens cxatninvd during this study (Figs. 12, 14, 1 5 ) . T h c slight creasing: of the surface in the organism shotvn in Fig. 11 may \\ell It? a n artifact.
l l a n y featurcs of ?‘i-itiicho,noncir foetus, a Tr-itrichotnonns aupustn-type organism is);, art’ secw i i i Fig. 16. Thc actual length (of thc almost fully extended anterior flagrlla and that of the fully extended frec postcricir flagellum, % longtar than the anterior ones, are evident. T h e periflagellar canal from \vhich c*niclrgethc atiterinr flagc~lla.thc \\.ell developed undulatiny mem-
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bratic, and the short “projecting” terminal segment of the axostyle can also be observed. T h e details of the anterior end of the trichomonad can be discerned in Fig. 17. From the periflagellar canal, of a relatively small diameter, the 3 anterior flagella appear to emerge in a bundle. T h e wall of the canal is reinforced by the pelta whose outlincs are cvident in scanning electronmicrographs (see also Fig. 16 j . T h e recurrent flagellum emerges from a n opening that pierces the \\.all of the canal, a t a right angle a n d dorsal to the anterior flagella. T h e former locomotor organelle forms the outer margin ( a in Fig. 1 7 ) of the well developed undulating membrane. T h e cord, coursing directly under and parallel to the recurrent flagellum, probably is the distal marginal lamella (b in Fig. 1 7 ) , which, together with the flagellum, constitutes the distal part of the undulating membrane. Finally, the proximal component of the membrane ( c in Fig. 17) corresponds to the proximal part of thc undulating membrane as identified with the aid of TEM ( 1 2 ) . Since the identification of all the parts of the undulating membrane is based on previous TEM observations ( I S ) , the details of this organelle depicted in Fig. 17 should he compared with those presented in Figs. 1, 2, 7, 31, 40 and 41 in Ref. 12. T h e posterior end of the undulating membrane a n d the proximal part of the frre posterior flagellum are shown in Fig. 18. I n thii figure, the short conical “projecting” part of the axostyle is alw evident. At the end of the projection there is a small spherical \tructure (Fig. 181, Lvhich r a n also be seen in other individuals (Fig. 1 6 ) . T h e possible nature of this structure will be considered in Discussion. T h e surface of the organism is generally sniooth (Figs. 16-18). The sniall pits seen in the surface of some cells (Fig. 1 7 ) may rcprvsent openings of pinocytotic canals.
c Figs. 11-15. [Pentatrichornonus hominis strain Hs3: S I H . ] 11. Dorsal view. T h e typical 4 anterior flagella (a.fl.) emerge from the periflagellar canal. The recurrent flagellum (r,fl.) emerges from the cell dorsal to the anterior flagella, evidently a short distance posterior to the periflagellar canal. T h e accessory filament (ac.f.) is seen emerghg beneath and to the right of the recurrent flagellum (r.fl.), which it parallels for the entire length of the membrane. x 13,500. 12. Right view. T h e typical 4 anterior flagella (a.fl.), independent flagellum (i.fl,); undulating membrane, as well as the quite hea\y terminal “projecting” segment of the axostyle (ax.) are visible. The posterior end of the accessory filament, which coincides with the posterior end of the undulating membrane, is indicated by a n arrowhead. [he recurrent flagellum extends beyond the end of the undulating membrane as a free poster~orflagellum(p.fl.). x 9,120. 13. From the anterior end of the body emerge the 4 typical anterior flagella, with apparently somewhat pointed ends. T h e independent flagellum ( i f l . ) emerges some distance from the other locomotor organelles. x 8,000. 14. T o p view. T h e areas of emergence of all 6 flagella are seen in this unique electronmicrograph. Note the periflagellar canal (P.c.) filled with the proximal parts of the anterior flagella. T h e recurrent flagellum (r,fl,) emerges somewhat posterior and dorsal to the canal ( t o the observer’s right) ; the accessory filament (ac.f.) is seen to the right of the flagellum. The outline of the pelta ( p e . ) is clearly evident on the ventral side (to the observer’s left) of the organism, its dorsal (upper) margin constituting the rim of the opening of the periflagellar canal. The independent flagellum (Lfl.) emerges ventral to the i>eIta a t -180” to the recurrent flagellum. x 18,600. 15. Terminal part of the undulating membrane. Note the spatial relationships of the recurrent flagellum (r.fl.) and the arcessory filament (ac.f.) whose posterior end coincides with that of the undulating membrane. The recurrent flagellum continues beyond the end of the membrane as a free posterior flagellum (p.fI.). T h e end of the projecting part of the axostyle (ax. 1 is also evident. x 12,750. ~
Figs. 16-18. [Tritrichomonus foetus strain KFT-l.1 16. Left and somewhat dorsal view of a n entire organism. Nearly all the structures discernible with SEM are evident in this preparation. S o t e the 3 subequal anterior flagella (a.fl.) emerging from the periflagellar canal (cf. Fig. 17); the wall of which is reinforced by the pelta ( p e . ) . T h e well developed undulating membrane (u.m.) originates posterior and dorsal to the anterior flagella coursing counterrlockwise from the anterior left toward the posterior right surface of the organism. The recurrent flagellum continues beyond the posterior end of the membrane as a long free posterior flagellum (p.fl.) (cf. Fig. 18). The coniral, short terminal segment (ax.) of the axostylar trunk “projects” from the posterior body surface. Note the “furry” or velvet-like appearance of the cell surface. x 4,000. 17. Anterior part of an organism in a left-dorsal view. The anterior flagella (a.fl.) emerge from well-defined perifagellar canal ( P . c . ) , in the wall of which is s-en the clearly defined outline of the pelta ( p e . ) . Note the lip-like projection of the pelta (arrow) whirh appears to mark its dorsal-right part. T h e recurrent flagellum (a),constituting the dorsal cord of the distal part of the undulating membrane, emerges from the body through a n opening in the wall of the periflagellar canal. A narrower structure ( b ) , which appears to correspond to the distal niar+nal lamella, described from transmission electronmicrographs (12), is seen roursinx between the rerurrent flagellum and a proximal thickening of the membrane ( c ) . This latter thickening, about as wide as the flagellurn. s e e m t o correspond to the proximal part of the undulating membrane as seen by T E M . T h e small depressions (arrowheads) in the flagellate‘s surface may be openings of pinocytotic canals. X 10,450. 18. Posterior part of an organism. T h e posterior end of the undulating membranc (arrow) coincides with the end of the proximal components of this organelle. T h e recurrent flagellum continues beyond thr end of thr inrrnhrane as a free posterior flagellum (p.fl.). The rime-like trrniinal segment of the axostyle (ax.), covered by the cell mcinbrarir. has :I sphcrical encliuq (for explanation of this structure. sec Discussion). T h e surfarc of the organism is smooth and has a “velvety” appi’arancr. ,X 9.000.
