Ditrichomonas
Ditrichomonas Farmer, 1993 (ref. ID; 7294 original paper)
[ref. ID; 7294]
Diagnosis; Round to ovoid flagellate with 2 anteriorly directed emergent flagella and 1 recurrent flagellum arising from a total of 4 basal bodies. One anterior flagellum (1.0-1.5 times body length) extends directly in front of cell and is often curved at its distal end. The second anterior flagellum (1.0 body length) extends in a more lateral position and beats actively. The recurrent flagellum (1.0 body length) is often associated with an undulating membrane near the point of flagellar emergence. A rigid axostyle protrudes from the posterior portion of the cell. Cells can form cysts with completely internalized flagella and an electron-dense cyst wall. Other features such as a parabasal apparatus and hydrogenosomes are like those reported for members of the Monocercomonadae. (ref. ID; 7294)
Etymology; The generic name derives from the two anterior flagella as opposed to the four anterior flagella found in Trichomonas. (ref. ID; 7294)
Type material; Type material is deposited at Department of Invertebrate Zoology, National Museum of Natural History W-325, Smithonian Institution, Washington, D.C. 20560. (ref. ID; 7294)
Type species; Ditrichomonas honigbergii (ref. ID; 7294)
- Ditrichomonas honigbergii Farmer, 1993 (ref. ID; 7294 original paper)
Diagnosis
Cell (4-5 um in diameter) are variable in shape ranging frr round or teardrop-shaped to nearly fusiform. In some cells the undulating membrane extends outwards producing a prominent flap along the cell's ventral/right surface. Cysts are round and 6-7 um in diameter. (ref. ID; 7294)
Descriptions
Ditrichomonas honigbergii is a round to oval cell with 3 emegent flagella, a small undulating membrane, and a prominent posterior extension. A great deal of variation is found in cultures of D. honigbergii and it is not uncommon to find highly elongate individuals lying next to rounder ones. The most distinct variations are in the size of the undulating membrane which in some individuals is nearly non-existent, whereas in others it protrudes widely from the cell's ventral right side forming a large wing or membranous keel.
The internal structure of Ditrichomonas honigbergii is similar to that of other trichomonads. Hydrogenosomes, food vacuoles, and the storage product glycogen are distributed throughout the cytoplasm. An anteriorly located Golgi dictyosome lies adjacent to the flagellar bases and immediately anterior to the nucleus. A prominent costa was not observed.
Ditrichomonas honigbergii has 3 flagella, 2 of which are directed anteriorly while the third is recurrent and is loosely associated with the undulating membrane. The three flagella originate from a flagellar apparatus composed of four basal bodies, several striated fibers, and a microtubular axostyle/pelta complex. The two anteriorly directed flagella are borne on parallel basal bodies near the anterior end of the cell. One of these flagella typically extends directly in front of the cell. The second anterior flagellum extends anteriorly but often beats in a near lateral fashion relative to the cell's body. The recurrent flagellum is brone on a basal body that lies orthogonal to the basal bodies of the anterior flagella. A fourth barren basal body lies parallel to the basal body of the recurrent flagellum and its imbrication indicates that it too is directed towards the surface of the cell. The fate of basal bodies during cell division was not determined and the basal body numbering scheme of Brugerolle (1976) was adopted with the recurrent flagellar base being labeled as "R" and the other basal bodies designated as either 1, 2, or 3 with the number "2" basal body being distingished by the presence of a sigmoidal fiber that contacts the pelta-axostyle junction. Based on its relative position the barren basal body corresponds to the number 3 basal body (Brugerolle 1976).
The basal bodies are embedded in an electron-dense material that connects them at their proximal ends. From this material several fibers that have a pronounced periodicity emerge. These fibers typically extend between the Golgi dictyosome and the nearby nucleus and are identical in appearance and position to the parabasal fibers found in trichomonad flagellates.
The recurrent flagellum is often associated with an undulating membrane, especially near the point of flagellar emergence. The undulating membrane is a variable structure and is often not in contact with the recurrent flagellum. In some cells the undulating membrane consists of nothing more than a tiny bit of plasma membrane that lies next to the recurrent flagellum near its point of emergence while in others the undulating membrane is an extensive structure that protrudes from the cell, forming either a paddle-shaped extension or in many cases a large membranous flap that just out from the cell's ventral/right surface. In those cells in which the undulating membrane is extensively developed it has a complex architecture in which its outermost portion is rounded and contains electron-dense material.
A few cells were fixed while phagocytosing a bacterium. Serial sectioning confirmed that the ingestion site was the cell's anterior/ventral surface immediately adjacent to the undulating membrane/recurrent flagellum complex. There were no microtubules or other cytoskeletal elaborations in the region of phagocytosis.
