Ultrastructural evidence for the biflagellate origin of the uniflagellate fungal zoospore

Summary The morphological similarities between the kinetosome and the second centriole of the zoospores of Phlyctochytrium kniepii and P. punctatum (Chytridiomycetes) suggest that the second centriole in the chytrid zoospore is a vestigial flagellum base. It is suggested that the term vestigial kinetosome may also be used when referring to the structure which is presently termed the second centriole of the chytrid zoospore. Morphological similarities between the chytrid zoospores of P. kniepii and P. punctatum and the zoospores of Rhizidiomyces apophysatus (Hyphochytridiomycetes) are noted. The possible biflagellate origin of fungi with uniflagellate zoospores is discussed. The third fiber (C fiber) of the kinetosome triplet is shown to form as an outgrowth of the B fiber of the kinetosome doublet.     

Study of DNA-containing structures in the nucleus and cytoplasm of the Crithidia flagellates using the Miller method

Weakly condensed interphase chromosomes made of chromatin, and the tightly packed kinetoplast DNA (kpDNA) of a single mitochondrion of Crithidia (Kinetoplastidea, Trypanosomatida) flagellates were studied on the Miller-type spread preparations by electron microscopy. Chromatin of organisms lysed at low ionic strength conditions for 5-10 min unfolds up to 10-nm nucleosomal filaments and 20-nm chromatin fibers. The initial indications of kpDNA decompaction become visible after a 10-13 min dispersion of lysed flagellates. However, at least a 15 min long procedure is required for well-defined identification of intrakinetoplast structures. In this case, the kinetoplast looks like a heterogeneous network disposed close to the kinetosome of a single flagellum. Cells with diameters of 162.5 nm and contour wall length of 510 nm dominated within the network. With the prolongation of the dispersion time up to 20 min both these parameters increased up to 218 and 686 nm, respectively. Further prolongation of the treatment up to 60 min results in wall disruption in many cells. Within these cells, some isolated circular kpDNA molecules appear with the contour length of 588-792 nm. The circles of this size correspond to individual minicircles of the Crithidia kpDNA. Partly unfolded maxicircles of the kpDNA can be found only at early stages of dispersion (10 min). Special features of compaction of DNA-containing structures in both the nucleus and cytoplasm of Crithidia are discussed.     

Ultrastructural aspects of the somatic and buccal infraciliature ofColeps amphacanthus Ehrenberg 1833

Summary Somatic and buccal infraciliature ofColeps amphacanthus Ehrenberg 1833 were studied by light and electron microscopy. The somatic kineties are composed of monokinetids and 2 dikinetids at the anterior end of each kinety. The monokinetids are associated with postciliary microtubules at triplet 9, a kinetodesmal fiber at triplet 5 and 7 and nearly radially arranged transverse microtubules at triplet 4. The associated fibrillar systems of the posterior kinetosome of the dikinetids are like those of the monokinetids. The anterior kinetosome is associated with transverse microtubules at triplet 4 and one or few postciliary microtubules at triplet 9. The anterior kinetosome bears only a short cilium.The oral ciliature is composed of a kinety of nearly circumorally arranged paroral dikinetids and 3 adoral organelles at the ventral left side of the oral opening. Nematodesmata arising from the oral ciliature form the major component of the cytopharyngeal apparatus which is lined by microtubular ribbons of postciliary origin. The buccal cavity is surrounded by oral papillae which often contain toxicysts.     

Effect of acriflavin on the kinetoplast of Leishmania tarentolae. Mode of action and physiological correlates of the loss of kinetoplast DNA

The loss of kinetoplast DNA in Leishmania tarentolae, which occurs in the presence of low concentrations of acriflavin, was found to be a result of selective inhibition of replication of this DNA. Nuclear DNA synthesis was relatively unaffected and cell and kinetoplast division proceeded normally for several generations. An approximately equal distribution of parental kinetoplast DNA between daughter kinetoplasts resulted in a decrease in the average amount of DNA per kinetoplast. The final disappearance of the stainable kinetoplast DNA occurred at a cell division in which all the remaining visible kinetoplast DNA was retained by one of the daughter cells. The selective inhibition of kinetoplast DNA synthesis was caused by a selective localization of acriflavin in the kinetoplast. The apparent intracellular localization of dye and the extent of uptake at a low dye concentration could be manipulated, respectively, by varying the hemin (or protoporphyrin IX) concentration in the medium and by adding red blood cell extract (or hemoglobin). Hemin and protoporphyrin IX were found to form a complex with acriflavin. During growth in acriflavin, cells exhibited an increasing impairment of colony-forming ability and rate of respiration. No change in the electrophoretic pattern of total cell soluble proteins was apparent. The data fit the working hypothesis that the loss of kinetoplast DNA leads to a respiratory defect which then leads to a decrease in biosynthetic reactions and eventual cell death. A possible use of the selective localization of acriflavin in the kinetoplast to photooxidize selectively the kinetoplast DNA is suggested.     