SEM
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DISCUSSION The picture seen with the aid of SEM is quite similar to that revealed by bright-field, phase contrast, and especially Nomarski interference optics. However, some features that cannot be resolved with certainty or actually are beyond the resolving power of the light microscope are brought out through the employment of SEM. It is those features that have been emphasized in the Results. The scanning electronmicrographs of the 4 species when viewed against the background of information derived from light microscopy (10, 16, 18, 20, 26, 30-34), and TEM (2, 11, 12, 24, 2 7 ) , provide additional insight into the actual appearance of, and spatial relationships among various, primarily surface, structures of these organisms. Thus it becomes apparent that there exists a certain sequence in the relationships between the emergence of the anterior and recurrent flagella from the cell which seems to be consistent with the evolutionary scheme of Trichomonadida elaborated by Honigberg ( 9 ) , after Kirby ( 1 7 ) , and subsequently accepted by many other workers, e.g. Brugerolle ( 3 ) or Levine ( 2 1 ) . In the representative species, Hypotrichomonas acosta, of the most primitive genus included in the present study, the recurrent flagellum emerges close to the anterior flagella from a common periflagellar canal; it can be differentiated from the latter organelles primarily by its direction both within and outside the canal (Fig. 4 ) . One can surmise, and we hope to confirm this assumption in future studies, that a similar situation obtains in the more primitive Monocercomonas, thought to be the basic genus of the order Trichomonadida (9, 1 7 ) . I n Trichomonas vaginalis, an organism belonging to the subfamily Trichomonadinae (the more primitive subgroup of the family Trichomonadidae) , the recurrent flagellum, aIthough it still emerges from the periflagellar canal, is separated, within the canal, from the anterior locomotor organelles by a strip of cytoplasm (Fig. 8 ) . One could have anticipated that a similar situation would prevail in P. hominis, a member of a genus apparently quite close to Trichomonas. Yet here the area of emergence of the recurrent flagellum is even more clearly separated from that of the anterior flagella, being some distance posterior to the latter, and probably outside the periflagellar canal (Figs. 11, 14). It would be of interest to ascertain if the situation in Tetrati-ichomonas, a genus, unlike Trichomonas, assumed to belong on the main evolutionary line of Trichomonadine and probably more closely related to Pentatrichomonas, is more similar to that found in the latter. If this relationship between the anterior and recurrent flagellum in Tetratrichomonas were more like that in Trichomonas, then in this characteristic Pcntatrichomonas would have to be considered a “more advanced” genus. I n Tritrichomonas foetus, a member of the presumably more advanced subfamily Tritrichomonadinae of Trichornonadidac ( 9 ) , the recurrent flagellum emerges from the area of the periflagellar canal through an opening piercing the left-dorsal part of the canal wall (Fig. 17). T h e foregoing sequence of events could not be seen by light microscopy and could perhaps be reconstructed by T E M only through the use of serial sections. Of special interest is the appearance of the poorly developed undulating membrane in H . acosta (Fig. 2 ) . Indeed, the entire membrane can be differentiated from the unattached Dart of the recurrent flagellum, i.e. the free posterior flagellum, only by the presence of the narrow accessory filament seen to the right of the attached segment of the recurrent flagellum. Clearly, an SEM and TEM study of a species of Monocercomonas with a relatively long proximal part of the recurrent flagellum attached to the body surface, such as M . moskowitzi Honigberg, would be of great
61
interest with regard to the morphologic evolution of the undulating membrane among trichomonads. Among the important observations of Trichornonadidac brought out by the present SEM study was the surface structure of the accessory filament and its spatial relationship to the attached part of the recurrent flagellum in the outer margin of the undulating membrane. Some light microscopists thought that the accessory filament was external (distal) to the rccurrerit flagellum in Trichomonas (32, 33) and Pentatrichffmonas (16, 31-33), and this interpretation was found, by TEM and now with SEM, to be more nearly correct than the view expressed on several occasions by the junior author (e.g. 7, 8 ) that in certain Trichomonadinae the recurrent flagellum was external to the filament. I t was demonstrated by TEM (1, 11, 13, 23, 2 7 ) that the accessory filament courses to one side of the flagellum projecting dorsally beyond its surface (Figs. 21, 22 in Ref. 11; Fig. 40 in Ref. 23; Fig. I I b in Ref. 27). The spatial relationship between these organelles can be visualized best with the aid of SEM (Figs. 7, 8, 11, 14, 15 in the present report). Apparently, the accessory filament emerges from the cell to the right of, and somewhat posterior to the area of emergence of the recurrent flagellum. The filament, seen as a fold in transmission electronmicrographs (1, 11, 13, 23, 2 7 ) , is more cordlike when viewed in SEM. I t parallels the recurrent flagellum for the length of the flagellum’s attachment to the cell body, coursing to the right of this locomotor organelle and, in many areas, folding over onto its surface. As might have been anticipated from light-microscopic (18, 3 4 ) , and T E M (12, 29) studies, the structure of the undulating membrane of Tritrichomonas foetus, when observed in SEM, differs greatly from that characteristic of Trichonionadinae. Indeed, in Tritrichomonas foetus and in all remaining species of the subfamily Tritrichomonadinae Honigberg, 1963, emend. Brugerolle, 1976 ( 3 ) , the recurrent flagellum, enclosed in a cytoplasmic cord, occupies the distal (dorsal) position (e.g. Figs. 1, 2, 31 in Ref. 1 2 ) . I n scanning electronmicrographs (Fig. 17, present report), the following component parts, going from the distal toward the proximal area, discernible in the anterior scgment of the undulating membrane, probably correspond to the expanded area of the distal part of the membrane that contains the axoneme of the recurrent flagellum, to the distal marginal lamella, and to the entire proximal part of the undulating mcmbrane (cf. Fig. 1 in Ref. 12, and Fig. 17, present