The microtubular axostyle originates adjacent to the basal bodies and consists of 20-25 microtubules that proceed posteriorly and form a semi-circular tube adjacent to the nucleus and eventually extends to the cell's posterior as a circular tube. Near their point of origin the microtubules of the axostyle overlap with those of the pelta. The pelta consists of 7-9 microtubules that extend anteriorly and then curve posteriorly, thus defining the cell's anterior end. Near the anteriormost portion of the cell the microtubules of the pelta overlie a small granular electron-dense body that appears not to be membrane bound. The granular nature of this anterior body and its invariant association with the pelt clearly distinguishes it from other organelles. Serial section microscopy indicates that the anterior body is spherical and approximately 0.4 um in diameter. The microtubules of the pelta terminate several micrometers posterior to this region.
As cultures approach maximum cell density many cells encyst. The spherical cysts are surrounded by an electron-dense wall composed of fine fibrils and is separated from the plasma membrane. Flagella are retained and internalized and the cyst cytoplasm is noticeably more granular than identically prepared trophozoites. Experiments in which cultures with no visible trophozoites were transferred into fresh medium yielded trophozoites within 3-4 days. The number of nuclei and internalized flagella in each cyst was not determined but at least seven flagellar profiles can be seen in a single section suggesting that more than three flagella are present per cyst. The presence of dividing trophozoites suggests that cyst formation is not necessary for cell division. (ref. ID; 7294)
Remarks
Parabasalia share a number of derived characters that distinguish them from other amitochondriate flagellates. These characters include a rigid microtubular axostyle and pelta complex, hydrogenosomes, and a set of striated fibers that link the flagellar bases with the anteriorly located dictyosome and together comprise the "parabasal apparatus" from which the group derives its name (Honigberg 1963). The presence of all of these features in D. honigbergii clearly supports it inclusion within this group. More specifically D. honigbergii belongs to the Monocercomonadae group of the Parabasalia, which together with the Trichomonadae form the order Trichomonadida (Honigberg 1963). Like other monocercomonads (Mattern et al. 1972) D. honigbergii has an undulating membrane that is attached to the anterior portion of the recurrent flagellum and lacks a prominent costa (Honigberg 1963). The presence of cysts (with elaborated cyst walls) in D. honigbergii and monocercomonads (Brugerolle 1973) as opposed to the pseudocysts of the Trichomonadae (Mattern and Daniel 1980; Mattern et al. 1973) also supports its inclusion in the Monocercomonadae.
The arrangement of the basal bodies of D. honigbergii is unique among the Trichomonadida (Honigberg 1963; Honingberg et al. 1971) in that only three basal bodies bear flagella, while the fourth is barren. The monoflagellate Histomonas is the only other parabasalid known to possess barren basal bodies (Rybicka et al. 1972). The typical flagellar apparatus of trichomonads consists of the three parallel basal bodies of the anterior flagella, and the orthogonally oriented basal body of the recurrent flagellum (Brugerolle 1991). In those species that contain more than four flagella the additional basal bodies lie parallel to the three anterior flagella (Brugerolle 1991). In contrast, the basal bodies of D. honigbergii ar arranged as two orthogonal pairs; the recurrent basal body with the F1 basal body and the barren F3 basal body with the F2 basal body. This basal body arrangement is similar to that found in the metamonad flagellates (Brugerolle 1973, 1977).
The fact that D. honigbergii was originally isolated from sediment samples of a freshwater lake does not preclude the posibility that it is a parasite of some metazoan host but does suggest that D. honigbergii is a free-living organism that inhabits microaerobic sediments. The only other reports of a free-living trichomonad are of Pseudotrichomonas keilini (Bishop 1935, 1939; Brugerolle 1991). Like P. keilini (Brugerolle 1991), D. honigbergii is a phagotroph that can ingest bacteria.
Ditrichomonas honigbergii is capable of completing all stages of the life cycle (excystment, binary fission, encystment) in relatively undefined culture media with no chemical induction. The ability of D. honigbergii to form walled cysts with internalized flagella is a character shared only with monocercomonads (Brugerolle 1973) and metamonads such as Giardia (Reiner et al. 1990) and Chilomastix (Brugerolle 1973). It is thought that cysts in these organisms aid in dispersal and infection of metazoan hosts but as there is presently no known host for D. honigbergii the exact role of its cyst is unclear.
Protargol preparations of D. honigbergii indicate that two nuclei are present in each cyst (T. Nerad, pers. commun.) as is the case with cysts of monocercomonads (Brugerolle 1973), but there is no indication that binucleate cysts represent a division stage of the life cycle. Culturing experiments in which cyst from late log cultures were added to fresh medium resulted in the re-emergence of trophozoites and suggest that the cyst of D. honigbergii is important for survival in adverse environmental conditions. Pseudocysts of Trichomonas muris are capable of infecting newborn hamsters (Mattern and Daniel 1980) and the accidental ingestion of cysts of free-living metamonads and parabasalids may have led to the origin of parasitic lifestyles for most known species of these groups.
