Recurrent Flagellum Research Articles

Morphology of Diplomonad Flagellates: Spironucleus torosa N. Sp. from Atlantic Cod Gadus morhua L., and Haddock Melanogrammus aeglefinus (L.) and Hexamita salmonis Moore from Brook Trout Salvelinus fontinalis (Mitchill)

A diplomonad flagellate, Spironucleus torosa n. sp. is described from Atlantic Cod Gadus morhua and haddock Melanogrammus aeglefinus. This is believed to be the 1st confirmed report of Spironucleus from a marine fish. Organisms swimming in the rectal lumen were broadly pyriform to elongate, and measured 10.5-18.6 microns long and 3.2-13.3 microns wide; other elongate organisms were attached to the rectal epithelium, via apical extensions appearing continuous with the microvilli. The posterior end of the body was extended into a caudal projection, on either side of which was a posteriolateral ring-shaped protrusion or torus, with a recurrent flagellum emerging from its centre. A symmetrical system of microtubules and lamellae, forming a "V" in protargol impregnated specimens, supported the flanges of the body surrounding the tori, the tori themselves and the caudal projection. Supranuclear microtubules were an inverted V to U shape in transverse section, and an electron dense band accompanied the cytostomes. Lightly staining homogenous cytoplasm was usually present in the anterior part of the body, the remainder being highly vacuolated with numerous dark granules. In swimming organisms, rough endoplasmic reticulum (RER) was present around the nuclei and cytostomes, and bacteria were occasionally seen in the cytoplasm. In "attached" organisms, RER was reduced, and bacteria were absent. Hexamita salmonis Moore from Salvelinus fontinalis was studied by light and scanning electron microscopy for comparison; its cytoplasm was not highly vacuolated. The two recurrent flagella emerged close together from the blunt posterior end of the body.

Growth, morphology and division of flagellates of the genusTrypanoplasma (Protozoa, Kinetoplastida) in vitro

Nine strains of trypanoplasms were grown in axenic culture. Cultures of Trypanoplasma borreli Laveran and Mesnil, 1901 from fish hosts Blicca bjoerkna, Cyprinus carpio, Scardinius erythrophthalmus and Tinca tinca and of T. guerneyorum Minchin, 1909 from Esox lucius and Trypanoplasma spp. from the leech Piscicola geometra were maintained in biphasic blood-agar medium SNB-9 supplemented with vitamins and antibiotics. In culture, the flagellates transformed into smaller, elongated stages with a little-developed undulating membrane and into short flagella that were morphologically similar to stages in the leech vector. The cultures were passaged weekly at 17-20 degrees C, but they also grew at 4 degrees C. The flagellates divided by binary fission, which was initiated by the formation of two new anterior flagella. The original anterior flagellum of the mother individual was gradually apposed to the cell surface and became the recurrent flagellum of one of the daughter individuals. In the meantime, nuclear division took place, followed by transverse cleavage of the kinetoplast. The division was completed by longitudinal fission of the mother individual into two offspring. Multiple fission that resulted in rosettes, which then cleaved into several daughter cells, was also observed, as well as some dyskinetoplastic and other anomalous forms. In cultures isolated from tenches with high parasitaemia, non-dividing, long filiform stages were observed. Culture stages were not infective for susceptible fishes.

Detection of Microtubules of the Flagellate Trichomonas vaginalis by Monoclonal Antibodies Specific for β‐Tubulin1