report). The position and the area of emergence of the indcpcndent flagellum in P. hominis, as seen in SEM, serrn to be consistent with the view based on studies of the division process in trichomonads (2, 2 2 ) that it may represent a precocious recurrent flagellum in this genus. The role played by the pelta in supporting the M-all of the periflagellar canal, as suggested by Mattern et al. (23) on thc basis of TEM observations, can be gleaned also from the various views of this crescent-shaped organelle whose outlines arc sren in the scanning electronmicrographs of the 4 trichornonad spvcies examined during the present study and shown in Figs. 4, 8, 14, and 17. As has been pointed out previously (9),when studied in protargol-stained preparations by light microscopy, Tritrichomonadinae appear to have poorly developed pcltas. I t is cvident, however, from T E M observations (3, 4, 12) that, althoiigh smaller in relation to the total body size, the peltas in the members of this subfamily are well developed. This vicw was fully confirmed by our SEM study (Fig. 1 7 ) . In their shape, size, and length, the “projecting” parts of the axostyle seen in the trichomonad species in the course of the present SEM study correspond quite closely to their descriptions from living (10, 15) and fixed and stained specimcns (10, 16, 18,
20, 26, 30-343. Of wnie intcvqt is the spherical ending of t h r conical axostylar projection noted in sonic trichomonads (Fig. 18 in the present s t u d y ) . I t is known that oftcn the trichomonads attach to the substratc~by nirans of thin c) toplasmic threads extending from the posterior cnd of the axostylar trunk (6; Honigberg, unpublished obsrrvariori.;'~. \Shen an organism dvrachcs frotn the substrate, the cytoplasmic. rstcmsion is rrtractrd toward the axostylar trunk. I t is likely that the spherical structures seen at the end of thr trunks in scanning electronmicrographs are thc filamcntous extensions that have bern I-etracted after thc cell dctarhcd itself from the substrate. Studies of other trichonionads \vhich appcar to occupy crucial positions in the evolution of the order should throw additional light on the kinships among various qcnera and species. Such investigations \vould aid also in a twtter understanding of spatial relationships of surfacr structures in tlic\e f1a~:c~llates. LITER.4TYRE C I T E D 1. Brugerolle G. 197 1. Ultrastructure du genre TrichomituJ Swezy 1915. Zooflagellata, Trichomonadida. Protistologica 7. 1 7 1-6. 2. ___ 1975. Etude de la cryptopleuromitose et de la morphogtnkse de division chez Trichomonas caginalzs et chez plusieurs genres de Trichonionadines primitives. Protistologica 11. 457-68. 3. __1976. Contribution ci l'itude cytologique des Protozoaires Zooflagelle's parasite.t: Proteromonadida, Retortamonadida, Diplomonadida, Oxymonadida, Trichonionndidu. Thesis presented for the degree of Doctcir of Natural Sciences. University of Clermont, Clermont-Ferrand, France. 246 pp. 4. Daniel WA, Mattern CFT. Honigberg BM. 1971. Fine structure of the mastigont system of Tritrichomona.\ n i u r i s (Grassi) . J. Protozool. 18. 575-86. 5. Diamond LS. 1957. T h e establishment of various trichomonads o f animals and man in axenic cultures. J. Parasitol. 43. 488-90. 6. Hogue MJ. 1947. The behavior of Trichomonas caginalis in tissue cultures: .4movie. 1.Parasitol. 33. 199-200. 7. Honigberg BM. 1951. Structure and morphogenesis of Trichomonas prou:azeki Alexeieff and Trichomonas brumpti Alexeieff. L'niu. Calif. (Berkeley) Publ. Zool. 5 5 , 337-94. 8. 1953. Structurc. taxonomir status, and host list of Tritrichomona.r batrachorum (Perty). J. Parasitol. 39. 191-208. 9. 1963. Evolutionary and systematic relationships in thc flagellate order Trichomonadida Kirby. J. Protozool. 10. 20-63. 10. _ _ . King \'M. 1964. Structure of Trichomonas r,aginalis Donne. J. Paratitol. 50, 345-64. 11. . Mattern CFT. Daniel iV.4. 1968. Structure of PentatrichomonaJ honiinis (Davaine I as revealcd by electron microscopy. ]. Protozool. 15, 419-30. 12, _ _ ~ 1971. Fine structure of the mastigont system in Tritrichomonoj f o e t w (Riedmiiller). J. Prolozoo!. 18. 183-98. 13. , Daniel iV.4, Mattern CFT. 1972. Fine structure of Trichomifut bntrachorum (Pertyi. J. Plotozool. 19. 446-53. ~
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~
~
I t . Kazanowska W, Jodczyk KJ, Kuczyliska K , Karpowicz MJ. 1977. Morphological variability of T . vaginalis in vivo and in vitro. It'iad. Parazytol. 23, 473-80. 15. Kirby H. 1943. Observations on a tric.homonad from the intestine of man. J. Parasitol. 29, 422-3. 16. ___- 1945. Th e structure of the common intestinal trichomonad of man. J. Parasitol. 31, 163-75. 1 7 . --~ 1947. Flagellate and host relationships of Trichomonad flagellates. J. Parasitol. 33, 2 14-28. 111. __ 195 1 . Observations of the trichomonad flagellate of the reproductive organs of cattle. J. Parasitol. 37, 445-59. 19. Kurnatowska A, Hajdukiewicz G. 1977. Trichomonas ;,aginalis variability in axenic cultures evaluated in scanning microscope. M'iad. Parazytol. 23, 481-7 (in Polish, English summary). 20. Lee JJ. 1960. Hypotrichomonas acosta (Moskowitz) gen. nov. from reptiles. I. Structure and division. J . Protozool. 7, 393-401. 21. Levine ND. 1973. Protozoan Parasites of Domestic Anim a l s and of M a n . 2nd ed., Burgess, Minneapolis. 22. Mattern CF T. 1973. Ultrastructmal events in the process of morphogenesis in trichomonads, in de Puytorac P., Grain J., eds.. Progress in Protozoology, Proc. IV. Int. Cong. Protozool., Clcrmcnt-Ferrand, 1973, Univ. Clermont Press, p. 273. 23. ___ . Honigberg BM, Daniel WA. 1967. The mastigont system of Trichomonas gallinae (Rivolta) as revealed by electron microscopy. J. Protozool. 14, 320-39. 24. ___ . Daniel WA, Honigberg BM. 1969. Structure of H1,potrichomonas acosta (Moskowitz) (Monocercomonadidae, Trichomonadida) as revealed by electron microscopy. J. Protozool. 16. 668-85. 25, McEntegart MG. 1952. Th e application of haemagglutination technique to the study of Trichomonas uaginalis infections. J. Clin. Pathol. 5: 275-80. 26. Moskowitz N. 1951. Observations on some intestinal flag,-llates from reptilian hosts (Squamata). J. Morphol. 89, 257-322. 27. Nielsen MH. Ludvik J, Nielsen R . 1966. O n the ultrastructure o f Trichomonas uaginalis Donne. ]. Microsc. 5, 229-50. 28. OvEinnikov NM, Delektorskij VV, Turanova EN, Yashkova G S . 1975. Further studies of Trichomonas uaginalis with transmission and scanning electron microscopy. Br. ]. Vener. Dis. 51, 1 57-75 .,-.
