Absolute orientation of the flagellar apparatus of Hibberdia magna comb. nov. (Chrysophyceae)

The first flagellum of Hibberdia magna comb. nov. bears mastigonemes that have both short and long lateral filaments attached to the tubular shaft. The second flagellum is very short (ca. 850 nm) and is directed posteriorly approximately 160° from the first flagellum. Three microtubular flagellar roots (R₁, R₂ and R₄) and a rhizoplast (= striated root) are present. The R₁ root consists of four microtubules that arise near the right surface of the first flagellum basal body; the R₁ root extends to the dorsal side of the cell and then curves back along the left side of the cell. Cytoskeletal microtubules are nucleated from the R₁ root including one loose cluster of cytoskeletal microtubules that extends down the left side of the cell adjacent to the contractile vacuole. The R₂ root is a single microtubule that arises along the left surface of the first flagellum basal body and extends to the left side of the cell. The R₄ root consists of three microtubules that arise along the left side of the second flagellum basal body. A helical band wraps around two microtubules at the proximal end of the R₄ root. Two of the three R₄ root microtubules extend along the left side of the second flagellum, curve around to the right side of that flagellum and terminate. No R₃ root was found. The orientation of the basal bodies of Hibberdia gen. nov. is similar to that of the Xanthophyceae and Oomycetes. There are apparent homologies in the R₁, R₂ and R₄ roots of Hibberdia and these and other protists, but only Hibberdia lacks a R₃ root. Three long flagella are present in preprophase but later one is endocytosized and the axoneme extends to the posterior of the cell. During metaphase the nuclear envelope is more or less intact except at the poles; the flagellar apparatuses are at the poles and the spindle microtubules originate near the basal bodies. Two stages are known in the life history: 1) a capsoidlike state with non-swimming flagellate cells inside a colonial gel, and 2) a free-swimming single-celled monad state. Vegetative cell division occurs in both stages. The flagellar apparatus, the cell division process and the life history combined with the previously described unique light-harvesting antheraxanthin make H. magna distinct from other algae. A new genus, Hibberdia gen. nov., a new family, Hibberdiaceae fam. nov. and a new order, Hibberdiales, ord. nov. are described.     

ANALYSIS OF MICROTUBULE STRUCTURE IN EUGLENA GRANULATA

The microtubules associated with the pellicle, reservoir and locomotory flagellum of Euglena granulata were subjected to rotational analysis using the technique of Markham et al. The results indicate that the microtubules have 11-14 subunits when viewed in transverse section. Rotational analysis of the entire locomotory flagellum was also conducted to better demonstrate the nature of the arms extending from the peripheral microtubules.     

Twist (and rotation?) of central-pair microtubules in flagella ofPoterioochromonas

Summary The chrysoflagellate algaPoterioochromonas bears two unequal flagella. There is a short naked one and a long flagellum with mastigonemes. Ultrastructural investigation reveals that the centralpair microtubules in both flagella have no fixed position with respect to the flagellar base and root system, or the mastigoneme rows in the long flagellum. The central-pair microtubules are twisted several times along the length of the flagellum. This might indicate active or passive rotation of the central-pair microtubules during flagellar beat.     

THE FLAGELLAR APPARATUS OF CHRYSOLEPIDOMONAS DENDROLEPIDOTA (CHRYSOPHYCEAE), A SINGLE-CELLED MONAD COVED WITH ORGANIC SCALES1

The flagellar apparatus of Chrysolepidomonas dedrolepidota Peters et Andersen is similar to that of other members of the Ochromonadales, Chrysophyceae. there are four microtubular roots (R_1-4) and a system II fiber (= rhizoplast). the R₁ root consists of three microtubules that nucleate many cytoplasmic microtubules. One compressed band of 10 or more cytoplasmic microtubules is directed black along the R1 root in an anti-parallel direction. The R₂ root consists of one to two microtubules, and it extends toward the distal end of the R₁ root. The R₃ root consists of six (?seven) microtubules near its proximal end. The “a” and “f” microtubules of the R₃ root are under the short flagellum, and the “f” microtubule loops back and under the basal body, extending down to the nucleus. The R₄ root consists of one to two microtubules extending along the left side of the shot flagellum and curving under the short flagellum where it terminates near the “a” microtubule of R₃ Both flagella have a transitional plate and a transitional helix with five gyres. There is a thin, second plate in the basal body at the level of the distal end of the “c” tubules of the basal body triplets. The tripartite flagellar hairs have long lateral filaments but lack short lateral filaments. We compare the flagellar apparatus with that of other members of the Ochromonadales and members of the Hydrurales and Hibberdiales.     