Monoclonal antibodies specific for mammalian beta-tubulin recognized the microtubule cytoskeleton of the flagellated protozoon Trichomonas vaginalis. Of seven antibodies, two demonstrated the axostyle, costa, recurrent flagellum, and anterior flagella by indirect immunofluorescence microscopy. The remaining five stained a hazy reticular pattern in the cytoplasm of formaldehydefixed, detergent-extracted organisms. Western immunoblots of whole T. vaginalis extracts treated with protease inhibitors and electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate showed a major band at molecular weight 50,000 when probed with only one of the antibodies which stained the axial cytoskeleton. The antibodies which stained only the cytoplasm showed a different western blot pattern with a major doublet band at MW 58,000-60,000. Another antibody, which stained both the axial cytoskeleton and the reticular cytoplasmic pattern showed major bands at MW 58,000-60,000 and also at MW 40,000-42,000. The recognition of microtubule populations in T. vaginalis by these monoclonal antibodies was different than we found earlier with Leishmania donovani and Toxoplasma gondii, where all seven antibodies recognize cytoskeletal microtubules and produce western blots characteristic of tubulin. Only one of these seven antibodies recognizes tubulin in T. vaginalis by immunoblot. The microtubules of T. vaginalis do not demonstrate all epitopes recognized by monoclonal antibodies specific for mammalian beta-tubulin; one of the antibodies appears to recognize an epitope which is morphologically associated with microtubules but does not have the characteristic MW of tubulin.

Tritrichomonas mobilensis n. sp. (Zoomastigophorea: Trichomonadida) from the Bolivian squirrel monkey Saimiri boliviensis boliviensis.

A trichomonad flagellate, Tritrichomonas mobilensis n. sp., is described from the large intestine of the squirrel monkey, Saimiri boliviensis boliviensis. The organism has a lanceolate body 7-10.5 micrometers in length; a well developed undulating membrane; a stout, tubular axostyle with periaxostylar rings that terminate in a cone-shaped segment projecting from the posterior end of the cell; and a moderately wide costa. The anterior flagella are about as long as the body, and the recurrent flagellum is of the acroneme type. All its characteristics suggest that the new species belongs in the Tritrichomonas augusta type of the subfamily Tritrichomonadinae.

An immunofluorescent study of the microtubule organization in Trichomonas vaginalis using antitubulin antibodies.

The flagellated protozoon Trichomonas vaginalis, parasite of the human urogenital tract, possesses a well developed microtubule system organized in highly differentiated structures. We have shown by immunoblotting that monospecific anti-sheep brain tubulin antibodies are able to react with the microtubular tubulin of T. vaginalis. These antibodies were used to study the microtubular system of T. vaginalis both in interphase and mitosis by indirect immunofluorescence. The interphase microtubular pattern, characterized by an axostyle, a pelta, four anterior flagella, and a recurrent flagellum, displayed remarkable changes at the onset of mitosis: the axostyle disappeared, and two pole bodies connected by a short spindle became evident; chromosomal fibers arose while pole-to-pole fibers elongated. The last phases of mitosis were marked by the disappearance of chromosomal fibers, the appearance of two small axostyles, and the depolymerization of the pole-to-pole bundle. At the end of mitosis, the normal interphase microtubule pattern was observed.

Tritrichomonas foetus: Cytochemical visualization of the endoplasmic reticulum-Golgi complex and lipids

The zinc iodide-osmium tetroxide technique was used to analyze the distribution of the endoplasmic reticulum-Golgi complex system of Tritrichomonas foetus. Interconnections between the cisternae of the endoplasmic reticulum as well as between cisternae of the Golgi complex were observed. The nuclear pores, as well as fenestrations in the Golgi complex, were evident. Three to four profiles of the endoplasmic reticulum were seen in the proximal marginal lamellae, related to the attachment of the recurrent flagellum to the protozoan body. No reaction product was seen in the costae, microtubules, glycogen particles, or hydrogenosomes. Imidazole-buffered osmium tetroxide solution was used to visualize lipids. Electron-dense materials were seen either free in the cytoplasm or within membrane-bounded cytoplasmic vesicles. A high contrast of some membranes, mainly of those which enclosed the hydrogenosomes, was observed in unstained sections.