29. Simpson CF, White F H . 1964. Structure of Trichomonas foetut a s revealed by electron microscopy. Am. J. Vet. Res. 25, t315-74 .,- - - .. 30. LVenrich DH. 1939. Th e morphology of Trichomonac t,aginalis. V o l . J u b . Sadao Yoshida 2, 65-76. 31. ___ 1944. Morphology of the intestinal trichomonad flagellates in man and of similar forms in monkeys, cats, dogs and rats. J. Morphol. 74, 189-21 I . 32. 1944. Comparative morphology of the trichomonad flagellates of man. A m . J . Trop. M e d . H y g . 24, 39-51. :33. 1947. Th e species of Trichomonas in man. J. Paratitol. 33. 177-88. 34. ~- . Emmerson MA. 1933. Studies on the morphology of Tritrichomonas foetus (Riedmiiller) (Protozoa, Flagellata) from .\merican cows. J. Morphol. 55, 193-206. 35. Yamin MA. 1979. The symbiotic flagellates Strebloniastix. Trichornitopsis, Trichonympha from the termite Zootermopsis: .4scanning electron microscope study. Trans. A m . Microsc. S O C .98. in press. ~
~
p11. 56-(iP 0 1979 by the Souety of I'roio~ot~loqi\ti
Structure of Trichomonads as Revealed by Scanning Electron Microscopy*?
SYNOPSIS. A scanning electron microscope ( S E M ) study of H1,potrichoiiionav acosta (Moskowitz), Trichornonas uaginalis Donne. Pentatrirhornona\ honiini.t (Davaine). and T r i t r i c h o m o n m loetus (Riedmiiller) provided new information about the structure of the periflagellar cnnal: emergence of the flagella froin the cell body: structure of the undulating membrane; and position, shape. and size of the pelta. Of special interest were the spatial relationships of the attached part of the recurrent flagrllum a n d the accessory filariient in Hypotrichornonns and in the members of Trichomonadinae. i.e. Trichomonas and Pentatrirhomonas. Index Key Words: H ~ p i , t i i c i i o ~ i i ~ ~ nrrcosta: ci.\ ?'richo~nonusilaginalis: Pentatricho~non(i.rhorninis ; Tritrichornotzas f o e t u s ; periflzigcllar c;inal: flasella : undulLrting mrnibrane : pelta : scanninq rlectron microscopy.
UC:II
h ; i \ h t ~ y ilearned ; i l ) o i i t \ti'iictui-e of 'Irirhoiiion;ididn, f i n t I ) \ light iiiici-o\(~q>~ ' s e ( ~IZvf. ! I ) and then 1)y trancniission rlectron iiiicroccop\ ' T E J I isre Krfs. 13, ?:I' ' I . Only a fc.\\- rrpnrts, ho\ve\ er. Iiavc, Ixwi t h r t result o f ohsrrvations b y qc,aiinirig c.Iwtrori riiic.rosc.opy II SEA1 and. ;I< far :IS ii-c k n o r t ~ , tiioct of thein \vcre roric.erncd \ \ ith ? ' / i r h o i i t o n ~ . s~ n g i n a l i sDonn6 14, 1 !), 28 . Preuumah1~-bccaust. of tec~hriical difficultit-? the rcwilts obraincd \\ ith this oi-C;iiii\iii \\err not spectacular. T h v 1-c-ccrit 2 rrlativcl!- lo\\ -~ria~iiific~atitrn wanning clcrtronmicrographs o f ?'l-irholl7ito~JJi.\ f c o p s i d i 5 ir sc)\wal tvecks, the exact time depending upon the numbers o f trichomonads found in cultures, the flagellates were used for thr preparation of specimens for SEM. Upon thawing, the strains of the remaining 3 sprcirs werc culti\.ated in T Y M f trypticase-yeast-iiialtose) inediuni ( 5 ) with 5% (\. Iv \, inactivated nornial horse seniin. After 2 serial transfers a t 37 C in T Y M , a t 48-h intervals, t h r strains were passaged twice, ayain c\.ert- 2 days a t 37 C , in the same niediiim, from which the asiir \t-a\ omittcd. Cultures obtained froin tht, 2nd transfer in the lattcr nicdiuni \\ere used for SEM studies. SE.\I .Methods.-In all instances the culturcs ivere ccntrifuged i i t -.XO g for 1 5 niin. T h e resulting pellets wen' fixed in 2% '\. '1.; glutaraldehyde in 0.1 11cacodylate buffer, pH 7.4, with 1%
56
SEM
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(w/v) sucrose. After 4 to 6 h of fixation a t 4 C, the flagellates were washed twice with the cacodylate buffer, then postfixed in 1% (w/v) OsO, in the same buffer for 1.5 h. Thereafter, the specimens were rinsed in several changes of the cacodylate buffer with sucrose. A Unipore ( 1 p m ) Polycarbonate membrane (BioRad Laboratories, Richmond CA) was placed in a Swinny syringe filter head (Millipore). T h e washed cells were then aspirated into a 10-ml syringe. T o this syringe was attached the Swinny filter with the Unipore membrane and the protozoa-containing suspension was expelled from the syringe. The flagellates adhered to the membrane which was then transferred to a special metal holder. This assembly was used during dehydration through a series of graded ethanols and acetone. Next, the material was subjected to the critical-point drying method using the acetone/ CO, system. Subsequently, the cells were coated with platinum and examined in a JEOL SEM-35 scanning electron microscope. RESULTS T o facilitate discussion, the 4 genera, represented by as many yxcies, except for Tritrichomonas foetus the type species, will be discussed separately. Since these species have been described in considerable detail on the basis of light microscopy (10, 16, 18, 20, 26, 30-34) and T E M (2, 11, 12, 24, 27, 29), we shall concern ourselves only with those morphologic feature? the understanding of which was improved with the aid of SEM.
Hypotrichomonas acosta The diameter of the single recurrent and 3 anterior flagella (Figy. 1, 3, 4 ) is as noted previously in living organisms examined by phase-contrast and Nomarski interference contrast optics (Honigberg, unpublished data). As seen in a polar view of the organism, all 4 flagella emerge from the periflagellar canal (Fig. 4 ) . The recurrmt flagellum can be differentiated from the anterior locomotor organelles, which emerge very close to one another, by its direction within and outside the canal (Fig. 4 ) . The wall of the periflagellar canal is reinforced by the pelta seen in scanning electronmicrographs as a cytoplasmic ridge around the canal (Fig. 4 ) . The feeblc undulating membrane appears to consist of the recurrent flagellum and a narrow strip, probably the poorly developed accessory filament (Fig. 2 ) reported by Mattern et al. (Figs. 27-30 in Ref. 24). The posterior end of the undulating
57
membrane is clearly evident in Fig. 2, in which it is indicated by an arrow. The posterior axostylar “projection” of medium diameter is seen in many specimens (Fig. 1 ) . T h e surface of the organism is quite smooth (Figs. 1-4).