Transformation of the flagella and associated flagellar components during cell division in the coccolithophoridPleurochrysis carterae

Summary Immunofluorescence microscopy, conventional and high voltage transmission electron microscopy were used to describe changes in the flagellar apparatus during cell division in the motile, coccolithbearing cells ofPleurochrysis carterae (Braarud and Fagerlund) Christensen. New basal bodies appear alongside the parental basal bodies before mitosis and at prophase the large microtubular (crystalline) roots disassemble as their component microtubules migrate to the future spindle poles. By prometaphase the crystalline roots have disappeared; the flagellar axonemes shorten and the two pairs of basal bodies (each consisting of one parental and one daughter basal body) separate so that each pair is distal to a spindle pole. By late prometaphase the pairs of basal bodies bear diminutive flagellar roots for the future daughter cells. The long flagellum of each daughter cell is derived from the parental basal bodies; thus, the basal body that produces a short flagellum in the parent produces a long flagellum in the daughter cell. We conclude that each basal body in these cells is inherently identical but that a first generation basal body generates a short flagellum and in succeeding generations it produces a long flagellum. At metaphase a fibrous band connecting the basal bodies appears and the roots and basal bodies reorient to their interphase configuration. By telophase the crystalline roots have begun to reform and the rootlet microtubules have assumed their interphase appearance by early cytokinesis.Abbreviations CR1, CR2 crystalline roots 1 and 2 - CT cytoplasmic tongue microtubules - DIC differential interference contrast light microscopy - H haptonema - HVEM high voltage transmission electron microscopy - IMF immunofluorescence microscopy - L left flagellum/basal body - M metaphase plate - MT microtubule - N nucleus - R right flagellum/basal body - R1, R2, R3 roots 1, 2, and 3 - TEM transmission electron microscopy     

CHARACTERIZATION AND PHYLOGENETIC POSITION OF THE ENIGMATIC GOLDEN ALGA PHAEOTHAMNION CONFERVICOLA: ULTRASTRUCTURE, PIGMENT COMPOSITION AND PARTIAL SSU rDNA SEQUENCE

The morphology, ultrastructure, photosynthetic pigments, and nuclear-encoded small subunit ribosomal DNA (SSU rDNA) were examined for Phaeothamnion confervicola Lagerheim strain SAG119.79. The morphology of the vegetative filaments, as viewed under light microscopy, was indistinguishable from the isotype. Light microscopy, including epifluorescence microscopy, also revealed the presence of one to three chloroplasts in both vegetative cells and zoospores. Vegetative filaments occasionally transformed to a palmelloid stage in old cultures. An eyespot was not visible in zoospores when examined with light microscopy, but small droplets, similar to eyespot droplets, were apparent beneath the shorter flagellum when cells were viewed with electron microscopy. Zoospores had two flagella that were laterally inserted in the cell approximately one-third of the cell length from the apex. The longer flagellum was directed anteriorly and the shorter flagellum was directed posteriorly. Electron microscopy revealed the presence of tubular tripartite flagellar hairs on the longer flagellum, but no lateral filaments were found on the tripartite hairs. The general organization of the flagellar root system was similar to that of zoospores belonging to the Xanthophyceae and Phaeophyceae. However, the transitional region of the flagella contained a transitional helix with four to six gyres. Microtubular root R₁ consisted of six microtubules at its proximal end and one microtubule at its distal end. Roots R₂ and R₄ consisted of one microtubule each and root R₃ consisted of two microtubules. No rhizoplast was found. Thin-layer chromatography revealed the presence of fucoxanthin, diadinoxanthin, neoxanthin, and heteroxanthin as well as chlorophylls a, c₁ and c₂. High-performance liquid chromatography revealed the presence of fucoxanthin, diadinoxanthin, diatoxanthin, heteroxanthin, and β,β-carotene as well as chlorophylls a and c. The complete sequence of the SSU rDNA could not be obtained, but a partial sequence (1201 bases) was determined. Parsimony and neighbor-joining distance analyses of SSU rDNA from Phaeothamnion and 36 other chromophyte algae (with two Öomycete fungi as the outgroup) indicated that Phaeothamnion was a weakly supported (bootstrap = <50%, 52%) sister taxon to the Xanthophyceae representatives and that this combined clade was in turn a weakly supported (bootstrap = <50%, 67%) sister to the Phaeophyceae. Based upon ultrastructural observations, pigment analysis, and SSU rDNA phylogenetic analysis, Phaeothamnion is not a member of the Chrysophyceae and should be classified as incertae sedis with affinities to the Xanthophyceae and Phaeophyceae.     