Tritrichomonas foetus: Localization of filipin-sterol complexes in cell membranes

The polyene antibiotic, filipin, was used as a probe for the detection of sterols in the freeze-fractured plasma membrane and the flagellar membranes of the pathogenic protozoa, Tritrichomonas foetus. A homogeneous distribution of filipin-sterol complexes was seen throughout the plasma membrane, and the membrane of the three anterior and the one recurrent flagella. No or very few filipin-sterol complexes were observed in some specialized regions such as the base of the flagella (necklace), the portion of the recurrent flagellum, and that part of the cell body to which the flagellum was attached. The density of filipin-sterol complexes varied from one cell to the other. In some cells, about 205 complexes/μm 2 were seen. A larger number of filipin-sterol complexes were observed on both faces of the membrane of cytoplasmic structures, probably corresponding to vacuoles. No complexes were seen in the nuclear membrane and in the membrane of the endoplasmic reticulum. Very few or no complexes were observed in the membrane of the hydrogenosomes. Treatment of living cells with filipin induced aggregation of filipin-sterol complexes at some points of the plasma membrane.

Fine Structure and Cytochemistry of the Hydrogenosome of Tritrichomonas foetus1

Fine structural studies of the hydrogenosomes of Tritrichomonas foetus using an improved fixative reveal that they are enclosed by two closely apposed 6 nm membranes, which separate at some regions forming a large intramembranous vacuole where Ca++-binding sites are located. Fixation of the cells in a glutaraldehyde solution containing 5 mM CaCl2 and postfixation in an osmium tetroxide-potassium ferrocyanide solution led to the appearance of a reaction product associated with certain regions of the membrane of the hydrogenosomes and in the cisternae of the endoplasmic reticulum, in the recurrent flagellum, and in the plasma membrane. Treatment of ultrathin sections with EGTA removed the reaction product. These results, in association with others previously described, indicate the existence of several similarities between the hydrogenosomes and the mitochondria.

Electron Microscopic Observations of the Bloodstream Form of Cryptobia salmositica Katz, 1951 (Kinetoplastida: Bodonina)1

ABSTRACTThe ultrastructure of the bloodstream form of Cryptobia salmositica in rainbow trout was examined during the acute phase of experimental infection. The arrangement of the major groupings of cytoplasmic microtubules originating near the basal bodies is similar to that in other bodonids. The cytostome is reinforced both by pellicular microtubules and an electron‐dense plaque. Certain microtubules associated with the flagellar pocket serve as nucleating sites for pellicular microtubules. A flagellar rootlet, consisting of two parallel fibers which are bound together intermittently by electron‐dense plaques, curves posteriorly from the basal body of the recurrent flagellum towards the kinetoplast. The basal body associated plaque on the kinetoplast membranes is duplicated at the same time as the basal bodies. Cytoplasmic microtubules are found in association with the plaque and the outer kinetoplastic membrane. A pulsatile vacuole, described for the first time in a hemoparasitic cryptobiid, lies adjacent to the post‐flagellar pit. Smaller, interconnected vesicles of the spongiome are continuous with the pulsatile vacuole. Since a pulsatile vacuole occurs not only in free‐living and ectoparasitic cryptobiids but in the hemoparasitic (=trypanoplasm) forms as well, this is no longer a character by which the genus Trypanoplasma may be separated from the genus Cryptobia. Possession of this osmoregulatory complex may allow the organism to survive outside of a host and fulfill a monoxenous life cycle, in addition to the usual heteroxenous cycle involving a leech as vector.

Tritrichomonas foetus: Ultrastructural localization of basic proteins and carbohydrates

The postformalin ammoniacal silver (AS) and the ethanolic phosphotungstic acid (E-PTA) techniques were used to localize basic proteins at the ultrastructural level in the protozoa, Tritrichomonas foetus. The periodic acid-thiosemicarbazide-silver proteinate technique was used to localize carbohydrates. With the AS technique reaction product was seen only in the hydrogenosomes. With the E-PTA technique, reaction product was seen in the microtubules that form the basal bodies, flagella, pelta, and axostyle, in the costa, in the hydrogenosomes, and at the region of the recurrent flagellum's adhesion to the cell body. Previous acetylation of the cells under conditions that block most free amino groups abolished (AS technique) or greatly reduced (E-PTA technique) staining. Carbohydrates were localized on the cell surface; in the membranes of the hydrogenosomes, Golgi complex, and cytoplasmic vesicles; and in the glycogen particles. The specificity of the AS and E-PTA techniques to detect basic proteins is based on observations made with T. foetus as well as with other cell types.