Trichomonas vaginalis The diameters of the anterior and recurrent flagella (Figs. 6, 7, 9, 10) correspond to those seen in living organisms ( 10) . The anterior flagella emerge from the periflagellar canal very close to one another (Figs. 5, 7-9). The recurrent flagellum emerges, independently of the anterior flagella, from the dorsal part of the canal (Figs. 7, 8). Within the canal, the former locomotor organelle appears to be separated from the latter flagella by a kind of partition (Fig. 8 ) . The wall of the periflagellar canal is reinforced by a well developed pelta whose outline is seen at the anterior end of the cell (Fig. 8). A comparison of members of a population reveals variability in length of the undulating membrane (Figs. 5-10). The spatial relationships between the recurrent flagellum and the accessory filament in the outer margin of the undulating membrane are illustrated in Figs. 7 and 8. The accessory filament originates very close and ventral to the recurrent flagellum. I t courses posteriad and to the right of, paralleling the flagellum and, in many areas, folding over onto its dorsal surface (Fig. 7 ) . T h e slender and, in some organisms, quite long terminal segment of the axostyle “projects” from the posterior surface of the cell (Figs. 6, 7, 9, 10). I n all urogenital trichomonads of man seen in the course of this study, the cell surface appeared to be rather smooth (Figs. 5-10). The small pits evident in the surface of some of the organisms (Fig. 8) may be openings of pinocytotic canals. Pseudopodia are present in some individuals (Fig. 10).
Pentatrichomonas hominis Except for its possession of an indepmdent flagcllum (the prime generic characteristic) (Figs. 12-14), a somewhat better developed and typically longer undulating nicmbranc (Figs. 11, 12, 15), a free posterior flagellum, and a heavier axostyle, P. hominis, as seen with SEM, is generally similar to Trichomonas vagina& (cf. Figs. 5-10 and 11-14). Pentatrichomonas is unique in the possession of the independent flagellum which leaves the cell ventrally and at some distance
+ All figures are scanning electronmicrographs of trichomonads. Figs. 1-4.[Hypotrichomonas acosta strain 6.1 1. Right and somewhat ventral view. The anterior flagella (a.fl.) emerge from the cell in a bundle-like arrangement. A part of the undulating membrane is evident, as are also the free posterior flagellum (p.fl.) and the terminal segment of the axostyle (ax.) “projecting” beyond the posterior surface of the organism. X 6,270. 2. Posterior part of the dorsal surfarc of an organism seen at a relatively high magnification. I n the feeble and rather short undulating membrane one can resolve the recurrent flagellum (r.fl.) and the poorly-developed accessory filament (ac.f.), coursing ventrally and somewhat to the right of the flagellum. The posterior end of the undulating membrane, coinciding with that of the accessory filament, is marked by an arrow, which indicates also the beginning of the free posterior flagellum. This latter flagellum tends, however, to adhere to the cell surface for some distance beyond the end of the undulating membrane (cf. Fig. 1). x 9,130. 3. Dorsal view. Note the bundle-like arrangement of the proximal parts of the anterior flagella, which appear to be at the end stage of the effective stroke (see Ref. 15). The recurrent flagellum is seen on the dorsal cell surface to the right of the anterior flagella. X 9,280. 4. Top view. The outline of the well developed pelta (pe.) is seen on the wall of the periflagellar canal (P.c.) from which emerge the 3 closely apposed anterior flagella (a.fl.) and, at about a right angle to them, the recurrent flagellum (r.fl.) . x 11,000. Figs. 5-10.Trichomonas vaginalis strain JH34A, dorsaI or nearly dorsal views. In all figures, note the anterior flagella (a.fl. in Figs. 8, 9 ) which emerge from the penflagellar canal closely apposed to one another. The recurrent flagellum (r.fl. in Fig. 8) emerges from the periflagellar canal (P.c. in Fig. 8 ) posterior and dorsal to the anterior locomotor organelles (Figs. 5 - 9 ) , the area of its emergence being evident most clearly in Figs. 7 and 8. The area of emergence of the recurrent flagellum appears to be separated by a narrow strip of cytoplasm from the major part of the periflagellar canal from which emerge the 4 anterior flagella (Figs. 7, 8 ) . The wall of the periflagellar canal is reinforced by the pelta, the outline of which is seen best in Fig. 8. The outer margin of the undulating membrane, which varies in length among members of a population (cf. Figs. 5, 7, 8 ) , consists of the recurrent flagellum (r.fl. in Fig. 8 ) and the accessory filament (ac.f. in Figs. 7, 8) ; details of the relationships of the 2 structures are shown in Figs. 7 and 8. Note the pseudopod (ps.) extending from the surface of the flagellate (Fig, 10). Fig. 5 , x 3,000; Fig. 6, x 4,720; Fig. 7, X 9,600; Fig. 8, X 12,600; Fig. 9, x 4,640; Fig. 10, X 5,800.
58
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59
-
from the periflagellar canal (Fig. 1 4 ; . Its arca of emergence is 180” anterior to the ventral margin of the prlta and a t from the rec‘urrrnt flagellum (Fig. 1 4 ) . In some specimens, one can s w the apparrntly some\\ hat pointed endings of the anterior flagella (Fig. 1 3 ) . T h e rrxcurrrnt flagclluni leaves thc cell posterior and dorsal to the 4 typical anterior flagella, and there is a strip of cytoplasm scparating the arcas of cnicrgencc of the former and the latter locomotor organellcs (Figs. 11, 1 4 ) . I t seems that the recurrent flagellum actually emerges outside the pcriflagcllar canal (Figs. 11, 14;. T h e outline of the c r t w r n t h p e d pelta is evident around thc ventral part of the c m a l (Fig. 1 1 , to the observer‘s left i. T h r accessory filament emergc.s from the ccll ventral to the recurrent flagcllum Figs. 11, 1-1-j. ?’he filament courses parallel and to the right of this flagellum, in man)- areas folding over onto its dorsal surface (Figs. 11, 15;. Nvar the posterior end of the organism, the recurrent flagellum, without the accessoIy filament, e s t m d s for some distance as the frec posterior flapcllum (Fig. 15 1. A rrlativclp heavy terminal scpment of the axost!-le “projects“ h y o n d the posterior body surface (Fig. 1 2 ;. T h e external surface of thc trichonionad \\-as smooth in the preponderant majority of specimens cxatninvd during this study (Figs. 12, 14, 1 5 ) . T h c slight creasing: of the surface in the organism shotvn in Fig. 11 may \\ell It? a n artifact.