The Morphology,Ultrastructure and SSU rRNA Gene Sequence of a New Freshwater Flagellate,Neobodo borokensis n. sp. (Kinetoplastea,Excavata)

A small free‐living freshwater bacteriotrophic flagellate Neobodo borokensis n. sp. was investigated by electron microscopy and analysis of its SSU ribosomal RNA gene. This protist has paraxonemal rods of typical bodonid structure in the flagella, mastigonemes on the proximal part of the posterior flagellum, two nearly parallel basal bodies, a compact kinetoplast, and discoid mitochondrial cristae. The flagellar pocket is supported by three microtubular roots (R1, R2 and R3) originating from the kinetosome. The cytopharynx is supported by the root R2, a microtubular prism, cytopharynx associated additional microtubules (CMT) and cytostome associated microtubules (FAS) bands. Symbiotic bacteria and small glycosomes were found in the cytoplasm. Cysts have not been found. The flagellate prefers freshwater habitats, but tolerates salinity up to 3–4‰. The overall morphological and ultrastructural features confirm that N. borokensis represents a new species of the genus Neobodo. Phylogenetic analysis of SSU rRNA genes is congruent with the ultrastructure and strongly supports the close relationship of N. borokensis to Neobodo saliens, N. designis, Actuariola, and a misidentified sequence of “Bodo curvifilus” within the class Kinetoplastea.     

ULTRASTRUCTURE OF SWARMERS IN THE LAMINARIALES (PHAEOPHYCEAE). II. SPERM1

The ultrastructure of sperm from 13 species in 11 genera of Laminariales collected in the northeast Pacific Ocean is unique in the brown algae. The sperm are elongate, and possess a nucleus, several mitochondria and two or three chloroplasts, but no eyespot. The anterior flagellum bears mastigonemes on the proximal half of its length; a distal “whiplash” portion lacks mastigonemes and is an extension of only the two central singlet microtubules of the axoneme. A peculiar feature of these sperm is the posterior flagellum, which is longer than the anterior flagellum and tapers distally as the doublet microtubules become singlets and decrease in number. This feature contrasts with the laminarialean zoospore, which possesses a short posterior flagellum with the usual “9 + 2” axoneme. The structure of these sperm differs from that reported for Chorda, the sperm of which resembles a primitive brown algal zoospore. The facts support the concept that Chorda is the most primitive member of the Laminariales.     