Tritrichomonas foetus: fine structure of freeze-fractured membranes.

Freeze-fracture techniques reveal differences in fine structure between the anterior three flagella of Tritrichomonas foetus and its recurrent flagellum. The anterior flagella have rosettes of 9-12 intramembranous particles on both the P and E faces. The recurrent flagellum lacks rosettes but has ribbon-like arrays of particles along the length of the flagellum, which may be involved in the flagellum's attachment to the cell body. This flagellum is attached to the membrane of the cell body along a distinct groove that contains few discernible particles. Some large intramembranous particles are visible on the P face of the cell body membrane at the point where the flagellum emerges from the cell body. The randomly distributed particles on the P and E faces of the plasma membrane have a particle density of 919/micron2 and 468/micron2 respectively, and there are areas on both faces that are devoid of particles. Freeze-fracture techniques also reveal numerous fenestrations in the membrane of the Golgi complex and about 24 pores per micron2 in the nuclear membrane.

Specializations in the Flagellar Membrane of Tritrichomonas foetus

Tritrichomonas foetus has three anterior flagella and one recurrent flagellum. The latter originate at the anterior portion of the protozoa and extends towards the posterior region of the cell adhering to the cell body. Freeze-fracturing studies showed in the flagellar membranes of the three anterior flagella, groups of nine to 12 intramembrane particles forming rosettes which could be observed on both the P- and the E-faces of the flagellar membrane. The rosettes are numerous and found along the length of the flagella. No such structure was seen in the membrane of the recurrent flagellum. The rosettes coexist with other particles in apparently random distribution.

Tritrichomonas muris in the hamster: pseudocysts and the infection of newborn.

Female golden hamsters, either in the last week of pregnancy or in the first weeks of nursing, excreted in their feces variable numbers of pseudocysts of Tritrichomonas muris. Pseudocysts examined by electron microscopy had internalization of the 3 anterior flagella and the undulating membrane with its recurrent flagellum. The undulating membrane and the associated marginal lamellae were characteristic of T. muris. Pseudocysts gradually become motile after 2 or more hours of incubation in medium. The "excysted" trophozoites were identified ultrastructurally as T. muris. Newborn hamsters were not infected with T. muris at 3 days of age, but by the 7th day essentially all were found to have infected ceca, concomitant with cecal enlargement and the appearance of adult-type feces.

Cryptobia branchialis Nie from fish gills: ultrastructural evidence of ectocommensal function

Abstract. Flagellates infesting the gills of freshwater fish are reviewed and Cryptobia branchialis Nie is differentiated from them. Gill filaments of two species of fish heavily infested with C. branchialis were examined with the transmission electron microscope. The flagellates are attached to the surface of the gill epithelium by their recurrent flagella. A prominent ridge on the flagellar membrane contacts the host cell membrane, forming a zone of gap junction and leaving a space of about 10 nm between the two membranes. The flagella do not penetrate into the epithelial cells. No other modifications of the cell membrane could be detected and the host cell reveals no signs of damage. Assumptions on the pathogenicity of C. branchialis are discussed in view of ultrastructural findings, which suggest that this flagellate is non‐pathogenic and ectocommensal.