l l a n y featurcs of ?‘i-itiicho,noncir foetus, a Tr-itrichotnonns aupustn-type organism is);, art’ secw i i i Fig. 16. Thc actual length (of thc almost fully extended anterior flagrlla and that of the fully extended frec postcricir flagellum, % longtar than the anterior ones, are evident. T h e periflagellar canal from \vhich c*niclrgethc atiterinr flagc~lla.thc \\.ell developed undulatiny mem-
-
bratic, and the short “projecting” terminal segment of the axostyle can also be observed. T h e details of the anterior end of the trichomonad can be discerned in Fig. 17. From the periflagellar canal, of a relatively small diameter, the 3 anterior flagella appear to emerge in a bundle. T h e wall of the canal is reinforced by the pelta whose outlincs are cvident in scanning electronmicrographs (see also Fig. 16 j . T h e recurrent flagellum emerges from a n opening that pierces the \\.all of the canal, a t a right angle a n d dorsal to the anterior flagella. T h e former locomotor organelle forms the outer margin ( a in Fig. 1 7 ) of the well developed undulating membrane. T h e cord, coursing directly under and parallel to the recurrent flagellum, probably is the distal marginal lamella (b in Fig. 1 7 ) , which, together with the flagellum, constitutes the distal part of the undulating membrane. Finally, the proximal component of the membrane ( c in Fig. 17) corresponds to the proximal part of thc undulating membrane as identified with the aid of TEM ( 1 2 ) . Since the identification of all the parts of the undulating membrane is based on previous TEM observations ( I S ) , the details of this organelle depicted in Fig. 17 should he compared with those presented in Figs. 1, 2, 7, 31, 40 and 41 in Ref. 12. T h e posterior end of the undulating membrane a n d the proximal part of the frre posterior flagellum are shown in Fig. 18. I n thii figure, the short conical “projecting” part of the axostyle is alw evident. At the end of the projection there is a small spherical \tructure (Fig. 181, Lvhich r a n also be seen in other individuals (Fig. 1 6 ) . T h e possible nature of this structure will be considered in Discussion. T h e surface of the organism is generally sniooth (Figs. 16-18). The sniall pits seen in the surface of some cells (Fig. 1 7 ) may rcprvsent openings of pinocytotic canals.
c Figs. 11-15. [Pentatrichornonus hominis strain Hs3: S I H . ] 11. Dorsal view. T h e typical 4 anterior flagella (a.fl.) emerge from the periflagellar canal. The recurrent flagellum (r,fl.) emerges from the cell dorsal to the anterior flagella, evidently a short distance posterior to the periflagellar canal. T h e accessory filament (ac.f.) is seen emerghg beneath and to the right of the recurrent flagellum (r.fl.), which it parallels for the entire length of the membrane. x 13,500. 12. Right view. T h e typical 4 anterior flagella (a.fl.), independent flagellum (i.fl,); undulating membrane, as well as the quite hea\y terminal “projecting” segment of the axostyle (ax.) are visible. The posterior end of the accessory filament, which coincides with the posterior end of the undulating membrane, is indicated by a n arrowhead. [he recurrent flagellum extends beyond the end of the undulating membrane as a free poster~orflagellum(p.fl.). x 9,120. 13. From the anterior end of the body emerge the 4 typical anterior flagella, with apparently somewhat pointed ends. T h e independent flagellum ( i f l . ) emerges some distance from the other locomotor organelles. x 8,000. 14. T o p view. T h e areas of emergence of all 6 flagella are seen in this unique electronmicrograph. Note the periflagellar canal (P.c.) filled with the proximal parts of the anterior flagella. T h e recurrent flagellum (r,fl,) emerges somewhat posterior and dorsal to the canal ( t o the observer’s right) ; the accessory filament (ac.f.) is seen to the right of the flagellum. The outline of the pelta ( p e . ) is clearly evident on the ventral side (to the observer’s left) of the organism, its dorsal (upper) margin constituting the rim of the opening of the periflagellar canal. The independent flagellum (Lfl.) emerges ventral to the i>eIta a t -180” to the recurrent flagellum. x 18,600. 15. Terminal part of the undulating membrane. Note the spatial relationships of the recurrent flagellum (r.fl.) and the arcessory filament (ac.f.) whose posterior end coincides with that of the undulating membrane. The recurrent flagellum continues beyond the end of the membrane as a free posterior flagellum (p.fI.). T h e end of the projecting part of the axostyle (ax. 1 is also evident. x 12,750. ~
Figs. 16-18. [Tritrichomonus foetus strain KFT-l.1 16. Left and somewhat dorsal view of a n entire organism. Nearly all the structures discernible with SEM are evident in this preparation. S o t e the 3 subequal anterior flagella (a.fl.) emerging from the periflagellar canal (cf. Fig. 17); the wall of which is reinforced by the pelta ( p e . ) . T h e well developed undulating membrane (u.m.) originates posterior and dorsal to the anterior flagella coursing counterrlockwise from the anterior left toward the posterior right surface of the organism. The recurrent flagellum continues beyond the posterior end of the membrane as a long free posterior flagellum (p.fl.) (cf. Fig. 18). The coniral, short terminal segment (ax.) of the axostylar trunk “projects” from the posterior body surface. Note the “furry” or velvet-like appearance of the cell surface. x 4,000. 17. Anterior part of an organism in a left-dorsal view. The anterior flagella (a.fl.) emerge from well-defined perifagellar canal ( P . c . ) , in the wall of which is s-en the clearly defined outline of the pelta ( p e . ) . Note the lip-like projection of the pelta (arrow) whirh appears to mark its dorsal-right part. T h e recurrent flagellum (a),constituting the dorsal cord of the distal part of the undulating membrane, emerges from the body through a n opening in the wall of the periflagellar canal. A narrower structure ( b ) , which appears to correspond to the distal niar+nal lamella, described from transmission electronmicrographs (12), is seen roursinx between the rerurrent flagellum and a proximal thickening of the membrane ( c ) . This latter thickening, about as wide as the flagellurn. s e e m t o correspond to the proximal part of the undulating membrane as seen by T E M . T h e small depressions (arrowheads) in the flagellate‘s surface may be openings of pinocytotic canals. X 10,450. 18. Posterior part of an organism. T h e posterior end of the undulating membranc (arrow) coincides with the end of the proximal components of this organelle. T h e recurrent flagellum continues beyond thr end of thr inrrnhrane as a free posterior flagellum (p.fl.). The rime-like trrniinal segment of the axostyle (ax.), covered by the cell mcinbrarir. has :I sphcrical encliuq (for explanation of this structure. sec Discussion). T h e surfarc of the organism is smooth and has a “velvety” appi’arancr. ,X 9.000.