Ultrastructure of the cytoplasm of the lower holotrichous infusorian Tracheloraphis prenanti]

The ciliature of T. prenanti Dragesco 1960 (forma oligocineta Raikov et Kovaleva, 1968) consists of 14-18 ventral and lateral longitudinal kineties with paired kinetosomes, carrying either two cilia or one cilium per kinetosome pair (in the latter case, the nonciliated kinetosome is always the posterior one). The ectoplasmic fibrillar system belongs to the postciliary type. A pair of kinetosomes shares a common basal plate. The anterior kinetosome gives rise to a short ribbon of transverse microtubules, the posterior one, to a poorly developed kinetodesmal filament and to a strong ribbon of postciliary microtubules. The latter proceeds backwards along 8 to 12 kinetosome pairs, being incorporated into a laminated postciliodesma which accompanies each kinety on its right side. Rows of Golgi elements, sending secretory vesicles and channels towards the body surface, exist beneath the kinetosome bases. Each kinety is accompanied on its left by a microfibrillar myoneme, surrounded by perimyary vesicles and underlain by a row of mitochondria. The median part of the dorsal surface is nonciliated; the cytoplasm here is rich of membrane systems, contains peripheral, electron-dense, extrusible inclusions and sometimes also bacteria. The electron-dense inclusions develop in the endoplasm, in close contact with mitochondria. The endoplasm contains also large microfibrillar spheres of unknown nature.     

Asexual reproduction in the suctorianDiscophrya collini

Summary Discophrya collini reproduces asexually through the formation of a ciliated swarmer by evaginative budding. This process is initiated by the repeated replication of a single subcortical kinetosome to form a kinetosome field. The epiplasm of the multilayered cortex covering this field becomes reduced in thickness and the whole cortex invaginates to produce an internal embryonic cavity. The kinetosomes become organised into rows, and each produces a cilium which projects into the cavity. On completion of the embryonic cavity its walls are extruded through the cavity opening to form an external ciliated swarmer connected to the parent by a thin bridge of cytoplasm. It is suggested that this evagination is induced by a rapid breakdown of supporting microtubules in the cavity wall and the subsequent hydrostatic pressure exerted by the body cytoplasm. The connecting bridge shows no specialised ultrastructural features and separation of swarmer from parent probably is achieved by the active movement of the swarmer. The cytoplasm of the swarmer is similar in structure to that of the adult cell but contains a number of primordia of tentacle axonemes. The infraciliature resembles that of other suctorian swarmers. On settling, the cilia of the swarmer are lost, at least some by resorption, a stalk may be secreted and the axoneme primordia are extended to form functional tentacles.     

Ultrastructural studies of the commensal suctorian,Choanophrya infundibulifera hartog

Summary The feeding tentacles of Choanophrya contain a central canal lined by microtubules. Only one tentacle develops during metamorphosis of the embryo into the adult, but others develop at intervals throughout adult life. Each tentacle forms adjacent to a solitary, subcortical kinetosome which lies parallel to the body surface, lacks accessory elements and never develops a cilium. Small condensations of electron-dense material and short bundles of microtubules form adjacent to the cartwheel region of the kinetosome. Initially these bundles are orientated randomly but later they become radially arranged and curved into prolamellae around a disc-shaped condensation centre, to form a paddlewheel-like tentacle primordium 0.8–1.1 m in diameter. The condensation centre consists of alternating concentric electron-dense and electron-transparent zones, and lies with its axis perpendicular to both the kinetosome and the cortex. The microtubules in each prolamella increase in number and pairs of short tip microtubules develop between adjacent prolamellae. Subsequently the developing lamellae become enclosed by a cylinder of ring microtubules. Once all the microtubule components of the tentacle primordium are established it increases in length by addition of material to the basal ends of the microtubules to form a short microtubule canal. As the canal elongates the epiplasm above it disappears and the pellicle membranes become uplifted around the protruding tentacle. An epiplasmic collar differentiates around the growing tentacle whilst spheroid vesicles and solenocysts begin to accumulate in the surrounding cytoplasm.This investigation was supported by the J.S. Dunkerley Fellowship in Protozoology, awarded by the University of Manchester.     