Cryptobia Sp. In the Snail Triadopsis Multilineata (Say): Fine Structure of Attached Flagellates and Their Mode of Attachment to the Spermatheca*

SYNOPSIS. A Cryptobia sp. that appears to differ structurally from Cryptobia helicis Leidy occurred as 2 distinct populations within the spermatheca of the land snail Triadopsis multilineata (Say). Attached flagellates were a resident population within the spermatheca while pleomorphic, free‐swimming forms were a transient population moving freely within the reproductive tract. Slender, free‐swimming flagellates were apparently the ones transmitted by a venereal route. It was observed by electron microscopy that attachment was achieved through an extensive morphologic transformation of the first 5 to 10 μm of the anterior flagellum which resulted in the formation of numerous pseudopodium‐like structures (“flagellapodia”) capable of invading the microvillus border of the host tissue. Flagellapodia were delimited by a single unit membrane, contained granular cytoplasm, and were long, branched, finger‐like structures which interdigitated extensively with host cell microvilli. Fine structure of the Cryptobia sp. is distinctly different from that reported for C. helicis. the differences include structure and position of the preoral ridge, the cytostome‐cytopharynx, the recurrent flagellum, and the presence of the flagellapodia.

Structure and Life Cycle of Trypanoplasma beckeri sp. n. (Kinetoplastida), a Parasite of the Cabezon, Scorpaenichthys marmoratus, in Oregon Coastal Waters*,†

SYNOPSIS. Structure of Trypanoplasma beckeri sp. n. from the cabezon, Scorpaenichthys marmoratus (Ayres), is described from living specimens and from both Giemsa‐, and protargol‐stained smears. Flagellates from fish blood were usually long and slender, averaging 109.0 × 6.5 fan. The anterior flagellum averaged 8.5 μm; the recurrent flagellum bordered the body and terminated as a very short free flagellum, 2.5 μm long on the average. No true undulating membrane was observed, but in living individuals the recurrent flagellum undulated rapidly near its point of origin. The oval nucleus, averaging 8.5 × 4.0 μm, was located near the anterior cad of the body. An argentophilic, aciculum‐like structure appeared to connect the nucleus to the area at the base of the flagella. The kinetoplast was not observed in fish blood forms. On the basis of laboratory experiments, the leech, Malmiana diminuta Burreson, was ascertained to be the vector for T. beckeri. Upon entry into the leech, flagellates became rounded, and division commenced within a few hours. Division continued for ˜48 h and the flagellates became progressively smaller until reaching a length of ˜10.0 μm. After 72 h they were found in high numbers in the proboscis sheath and also in the anterior crop of the leech. When infected leeches fed on an uninfected fish, flagellates were first observed in the fishes’ peripheral circulation 8 days later.

Structure of Trichomonads as Revealed by Scanning Electron Microscopy*†

A scanning electron microscope (SEM) study of Hypotrichomonas acosta (Moskowitz), Trichomonas vaginalis Donné, Pentatrichomonas hominis (Davaine), and Tritrichomonas foetus (Riedmüller) provided new information about the structure of the periflagellar canal; emergence of the flagella from 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 flagellum and the accessory filament in Hypotrichomonas and in the members of Trichomonadinae, i.e. Trichomonas and Pentatrichomonas.

Ultrastructure of the genus Enteromonas da Fonseca (Zoomastigophorea) and revision of the order of Diplomonadida Wenyon