SEM
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DISCUSSION The picture seen with the aid of SEM is quite similar to that revealed by bright-field, phase contrast, and especially Nomarski interference optics. However, some features that cannot be resolved with certainty or actually are beyond the resolving power of the light microscope are brought out through the employment of SEM. It is those features that have been emphasized in the Results. The scanning electronmicrographs of the 4 species when viewed against the background of information derived from light microscopy (10, 16, 18, 20, 26, 30-34), and TEM (2, 11, 12, 24, 2 7 ) , provide additional insight into the actual appearance of, and spatial relationships among various, primarily surface, structures of these organisms. Thus it becomes apparent that there exists a certain sequence in the relationships between the emergence of the anterior and recurrent flagella from the cell which seems to be consistent with the evolutionary scheme of Trichomonadida elaborated by Honigberg ( 9 ) , after Kirby ( 1 7 ) , and subsequently accepted by many other workers, e.g. Brugerolle ( 3 ) or Levine ( 2 1 ) . In the representative species, Hypotrichomonas acosta, of the most primitive genus included in the present study, the recurrent flagellum emerges close to the anterior flagella from a common periflagellar canal; it can be differentiated from the latter organelles primarily by its direction both within and outside the canal (Fig. 4 ) . One can surmise, and we hope to confirm this assumption in future studies, that a similar situation obtains in the more primitive Monocercomonas, thought to be the basic genus of the order Trichomonadida (9, 1 7 ) . I n Trichomonas vaginalis, an organism belonging to the subfamily Trichomonadinae (the more primitive subgroup of the family Trichomonadidae) , the recurrent flagellum, aIthough it still emerges from the periflagellar canal, is separated, within the canal, from the anterior locomotor organelles by a strip of cytoplasm (Fig. 8 ) . One could have anticipated that a similar situation would prevail in P. hominis, a member of a genus apparently quite close to Trichomonas. Yet here the area of emergence of the recurrent flagellum is even more clearly separated from that of the anterior flagella, being some distance posterior to the latter, and probably outside the periflagellar canal (Figs. 11, 14). It would be of interest to ascertain if the situation in Tetrati-ichomonas, a genus, unlike Trichomonas, assumed to belong on the main evolutionary line of Trichomonadine and probably more closely related to Pentatrichomonas, is more similar to that found in the latter. If this relationship between the anterior and recurrent flagellum in Tetratrichomonas were more like that in Trichomonas, then in this characteristic Pcntatrichomonas would have to be considered a “more advanced” genus. I n Tritrichomonas foetus, a member of the presumably more advanced subfamily Tritrichomonadinae of Trichornonadidac ( 9 ) , the recurrent flagellum emerges from the area of the periflagellar canal through an opening piercing the left-dorsal part of the canal wall (Fig. 17). T h e foregoing sequence of events could not be seen by light microscopy and could perhaps be reconstructed by T E M only through the use of serial sections. Of special interest is the appearance of the poorly developed undulating membrane in H . acosta (Fig. 2 ) . Indeed, the entire membrane can be differentiated from the unattached Dart of the recurrent flagellum, i.e. the free posterior flagellum, only by the presence of the narrow accessory filament seen to the right of the attached segment of the recurrent flagellum. Clearly, an SEM and TEM study of a species of Monocercomonas with a relatively long proximal part of the recurrent flagellum attached to the body surface, such as M . moskowitzi Honigberg, would be of great
61
interest with regard to the morphologic evolution of the undulating membrane among trichomonads. Among the important observations of Trichornonadidac brought out by the present SEM study was the surface structure of the accessory filament and its spatial relationship to the attached part of the recurrent flagellum in the outer margin of the undulating membrane. Some light microscopists thought that the accessory filament was external (distal) to the rccurrerit flagellum in Trichomonas (32, 33) and Pentatrichffmonas (16, 31-33), and this interpretation was found, by TEM and now with SEM, to be more nearly correct than the view expressed on several occasions by the junior author (e.g. 7, 8 ) that in certain Trichomonadinae the recurrent flagellum was external to the filament. I t was demonstrated by TEM (1, 11, 13, 23, 2 7 ) that the accessory filament courses to one side of the flagellum projecting dorsally beyond its surface (Figs. 21, 22 in Ref. 11; Fig. 40 in Ref. 23; Fig. I I b in Ref. 27). The spatial relationship between these organelles can be visualized best with the aid of SEM (Figs. 7, 8, 11, 14, 15 in the present report). Apparently, the accessory filament emerges from the cell to the right of, and somewhat posterior to the area of emergence of the recurrent flagellum. The filament, seen as a fold in transmission electronmicrographs (1, 11, 13, 23, 2 7 ) , is more cordlike when viewed in SEM. I t parallels the recurrent flagellum for the length of the flagellum’s attachment to the cell body, coursing to the right of this locomotor organelle and, in many areas, folding over onto its surface. As might have been anticipated from light-microscopic (18, 3 4 ) , and T E M (12, 29) studies, the structure of the undulating membrane of Tritrichomonas foetus, when observed in SEM, differs greatly from that characteristic of Trichonionadinae. Indeed, in Tritrichomonas foetus and in all remaining species of the subfamily Tritrichomonadinae Honigberg, 1963, emend. Brugerolle, 1976 ( 3 ) , the recurrent flagellum, enclosed in a cytoplasmic cord, occupies the distal (dorsal) position (e.g. Figs. 1, 2, 31 in Ref. 1 2 ) . I n scanning electronmicrographs (Fig. 17, present report), the following component parts, going from the distal toward the proximal area, discernible in the anterior scgment of the undulating membrane, probably correspond to the expanded area of the distal part of the membrane that contains the axoneme of the recurrent flagellum, to the distal marginal lamella, and to the entire proximal part of the undulating mcmbrane (cf. Fig. 1 in Ref. 12, and Fig. 17, present report). The position and the area of emergence of the indcpcndent flagellum in P. hominis, as seen in SEM, serrn to be consistent with the view based on studies of the division process in trichomonads (2, 2 2 ) that it may represent a precocious recurrent flagellum in this genus. The role played by the pelta in supporting the M-all of the periflagellar canal, as suggested by Mattern et al. (23) on thc basis of TEM observations, can be gleaned also from the various views of this crescent-shaped organelle whose outlines arc sren in the scanning electronmicrographs of the 4 trichornonad spvcies examined during the present study and shown in Figs. 4, 8, 14, and 17. As has been pointed out previously (9),when studied in protargol-stained preparations by light microscopy, Tritrichomonadinae appear to have poorly developed pcltas. I t is cvident, however, from T E M observations (3, 4, 12) that, althoiigh smaller in relation to the total body size, the peltas in the members of this subfamily are well developed. This vicw was fully confirmed by our SEM study (Fig. 1 7 ) . In their shape, size, and length, the “projecting” parts of the axostyle seen in the trichomonad species in the course of the present SEM study correspond quite closely to their descriptions from living (10, 15) and fixed and stained specimcns (10, 16, 18,