Pellicular Microtubules in the Family Trypanosomatidae*

SYNOPSIS. Flagellates of 7 species from 5 genera of the family Trypanosomatidae were prepared for electron microscopy using a combination of surface tension spreading on a simplified Langmuir trough followed by critical point drying. These technics opened the cells and showed the microtubular complex in its 3-dimensional configuration or in a flat sheet with the inside surface facing-up. Tears originated near the flagellar end and proceeded in a posterior direction usually paralleling the tubules. Major differences in the spreading pattern were species-specific. A connection between the flagellum and microtubules was not shown but strongly indicated by their constant association in all degrees of spreading. Most microtubules follow a spiral course from the flagellar end toward the posterior apical area and terminate short of the apex. It is suggested that the concept of antero-posterior orientation in the genus Trypanosoma be reconsidered. Cells of Leptomonas collosoma showed, without exception, a clockwise spiral as the tubules approached the posterior apex. Therefore, both the direction of spiraling and the manner of spreading were consistent. The number of microtubules varied among cells of the same species as well as on different levels of the same cell. Highest counts coincided with the level of greatest circumference. No connections or anastomoses between tubules were found. The microtubules are believed to have limited flexibility. Changes in diameter were interpreted to mean flattening had occurred. An intertubular substance, believed to surround the microtubules in the living cell, was present in all species.     

Ultrastructure,Behavior, and Algal Flagellate Affinities of the Helioflagellate Ciliophrys marina,and the Classification of the Helioflagellates (Protista,Actinopoda, Heliozoea)1

Ciliophrys marina is a small marine helioflagellate, with a central nucleus, which is capable of reversibly transforming from a rapidly swimming flagellate cell with no axopodia to the structure of a heliozoan with a flagellum that beats only a few times a minute. When in the flagellate form, the flagellum acts as a tractellum due to the tubular mastigonemes found along its length. When the rapidly swimming flagellate strikes a piece of debris, the flagellum goes through a very characteristic shock-induced avoidance reaction. Similarly, when a mechanical shock is delivered to the cell in its heliozoan form, the axopodia are contracted in less than 20 msec. Both reactions are inhibited in low calcium seawater. Transformation from the heliozoan to the flagellate form is accomplished by slow retraction and absorbance of the axopodia and activation of the flagellum. Ultrastructurally, each axopodium is found to contain three microtubules which attach to the outer nuclear membrane of the central nucleus at sites that this study characterizes by electron microscopy of thin sections and freeze fracture preparations. The mitochondria have tubular cristae, each containing an intracristal filament. Finally, a taxonomic review of the helioflagellates is presented, and it is suggested that C. marina is derived from the chrysomonads. An argument is also made for classifying C. marina with the heliozoan order Actinophryida, as a recently published classification of the protozoa does.     

Ultrastructure of Euglena anabaena var. minor (Euglenophyceae)

The freshwater green euglenoid Euglena anabaena var. minor has a pellicle with groove‐ridge articulation, a chloroplast with pyrenoids doubly sheathed by two paramylon caps, and a nucleus with permanently condensed chromosomes and nucleolus. The flagellar apparatus basically resembles that of Euglena. The dorsal root (DR) originates at the dorsal basal body of the emergent flagellum, while both the intermediate root (IR) and ventral root (VR) originate at the ventral basal body of the non‐emergent flagellum. The cytoplasmic pocket is associated with the ventral root/ reinforcing microtubular band. However, ultrastructural characterization of E. anabaena var. minor shows the pocket to consist of five to seven microtubules, and flagellar roots with microtubule configuration of 3–4–6 in the DR‐IR‐VR. The dorsal band microtubules pair at the reservoir‐canal transition level. The doublet microtubules are formed into triplets and doublets at the lower canal level and then make pellicular microtubules at the upper canal level.     

Fine Structure of Trypanosoma cyclops in Noncellular Cultures*

The fine structure of the epimastigotes of Trypanosoma cyclops maintained in blood agar medium at 25 C is described. This organism was isolated from the Malaysian primates Macaca nemestrina and Macaca ira. A distinctive feature of T. cyclops is that it is pigmented when grown in the presence of hemoglobin. The pigment bodies apparently lack a substructure and are electron dense even in unstained sections. Most of the pigment is located posterior to the kinetoplast region but some is found adjacent and anterior to the kinetoplast. Cells from control cultures grown in medium lacking hemoglobin did not possess this type of pigment body. Similarly, pigment was not found in cells of an Indonesian trypanosome grown in medium containing hemoglobin. The cytoplasm of T. cyclops is bounded by a unit membrane which is specialized where it makes contact with the flagellum. A cytostome extends from the region of the flagellar pocket. The kinetoplast and nucleus are immediately posterior to the base of the flagellum. Transverse sections in the region of the flagellar pocket and flagellar base often reveal a group of 3 microtubules which are distinct from the pellicular microtubules.     