The fine structure of the paralabial organelle in the rumen ciliate Ophryoscolex purkinjei Stein, 1858

The paralabial organelle of the rumen ciliate Ophryoscolex purkinjei, located on the ventral side of the ciliophor, is a highly specialized part of the somatic cortex. It consists of alternating rows of short modified cilia and thin pellicular folds which form a ridge-like structure. The central "top kinety" is composed of monokinetids which bear cilia with 9 + 2 axonemes and 2 microns in length. The top kinety is accompanied by a comb-shaped fold on its distal side and by a broad wedge-shaped fold on its proximal side. To both sides there follow two or three lateral kineties made of dikinetids. The anterior kinetosome of each pair bears a clavate cilium, only 0.5-0.7 micron in length and with a 9 + 0 axoneme while the cilium of the posterior kinetosome is even shorter. Lateral folds with numerous microtubules cover these lateral kineties and rows of barren basal bodies. The fine structure of this supposed sensory organelle show a basic pattern in four other ophryoscolecids, and its increasing complexity parallels the suggested phylogenetic line of evolution of these ciliates.     

THALASSOCHYTRIUM GRACILARIOPSIDIS (CHYTRIDIOMYCOTA), GEN. ET SP. NOV., ENDOSYMBIOTIC IN GRACILARIOPSIS SP. (RHODOPHYCEAE)

Thalassochytrium gracilariopsidis gen. et sp. nov. is an endosymbiotic, polycentric, zoosporic fungus that infected cultures of the red alga Gracilariopsis sp. Based on the posteriorly uniflagellate zoospore and the platelike cristae of the mitochondria, the fungus is placed in the Chytridiomycota. Ultrastructurally, the fungal zoospore is distinguished by the anterior position of the kinetosome, a unique microbody-lipid globule complex, an electron-opaque helix associated with the kinetosome and lipid globules, and a beaked nucleus. Zoospores are positively phototactic, and the unusual helix might constitute part of the photosensory apparatus. Zoospores lack certain taxonomically important structures, such as a rumposome, props, a nonflagellated kinetosome, and flagellar roots. The organism does not fit into any described genus, and the features of its zoospore differ from those of any described order. The fungal thallus is polycentric with multinucleate, septate hyphae. Haustoria form within the algal cells. The fungus does not appear to cause major harm to its host and seems to be host specific. However, during intense sporulation of the fungus, degradation of host chloroplasts was observed in medullary cells.     

The sequence-directed bent structure in kinetoplast DNA is recognized by an enzyme from Crithidia fasciculata

Crithidia fasciculata nicking enzyme (Shlomai, J., and Linial, M. (1986) J. Biol. Chem. 261, 16219-16225) interrupts a single phosphodiester bond in duplex DNA circles from various sources, only in their supercoiled form, but not following their relaxation by DNA topoisomerases. However, this requirement for DNA substrate supercoiling was not observed using the natural kinetoplast DNA as a substrate. Relaxed kinetoplast DNA minicircles, either free or topologically linked, were efficiently nicked by the enzyme. Furthermore, bacterial plasmids, containing a unit length kinetoplast DNA minicircle insert, were used as substrates for nicking in their relaxed form. This capacity to activate a relaxed DNA topoisomer as a substrate for nicking is an intrinsic property of the sequence-directed bend, naturally present in kinetoplast DNA. The 211-base pair fragment of the bent region from C. fasciculata kinetoplast DNA could support the nicking of a relaxed DNA substrate in a reaction dependent upon the DNA helix curvature.     

The Fine Structure of the Paralabial Organelle in the Rumen Ciliate Ophryoscolex purkinjei Stein, 1858

The paralabial organelle of the rumen ciliate Ophryoscolex purkinjei , located on the ventral side of the ciliophor, is a highly specialized part of the somatic cortex. It consists of alternating rows of short modified cilia and thin pellicular folds which form a ridge-like structure. The central "top kinety" is composed of monokinetids which bear cilia with 9 + 2 axonemes and 2 μm in length. The top kinety is accompanied by a comb-shaped fold on its distal side and by a broad wedge-shaped fold on its proximal side. To both sides there follow two or three lateral kineties made of dikinetids. The anterior kinetosome of each pair bears a clavate cilium, only 0.5–0.7 μm in length and with a 9 + 0 axoneme while the cilium of the posterior kinetosome is even shorter. Lateral folds with numerous microtubules cover these lateral kineties and rows of barren basal bodies. The fine structure of this supposed sensory organelle show a basic pattern in four other ophryoscolecids, and its increasing complexity parallels the suggested phylogenetic line of evolution of these ciliates.     