The absence of mitochondria and peroxisomes in some protists led Cavalier-Smith to originally propose that metamonads (diplomonads and retortamonads), microsporidians, and parabasalids together with the archamoebae formed the eukaryotic subkingdom Archezoa (Cavalier-Simth 1983). The Archezoa are considered more primitive than the Metakaryota (eukaryotes that contain mitochondria). More recently Cavalier-Smith has altered this scheme by removing the parabasalids from the Archezoa and placing them in the Metakaryota (Cavalier-Smith 1987, 1991). Cavalier-Smith cites three reason for this change; 1) hydrogenosomes, which are present in all parabasalids, may be derived from mitochondria; 2) parabasalids have easily recognizable Golgi dictyosomes that are not seen in any metamonads, archamoebae, or microsporidians; and 3) parabasalids have an extranuclear mitotic spindle whereas other members of the Archezoa have intranuclear spindles (Cavalier-Smith 1991). I believe that these differences are not significant enough to warrant the exclusion of the parabasalids from the Archezoa and that their relatedness to the metamonads is closer that previously believed.
Some microaerobic ciliates species possess similar organelles that have been termed "hydrogenosomes" (Yarlett et al. 1981, 1982). The suggestion that the hydrogenosomes of anaerobic ciliates are derived from modified mitochondria is plausible and supported by the occurrence of cristae-like membranes in the hydrogenosomes (Finlay and Fenchel 1989). Unlike the ciliates, there are no known examples of mitochondrial bearing parabasalids. It has been argued that the presence of two membranes around each hydrogenosome is an indication of their mitochondrial origin (Cavalier-Smith 1991; Finlay and Fenchel 1989) but this may simply be an indication that the hydrogenosome is the result of a symbiosis between a trichomonad ancestor and a different prokaryote from the one that gave rise to mitocondria (Muller 1988). Finally, the biochemical conversion of a mitochondrion to a hydrogenosome is not a trivial matter. Hydrogenosomes lack a complete tricarboxylic acid cycle, cytochromes, cytochrome oxidase, and electron transport linked phosphorylation (Cerkasov et al. 1978; Lloyd et al. 1979) while these all occur in mitochondria. A separate origin of mitochondria and parabasalian hydrogenosomes is entirely possible (Muller 1988).
Cavalier-Smith's second reason for excluding the parabasalids from the Archezoa is the presence of well-defined Golgi dictyosomes in trichomonads. Unlike discrete cellular organelles such as mitochondria, chloroplasts, nucleus, and even hydrogenosomes, the Golgi apparatus is a more difficult structure to define. The Golgi dictyosome, a collection of membranous sacs used in the glycosylation of proteins and synthesis of complex polysaccharides, is typically thought of as a distinct stack of membranous cisternae yet a Golgi apparatus may have only a single cisternum. The presence or absence of a dictyosome is often difficult to determine. Cavalier-Smith (1991) also makes the argument that mitochondria predate dictyosomes and cites the example Percolomonas a tetrakont flagellate that has mitochondria but no visible dictyosomes (Fenchel and Patterson 1986). It is possile that dictyosomes in Percolomonas and other heteroloboseans have simply been overlooked or that they are only present during a certain phase of the life cycle. This is the case in the eustigmatophyte Vischeria in which Golgi dictyosomes have only recently been discovered in a uniflagellate zoospore (Santos and Leedale 1991). Furthermore, studies of the metamonad Giardia have revealed what appear to be Golgi dictyosomes in cells that are undergoing encystment (Gillin et al. 1991; Reiner et al. 1990) as well as in trophozoites (F. Gillin, pers. commun). If dictyosomes are indeed present in Giardia it wil require a redefinition of the Archezoa to accommodate organisms that have dictyosomes or dictyosome-like organelles.
Cavalier-Smith's third reason for removing the parabasalids from the Archezoa is the presence of an extranuclear spindle in the trichomonads. Trichomonad "pleuromitosis' is unique from the types of division spindles found in microsporidians, metamonads and Archamoebae (Brugerolle 1975, 1991; Cavalier-Smith 1991). Despite this, the question of whether all metamonads and Archamoebae have intranuclear spindles is unclear. In the retortamonads there is evidence of a closed intranuclear spindle (Brugerolle 1977) and mitosis of diplomonads is of a semi-open nature (Brugerolle 1991) but knowledge of both groups is very incomplete. Even less is known about mitosis in the Archamoebae as there are no electron micrographic studies and only one report of division in Pelomyxa (Hollande 1945). Other protistan groups such as the distantly related dinoflagellates have extranuclear spindles (Heath 1980). While this may be a useful character for defining the Parabasalia as a distinct taxon I would be hesitant to make "presence of an internuclear spindle" a criterion for inclusion within the Archezoa.