Spermatology of Some Pycnogonida (Arthropoda), with Special Reference to a Microtubule-Nuclear Envelope Complex

The sperm cells of Nymphon leptocheles and N. rubrum are of the primitive type, which is a remarkable condition among arthropods. The motile sperm consist of a somewhat elongated head, a kind of midpiece and a long tail. An acrosome is absent. The nucleus is surrounded by longitudinally oriented microtubules running in furrows in the nuclear envelope. These microtubules are not interconnected by links or connected to the nuclear envelope; they persist in the mature sperm. No appreciable chromatin condensation takes place. The midpiece contains some unmodified mitochondria and a centriole. The tail is a simple, free flagellum. The results are in particular discussed in relation to other known microtubule-nuclear envelope complexes in sperm cells. The sperm cells of Pycnogonum littorale are, on the other hand, highly aberrant. They are unmotile, elongated cells containing a very high number (often more than 1000) of longitudinal microtubules arranged in complex patterns. Some folded membranes may represent the nuclear envelope. Other organelles are unidentificable or may be absent.     

Flagellum retraction and axoneme depolymerisation during the transformation of flagellates to amoebae inPhysarum

Summary The transformation ofPhysarum polycephalum flagellates to myxamoebae is characterised by disappearance of the flagellum. This transition, from the flagellate to the myxamoeba was observed by phase contrast light microscopy and recorded by time lapse video photography to determine whether flagellates shed their flagella or they are absorbed within the cell. In addition, the kinetics of flagellum disappearance were also studied. Our observations indicate that the flagellum was absorbed within the cell; the process occurred within seconds. Flagellum resorbtion was preceded by typical morphological cell changes. The shape of the nucleus altered and its mobility within the cell decreased. It was not possible to observe the flagellum within the cell with phase contrast video recordings. Thin section electron microscopy was used to study this intracellular phenomenon. Several stages of flagellum dissolution could be identified within the cell. The two most important stages were: an axoneme surrounded by the flagellar membrane within a plasma membrane lined pocket or vacuole and the naked axoneme without its membrane, free within the cell cytoplasm. The existence of cytoplasmic microtubules prevented identification of any further dissolution stages of the flagellum. A group of microtubules adjacent to the flagellum but within the cytoplasm was observed in flagellates and also in those cells which possesed enveloped axonemes. The flagellum did not dissociate from the kinetosomes before resorbtion.Immunofluorescence studies with the 6-11-B-1 monoclonal antibody indicated that acetylated microtubules exist in myxamoebae after transformation from flagellates for up to 40 min. Acetylated tubulin is not limited to the centrioles in these cells.     

CRYPTOGLENA PIGRA: A EUGLENOID WITH ONE CHLOROPLAST12

The taxonomic status of Cryptoglena pigra Ehrb., interpreted from observations based on bright-field microscopy, has been uncertain. Examination with the electron microscope of a clone of C pigra isolated by E. G. Pringsheim reveals certain features which, collectively, are distinctly euglenoid: periplast associated with muciferous bodies and subpellicular microtubules; canal and reservoir with microtubules; one flagellum with a swelling and emergent through a canal, and a second flagellum without a swelling and nonremergent; stigma (eyetpot) closely apprrssed to but not part of the chloroplast; nucleus with permanently condensed chromosomes attached to the inner nuclrar membrane; mitochondria with disc-shaped cristae constricted at the base; chloroplast with thylakoids often in triplets; and paramylon grains in the cytoplasm. Unlike most euglenoids, C. pigra possesses a single chloroplast that in transverse thin sections is U-shaped.     