AN ELECTRON MICROSCOPE STUDY OF CORTICAL STRUCTURES OF OPALINA OBTRIGONOIDEA

The pellicular framework of Opalina obtrigonoidea consists of numerous longitudinal ribs parallel to the kineties. These ribs lie erect on the cell surface, and each is composed of striated longitudinal fibers. A membrane covers the ribs and the ectoplasm between them. Flagella, of conventional structure, emerge from the ectoplasm between the ribs. The two central fibers of each flagellum end at the cell surface; the nine peripheral fibers continue for about 400 mµ into the cell to form an open tubular kinetosome. From the anterolateral curvature of each kinetosome arise two rows of fibrils, each fibril oriented perpendicular to the cell surface and about 150 A in diameter. The two rows converge anteriorly and probably meet the next adjacent kinetosome. Minute granules or tubules, arranged in oblique rows and at least sometimes accompanied by very fine fibers, lie at the surface of the ectoplasm but show no detectable connection with the kinetosomes. The whole flagellar apparatus of Opalina thus bears a general resemblance to the infraciliature of some holotrich ciliates, but the degree of evolutionary relationship between them remains uncertain.     

On the sequence determinants and flexibility of the kinetoplast DNA fragment with abnormal gel electrophoretic mobilities

A 410 base-pair (bp) Sau3A restriction fragment derived from a Leishmania tarentolae kinetoplast DNA minicircle, which is known to have slower than expected electrophoretic mobilities in polyacrylamide gels, has been cloned in a plasmid and deletions from one end of the cloned segment have been constructed. Analysis of the gel electrophoretic mobility data of a large number of restriction fragments derived from the kinetoplast DNA clone and its deletion subclones has led to the conclusion that two sequences, one in the region bp 100 to 170 and the other bp 190 to 250, both numbered from one end of the 410 bp kinetoplast DNA segment, are important for the abnormal gel electrophoretic behavior of the kinetoplast DNA fragment. One common feature of these sequences is the periodic presence of short runs of A residues (3 to 6 As in each); auto-correlation analysis of these runs of A residues shows a strong harmonic component with a period around 11 bp. These results support and extend the previous analysis of Wu & Crothers (1984). The abnormal electrophoretic behavior is accentuated at low temperature and by the addition of Mg2+ to the electrophoresis buffer; addition of Na+ has the opposite effect. Insertion of sequences derived from the kinetoplast DNA fragment into nicked circular DNA causes no unexpected change in its electrophoretic mobility in agarose gel, suggesting that the 410 bp sequence, or segments of it, has no significant spatial writhe. Abnormal shifts in agarose gel mobilities are observed, however, when certain segments of the kinetoplast DNA are inserted into positively or negatively supercoiled DNA topoisomers. These results are consistent with a bent structure of the kinetoplast DNA in which the bend has zero writhe in its undistorted form but is easily distorted.     

A Reexamination of the Ultrastructure of Didinium nasutum and a Reanalysis of the Phylogeny of the Haptorid Ciliates

ABSTRACT. The cilia of Didinium nasutum are restricted to two girdles encircling the cell. Each row of cilia in both girdles is made up of two to three anterior pairs of kinetosomes followed by several single kinetosomes. Each single kinetosome has two sets of transverse microtubules, an overlapping postciliary microtubular ribbon, and a laterally directed kinetodesmal fiber. The pairs of kinetosomes are homologous to the oral dikinetids of other haptorians: the nonciliated kinetosome of the pair has a transverse microtubular ribbon that extends to line the membrane of the proboscis, a single short postciliary microtubule, and a nematodesma; the ciliated kinetosome has a ribbon of postciliary microtubules and two sets of transverse microtubules. The presence of these characters in Didinium invalidates Leipe & Hausmann's conclusion that the Didiniidae should be removed from the subclass that contains the other haptorians (Leipe, D. D. & Hausumann, K. 1989. Somatic infraciliature of the haptorid ciliate Homalozoon vermiculare (Kinetofragminophora, Gymnostomata) Ditransversalia n. subcl. and phylogenetic implications. J. Protozool. , 36 :280–289). In light of this, the justification for a subclass Ditransversalia is challenged and shown to be unnecessary.     