Fine structure of 2 species of Enteromonas, one from the intestine of the salamander, Triturus vulgairs, and another from the feces of domestic rabbit, Oryctolagus cuniculi, is described. The pyriform cell has an anteriorly located nucleus. The 4 flagella originate from an area near the anterior end of the nucleus. The recurrent flagellum (R) is lodged in a ventral depression or cytostome. The kinetosomes, arranged into 2 pairs, anterior (no. 1, no. 2) and posterior ("3, R), are interconnected by microfibrils. One microtubular fiber, connected to kinetosome "1, is situated near the anterior surface of the nucleus. Another, subnuclear, microtubular fiber is homologous to the "crossed" fiber found in Diplozoa. The cytostome is bordered by 2 lips: the preeminent left lip is equipped with several rows of microtubules, while the right lip contains only a thin microtubular fiber associated with microfibrils. The cytostome occupies 2/3 of the ventral surface. The recurrent flagellum passos over the anterior surface of the cell and then comes to lie in the cytostome. The bacteria are phagocytosed in the bottom part of the cytostome between the 2 distended lips. They are digested in numerous vacuoles. The undigested residual bodies are evacuated by a rupture of the cell membrane. The ergastoplasm is concentrated near the cell periphery. Mitochondria and the Golgi apparatus are absent. In the cyst stage, the multinucleate cell is enclosed in a microfibrillar membrane; the axonemes lie free in the cytoplasm. Diplomonad forms of Enteromonas resembling Hexamita are numerous, except that the cytostome is different in these 2 genera. In such forms, the arrangement of the 2 individuals often has binary axial symmetry, but on occasion they are associated in a more anarchic fashion. The mastigont of Enteromonas is organized like that of a single zooid of a diplozoon. It is possible that the genus Enteromonas is ancestral to Diplomonadida and that the diplomonad state, transitory in Enteromonas, became permanently established in Diplomonadida. Enteromonas appears to be more primitive than the other genera of Diplomonadida. Thus we propose 2 suborders: Enteromonadina, subord. nov. with the genus Enteromonas, and Diplomonadina Wenyon, emend., with the genera Trepomonas, Trigonomonas, Hexamita, Spironucleus, Octomitus, Giardia. The arrangement of the kinetosomes and the existence of a cytostome are the principal characters common to Enteromonas and Retortamonadida, while their "accessory" fibers are not homologous. A more complete study of division of the 2 zooflagellate orders is necessary for the presentation of a more detailed evolutionary scheme of these groups.

The fine structure of the colonial kinetoplastid flagellate Cephalothamnium cyclopum Stein.

SYNOPSISCell structure, cell adhesion, and stalk formation have been examined by electron microscopy in the colonial flagellate, Cephalothamnium cyclopum. Each cell is obconical or spindle‐shaped, pointed posteriorly and truncated anteriorly. The cell membrane is underlain by epiplasm 0.1 μm thick in the posterior region, but bands of microtubules support the anterior region which is differentiated into a flagellar pocket, oral apparatus and contractile vacuole. Each of 2 flagella, joined a short way above their bases by an interflagellar connective, has a paraxial rod and mastigonemes. One flagellum is free and is important in food gathering while the other is recurrent and lies in a shallow groove on the ventral cell surface but projects posteriorly into the stalk. The basal bodies of these flagella are bipartite structures connected by a pair of striated rootlets with accessory microtubular fibers. The oral apparatus consists of a funnel‐shaped buccal cavity and cytostome. It is supported by helical and longitudinal microtubules and also has nearby striated and microtubular fibers. Possible roles of associated oral vesicles in relation to ingestion are discussed. A reticulate mitochondrion houses a massive kinetoplast which has a fibrillar substructure resembling that of dinoflagellate chromosomes. Adjacent flagellates adhere by laminate extensions of their posterior regions and attach by their recurrent flagella to a communally secreted stalk composed of finely fibrillar material. This study indicates that Cephalothamnium belongs in the order Kinetoplastida, and has many features in common with members of the family Bodonidae.

Fine-structural changes associated with pseudocyst formation in Trichomitus batrachorum.

SYNOPSIS. Several fine‐structural changes were observed during formation of the so‐called pseudocysts of Trichomitus batrachorum grown in an agar‐free medium. Among these changes the most frequent were those of the undulating membrane complex. Internalization of the marginal lamella combined with disappearance of the fin‐like, membranous dorsal fold occurred with the highest frequency. In many organisms these alterations were accompanied by internalization of the recurrent flagellum, which, however, remained external in other, presumably earlier stages of pseudocyst formation. In some of the internalized and even still external recurrent flagella the microtubules were in various states of disassembly. The often distended flagellar membrane enclosed large accumulations of filaments 5 nm in diameter. Internalization of the anterior flagella also was noted. Changes similar to those characteristic of the recurrent flagella were not seen, however, among the microtubules of the anterior flagella or among those of the pelta or the axostyle.