20, 26, 30-343. Of wnie intcvqt is the spherical ending of t h r conical axostylar projection noted in sonic trichomonads (Fig. 18 in the present s t u d y ) . I t is known that oftcn the trichomonads attach to the substratc~by nirans of thin c) toplasmic threads extending from the posterior cnd of the axostylar trunk (6; Honigberg, unpublished obsrrvariori.;'~. \Shen an organism dvrachcs frotn the substrate, the cytoplasmic. rstcmsion is rrtractrd toward the axostylar trunk. I t is likely that the spherical structures seen at the end of thr trunks in scanning electronmicrographs are thc filamcntous extensions that have bern I-etracted after thc cell dctarhcd itself from the substrate. Studies of other trichonionads \vhich appcar to occupy crucial positions in the evolution of the order should throw additional light on the kinships among various qcnera and species. Such investigations \vould aid also in a twtter understanding of spatial relationships of surfacr structures in tlic\e f1a~:c~llates. LITER.4TYRE C I T E D 1. Brugerolle G. 197 1. Ultrastructure du genre TrichomituJ Swezy 1915. Zooflagellata, Trichomonadida. Protistologica 7. 1 7 1-6. 2. ___ 1975. Etude de la cryptopleuromitose et de la morphogtnkse de division chez Trichomonas caginalzs et chez plusieurs genres de Trichonionadines primitives. Protistologica 11. 457-68. 3. __1976. Contribution ci l'itude cytologique des Protozoaires Zooflagelle's parasite.t: Proteromonadida, Retortamonadida, Diplomonadida, Oxymonadida, Trichonionndidu. Thesis presented for the degree of Doctcir of Natural Sciences. University of Clermont, Clermont-Ferrand, France. 246 pp. 4. Daniel WA, Mattern CFT. Honigberg BM. 1971. Fine structure of the mastigont system of Tritrichomona.\ n i u r i s (Grassi) . J. Protozool. 18. 575-86. 5. Diamond LS. 1957. T h e establishment of various trichomonads o f animals and man in axenic cultures. J. Parasitol. 43. 488-90. 6. Hogue MJ. 1947. The behavior of Trichomonas caginalis in tissue cultures: .4movie. 1.Parasitol. 33. 199-200. 7. Honigberg BM. 1951. Structure and morphogenesis of Trichomonas prou:azeki Alexeieff and Trichomonas brumpti Alexeieff. L'niu. Calif. (Berkeley) Publ. Zool. 5 5 , 337-94. 8. 1953. Structurc. taxonomir status, and host list of Tritrichomona.r batrachorum (Perty). J. Parasitol. 39. 191-208. 9. 1963. Evolutionary and systematic relationships in thc flagellate order Trichomonadida Kirby. J. Protozool. 10. 20-63. 10. _ _ . King \'M. 1964. Structure of Trichomonas r,aginalis Donne. J. Paratitol. 50, 345-64. 11. . Mattern CFT. Daniel iV.4. 1968. Structure of PentatrichomonaJ honiinis (Davaine I as revealcd by electron microscopy. ]. Protozool. 15, 419-30. 12, _ _ ~ 1971. Fine structure of the mastigont system in Tritrichomonoj f o e t w (Riedmiiller). J. Prolozoo!. 18. 183-98. 13. , Daniel iV.4, Mattern CFT. 1972. Fine structure of Trichomifut bntrachorum (Pertyi. J. Plotozool. 19. 446-53. ~
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I t . Kazanowska W, Jodczyk KJ, Kuczyliska K , Karpowicz MJ. 1977. Morphological variability of T . vaginalis in vivo and in vitro. It'iad. Parazytol. 23, 473-80. 15. Kirby H. 1943. Observations on a tric.homonad from the intestine of man. J. Parasitol. 29, 422-3. 16. ___- 1945. Th e structure of the common intestinal trichomonad of man. J. Parasitol. 31, 163-75. 1 7 . --~ 1947. Flagellate and host relationships of Trichomonad flagellates. J. Parasitol. 33, 2 14-28. 111. __ 195 1 . Observations of the trichomonad flagellate of the reproductive organs of cattle. J. Parasitol. 37, 445-59. 19. Kurnatowska A, Hajdukiewicz G. 1977. Trichomonas ;,aginalis variability in axenic cultures evaluated in scanning microscope. M'iad. Parazytol. 23, 481-7 (in Polish, English summary). 20. Lee JJ. 1960. Hypotrichomonas acosta (Moskowitz) gen. nov. from reptiles. I. Structure and division. J . Protozool. 7, 393-401. 21. Levine ND. 1973. Protozoan Parasites of Domestic Anim a l s and of M a n . 2nd ed., Burgess, Minneapolis. 22. Mattern CF T. 1973. Ultrastructmal events in the process of morphogenesis in trichomonads, in de Puytorac P., Grain J., eds.. Progress in Protozoology, Proc. IV. Int. Cong. Protozool., Clcrmcnt-Ferrand, 1973, Univ. Clermont Press, p. 273. 23. ___ . Honigberg BM, Daniel WA. 1967. The mastigont system of Trichomonas gallinae (Rivolta) as revealed by electron microscopy. J. Protozool. 14, 320-39. 24. ___ . Daniel WA, Honigberg BM. 1969. Structure of H1,potrichomonas acosta (Moskowitz) (Monocercomonadidae, Trichomonadida) as revealed by electron microscopy. J. Protozool. 16. 668-85. 25, McEntegart MG. 1952. Th e application of haemagglutination technique to the study of Trichomonas uaginalis infections. J. Clin. Pathol. 5: 275-80. 26. Moskowitz N. 1951. Observations on some intestinal flag,-llates from reptilian hosts (Squamata). J. Morphol. 89, 257-322. 27. Nielsen MH. Ludvik J, Nielsen R . 1966. O n the ultrastructure o f Trichomonas uaginalis Donne. ]. Microsc. 5, 229-50. 28. OvEinnikov NM, Delektorskij VV, Turanova EN, Yashkova G S . 1975. Further studies of Trichomonas uaginalis with transmission and scanning electron microscopy. Br. ]. Vener. Dis. 51, 1 57-75 .,-.
29. Simpson CF, White F H . 1964. Structure of Trichomonas foetut a s revealed by electron microscopy. Am. J. Vet. Res. 25, t315-74 .,- - - .. 30. LVenrich DH. 1939. Th e morphology of Trichomonac t,aginalis. V o l . J u b . Sadao Yoshida 2, 65-76. 31. ___ 1944. Morphology of the intestinal trichomonad flagellates in man and of similar forms in monkeys, cats, dogs and rats. J. Morphol. 74, 189-21 I . 32. 1944. Comparative morphology of the trichomonad flagellates of man. A m . J . Trop. M e d . H y g . 24, 39-51. :33. 1947. Th e species of Trichomonas in man. J. Paratitol. 33. 177-88. 34. ~- . Emmerson MA. 1933. Studies on the morphology of Tritrichomonas foetus (Riedmiiller) (Protozoa, Flagellata) from .\merican cows. J. Morphol. 55, 193-206. 35. Yamin MA. 1979. The symbiotic flagellates Strebloniastix. Trichornitopsis, Trichonympha from the termite Zootermopsis: .4scanning electron microscope study. Trans. A m . Microsc. S O C .98. in press. ~
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