Food uptake and fine structure of Cryothecomonas longipes sp. nov., a marine nanoflagellate incertae sedis feeding phagotrophically on large diatoms

Cryothecomonas longipes Schnepf and Kühn sp. nov. is a colourless biflagellate organism, 9–14 μm long and 7–9 μm wide when not filled with food vacuoles. It was detected in the North Sea, feeding with pseudopodia on diatoms. It penetrates the host shell, while the main body of the flagellate remains outside the frustule. Cells are covered with a multilayered theca. The pseudopodium protrudes through a preformed slit in the theca. Each flagellum also emerges through a pit in which the theca forms a funnel of complex structure that girdles each flagellum. The anterior flagellum is 9–15 μm long and oriented forward; the ventral flagellum, posteriorly directed, is 20–24 μm long and bears fine hairs. The flagellar roots consist of microtubules that emerge at satellites around the basal bodies and run along the flagellar pits. In addition, the ventral flagellum is accompanied by a band of six microtubules. It is proximally attached to a small fibrillar band, which interconnects the basal bodies. Cryothecomonas longipes has two or three types of extrusomes which pierce the theca when discharged. Their mode of discharge is discussed. Microbody-like vesicles containing small tubules are closely associated with older digestion vacuoles. Cryothecomonas longipes is compared with other species of the genus and a diagnosis is given. Received: 4 March 1999 / Received in revised form: 28 July 1999 / Accepted: 30 August 1999     

A light and electron microscopic study of the flagellate-to-ameba conversion in the MyxomyceteStemonitis pallida

Summary The flagellate-to-ameba conversion process of the MyxomyceteStemonitis pallida was investigated with Nomarski optics and electron microscopy. The flagellate has two flagella, a long and a short one. When the water film containing the flagellates becomes very thin, they retract their flagella, usually the short one first and then the long one. The short flagellum is retracted by only one method, in which the sheath membrane of the flagellum fuses with the cell membrane, consequently causing the axoneme to be absorbed into the cytoplasm. Retraction of the long flagellum can be divided into four types. In all cases, fusion of the sheath membrane and the cell membrane takes place. The retracted axoneme of the long flagellum sometimes beats convulsively for about 10 minutes after retraction, and after 10–15 minutes it became indistinguishable as it was detached from the blepharoplast.Analysis of thin sections shows that the retracted axonemes disintegrate in the following squence: B-tubules, A-tubules, spokes, central microtubules. In almost all cells the degradation begins immediately after retraction and is completed within 90 minutes. Only on rare occasions, structures which seem to have been derived from retracted axonemes are observed in the ameba about 90 minutes after conversion. The basal bodies and cytoplasmic microtubules are a little more stable than the retracted axonemes. Some basal bodies of the short flagellum, whose C-tubules are affected, are present in the amebae more than 90 minutes after conversion. Cytoplasmic microtubules decrease in number and become shorter in the amebae after about 24 hours, when newly formed regions filled with flocculent material appear.     

The Trypanosoma brucei cytoskeleton: Ultrastructure and localization of microtubule-associated and spectrin-like proteins using quick-freeze, deep-etch, immunogold electron microscopy

We have used a combination of quick-freezing/deep-etching and colloidal gold immunocytochemistry (i) to analyze the molecular organization of the microtubular membrane skeleton and the flagellum of Trypanosoma brucei, and (ii) to localize two defined cytoskeletal proteins within these structures. The cell body of trypanosomatids is enveloped by a membrane skeleton consisting of a tightly packed array of microtubules which are closely associated with the cell membrane. The membrane-oriented face of these microtubules is richly decorated with microtubule-associated proteins, which form intermicrotubule and microtubule-membrane linkers. In contrast, the cytoplasmic faces of the microtubules have a smooth, nondecorated appearance. A previously identified, highly repetitive microtubule-associated protein is confined to the membrane-oriented face of the microtubular array, suggesting that the function of this protein may be that of a microtubule-membrane linker. Quickfreezing has also been used to reveal the geometric organization of the paraflagellar rod structure in the flagellum, its interaction with the cell body, and a unique series of fleur-de-lis-like molecules which link this organelle to axonemal microtubules. Immunohistochemistry using an antibody against human erythrocyte spectrin suggests that these linker structures may contain ancestral spectrin-like molecules.