On the contrary, I believe that the monocercomonad parabasalids have a number of striking similarities with the archezoan metamonads, in particular the retortamonads. First, the tetrakont flagellar arrangement (one recurrent, three anterior) is very similar to that of the retortamonad Chilomastix (Brugerolle 1973; Brugerolle and Mignot 1990) and other diplomonads. Second, the general cell morphology (teardrop-shaped body with posterior projection) is nearly identical in monocercomonads Chilomastix (Brugerolle 1973; Brugerolle and Mignot 1990). Third, the monocercomonads and the metamonads are the only two protistan groups known to form cysts with completely internalized flagella (Brugerolle 1973; Reiner et al. 1990). Fouth, both monocercomonads and metamonads are comprised entirely of anaerobic or microaerophilic species that lack mitochondria and peroxisomes. Fifth, the site of bacterial ingestion in D. honigbergii (anterior ventral surface) corresponds exactly with the location of the retortamonad cytostome.
This last observation offers a possible explanation for the origin of the trichomonad undulating membrane. The cytostome of the retortamonad Chilomastix is an asymmetrical structure designed for the ingestion of bacterial prey. On the right ventral side of the retortamonad cytostome is a protruding flap of membrane that is supported by an internal fibrous structure (Brugerolle 1973, 1977). The recurrent flagellum of retortamonads lies in the cytostome adjacent to this flap (Brugerolle 1973, 1977) and beats in an undulating fashion. A similar-appearing fibrous structure is present in the undulating membrane of D. honigbergii and other trichomonads (Brugerolle 1991; Honigberg and Brugerolle 1991). Living D. honigbergii cells often distend the right ventral portion of the cell and this extended flap may concentrate bacterial prey to region of the cell that is capable of phagocytosis. Thus in terms of its position, function, and structure the undulating membrane of trichomonads bears an interesting resemblance to the specialized right side of the retortamonad cytostome, an observation that has previously been noted by Kofoid & Swezy (1920). Recent studies using antibodies generated against the fibers of the undulating membranes of different trichomonads have shown that distinct classes within the group can be recognized (Brugerolle, G. Evolution and diversity of amitochondrial zooflagellates, J.Euk.Microbiol., 40: 616-618). It would be interesting to see if biochemical studies revealed a similarity between the composition of monocercomonad undulating membrane and the right side cytostomal fiber of retortamonads.
Several studies that have utilized sequence data for 28S ribosomal RNA suggest that Trichomonas vaginalis diverged from the eukaryotic lineage after the occurrence of mitochondrial bearing protists (Baroin et al. 1988; Perasso et al. 1989). In contrast, a study of 16S-like rRNA sequences suggests that Tritrichomonas vaginalis, the metamonad Giardia, and the microsporidian Vairimorpha, form the earliest eukaryotic branch points and immediately precede the divergence of the mitochondria-possessing euglenozoa (Sogin 1989). All of these studies generated trees with unusally long branch lengths for the basal organisms suggesting that due caution be used in interpreting their relative branch positions (Baroin et al. 1988; Perasso et al. 1989; Sogin 1989).
A re-examination of the structural data from D. honigbergii and other trichomonads supports the belief that parabasalids are descended from a free-living amitochondrial ancestor that was closely related to the retortamonad Chilomastix or a common ancestor of both the retortamonads and monocercomonads. The differences that exist between the two groups today (mitotic apparatus, hydrogenosomes, dictyosome with parabasal apparatus, etc.) certainly warrant maintaining two separate taxonomic orders but are not so profound as to obscure a closer evolutionary relationship that previously been recognized. Such an interpretation suggests that the Golgi dictyosome evolved before the acquisition of mitochondria by eukaryotes and that the hydrogenosomes of parabasalians are the result of the a separate endosymbiotic event between a prokaryote and a eukaryote. Thus I prefer the original curcumscription of the Archezoa that included the Parabasalia (Cavalier-Smith 1983). (ref. ID; 7294)
Etymology
The specific epithet honigbergii is given in recognition of the late Bronislaw M. Honigberg, whose outstanding contributions to our understanding of trichomonad flagellates and the field of protozoology will continue to influence researchers for decades to come. (ref. ID; 7294)
Type locality
Isolates were collected from sediments of Lake Enriquillo, a freshwater lake in the Dominican Republic.
There are no known metazoan hosts. (ref. ID; 7294)