Kinetoplast morphology and segregation pattern as a marker for cell cycle progression in Leishmania donovani

Trypanosomatids are typified by uniquely configured mitochondrial DNA--the kinetoplast. The replication timing of kinetoplast DNA (kDNA) is closely linked to nuclear S phase, but nuclear and kinetoplast compartments display staggered timing of segregation, post-replication. Kinetoplast division is completed before nuclear division in Trypanosoma species while nuclear division is completed first in Crithidia species. Leishmania donovani is the causative agent of visceral leishmaniasis, a form of leishmanial infection that is often fatal. Cell cycle related studies in Leishmania are hampered by difficulties in synchronizing these cells. This report examines the replication/segregation pattern and morphology of the kinetoplast in L. donovani with the aim of determining if these traits can be used to assign cell cycle stage to individual cells. By labeling replicating cells with bromodeoxyuridine after synchronization with hydroxyurea, we find that although both nuclear and kDNA initiate replication in early S phase, nuclear division precedes kinetoplast segregation in 80% of the cells. The kinetoplast is roundish/short rod-like in G1 and in early to mid-S phase, but prominently elongated/bilobed in late S phase and early G2/M. These morphological traits and segregation pattern of the kinetoplast can be used as a marker for cell cycle stage in a population of asynchronously growing L. donovani promastigotes, in place of cell synchronization procedures or instead of using antibody staining for cell cycle stage marker proteins.     

On the Encystment of Leptomonas sp. (Kinetoplastida: Trypanosomatidae), a Parasite of the Silkworm, Bombyx mori Linnaeus

SYNOPSIS. Promastigotes of Leptomonas sp., a flagellate parasite of the silkworm, Bombyx mori , multiplied by binary fission with the following sequence of events: duplication of the flagellum; division of the kinetoplast and the nucleus; spatial separation of the kinetoplast: and cytokinesis resulting in the formation of 2 daughter promastigotes. In the early stages of encystment, promastigotes aggregated in a rosette and assumed a stumpy form. The nucleus and kinetoplast of the stumpy promastigotes were double, suggesting a possibility of fusion of the organism in the rosette. When most of the promastigotes in the cluster became stumpy, each individual was isolated from the cluster and acquired a thick coat with an acidophilic substance, thus forming a cyst.     

Effect of hydroxystilbamidine on the kinetoplast of Trypanosoma gambiense in mice.

Hydroxystilbamidine (OHSA), an inhibitor of DNA synthesis, was shown to induce the dyskinetoplastic forms (akinetoplastic forms, AK forms) of Trypanosoma gambiense. The mode of appearance of the AK forms after injection of various doses of OHSA into infected mice was examined. The results suggested that production of the AK form is due to the selective inhibition of kinetoplast duplication of the drug without any effect on nuclear and cytoplasmic multiplication. When the parasites were treated with moderate doses of OHSA, segmenting forms without stainable kinetoplasts, were occasionally seen but attempts to establish a clone of akinetoplastic parasite were unsuccessful. Electron microscopy of parasites obtained after OHSA treatment showed not only irregular division of the kinetoplast but also the disorganization of kinetonucleus with disappearance of its envelope. Therefore, it was concluded that the AK forms were also produced by OHSA through disappearance of the kinetoplast.     

Characterization by sedimentation analysis of kinetoplast DNA from Trypanosoma cruzi at different stages of culture.

The kinetoplast DNA networks of Trypanosoma cruzi exist under two forms which have been studied by equilibrium density centrifugation in CsCl gradients containing ethidium bromide and by band sedimentation analysis. The relative proportion of the two forms has been measured and varies significantly between the exponential and stationary phase of growth, suggesting that one of these forms is a replicative intermediate. Both forms exhibit very high sedimentation coefficients. The sedimentation velocity ethidium titration was used to measure the superhelix density of the kinetoplast DNA after having established the validity of the method with in vitro closed DNA networks. The superhelix density of the native form of the kinetoplast DNA minicircles is very low and varies according to the physiological state of the trypanosomes. Furthermore, we observed a significant increase of the superhelix density of the kinetoplast DNA of trypanosomes grown in the presence of ethidium.     

THE FINE STRUCTURE OF BLASTOCLADIELLA EMERSONII ZOOSPORES

The zoospore of Blastocladiella emersonii has been re-examined with the electron microscope. The following new findings were made. A double unit-membrane system surrounds all cell organelles except γ-bodies, vacuoles and a few fragments of membranes. Lipid granules on one side of the large mitochondrion alternate with vesicles. The kinetosome of the posterior flagellum does not have any central fibrils as previously reported; a small, cylindrical structure is found within its anterior end. An associated centriole is located next to the kinetosome. Three striated rootlets pass from the kinetosome by separate channels through the mitochondrion. There appears to be no connection between the striated rootlets and the mitochondrion. Microtubules originating at the anterior end of the kinetosome pass into the cytoplasm between the mitochondrion and the nuclear cap. Long, dense strands were observed in some nuclei. The axoneme is taken up into the spore during encystment and is found in the freshly encysted spore. No trace of the flagellar sheath has been found in the encysted spore.