DISTINCTIVE LECTIN LABELING OF THE FLAGELLAR APPARATUS OF TETRASELMIS (PRASINOPHYCEAE) AND DUNALIELLA (CHLOROPHYCEAE)1

Fluorescent labeling of the flagellar apparatus of Tetraselmis (Prasinophyceae) and Dunaliella (Polyblepharidaceae, Chlorophyceae) were successfully performed using fluorescein isothiocyanate–labeled lectins from Arachis hypogaea and Glycine maxima. These lectins specifically bound to the flagella and kinetosome of the cell but did not bind to the cell surface. Lectin binding on the flagellar apparatus remained constant under different culture media, temperatures, irradiances, cell division cycle, and culture aging. All the Tetraselmis and Dunaliella analyzed (five species, 20 clones) showed intense labeling of the flagellar apparatus. In contrast, no other species analyzed (46 clones of 25 species from four classes) showed binding to their flagellar apparatus. After the lectin treatment, many cells remained alive, and they were able to swim with the flagellar apparatus intensely labeled. The lectin binding indicates that the flagella and kinetosome of Tetraselmis are rich in Gal and GalNH₂ moieties and that the flagella of Dunaliella are rich in Gal and GalNAc moieties. Apparently, this feature seems to be specific to these species.     

DEVELOPMENT OF THE FLAGELLAR APPARATUS AND FLAGELLAR POSITION IN THE COLONIAL GREEN ALGA PLATYDORINA CAUDATA (CHLOROPHYCEAE)1

The ultrastructure of the flagellar apparatus in pre-inversion and inversion stages of Platydorina resembles that of Chlamydomonas in having 180° rotational symmetry and clockwise absolute orientation. Basal bodies are in a “V” configuration and connected by one distal and two proximal fibers. Alternating two- and four-membered microtubular rootlets are cruciately arranged. During maturation, the basal bodies rotate and separate, and 180° rotational symmetry is lost. Simultaneously, each proximal fiber detaches from one of the functional basal bodies, and the distal fiber detaches from both. The mature apparatus has widely separated and nearly parallel basal bodies. Flagellar orientation in Platydorina is completed just after inversion and a flattening of the colony called intercalation, resulting in the pairs of flagella of neighboring cells extending from the colony in opposite directions in an alternating fashion. Flagellar orientation and separated basal bodies minimize the interference between the flagella of neighboring cells. Basal bodies and rootlets of the two intercalated halves of a colony rotate, resulting in the effective strokes of the flagella of every cell being towards the colonial posterior. The flagella of each cell beat with an effective stroke in the direction of the two inner rootlets. The flagella have an asymmetrical ciliary type beat. The rotated, separated, and parallel basal bodies, together with the nearly parallel rootlets probably are adaptations for movement of this colonial volvocalean alga. The flagellar apparatus in immature stages of Platydorina lends support to the suggestion that the alga has evolved from a Chlamydomonas-like ancestor.     

Coordinated Flagellar and Ciliary Beating in the Protozoon Tritrichomonas foetus1

ABSTRACT. Tritrichomonas foetus is a flagellated protozoon found in urogenital tract of cattle. Its free movement in liquid medium is powered by the coordinated movement of three flagella projecting towards the anterior region of the cell, and one recurrent flagellum that forms a junction with the cell body and ends as a free projection in the posterior region of the cell. We have used video microscopy and digital image processing to analyze the relationships between the movements of these flagella. The anterior flagella beat in a ciliary type pattern displaying effective and recovery strokes, while the recurrent flagellum beats in a typical flagellar wave form. One of the three anterior flagella has a distinctive pattern of beating. It beats straight in its forward direction as opposed to the ample beats performed by the others. Frequency measurements obtained from cells swimming in a viscous medium shows that the beating frequency of the recurrent flagelium is approximate twice the frequency for the three anterior flagella. We also observed that the costa and the axostyle do not show any active motion. On the contrary, they form a cytoskeletal base for the anchoring and orientation of the flagella.     

Swimming behavior and ultrastructure of sperm of<Emphasis Type="Italic"> Lygodium japonicum</Emphasis> (Pteridophyta)

Swimming behavior of the sperm of Lygodium japonicum (Pteridophyta) and the associated ultrastructure of the flagellar apparatus were studied by video microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The sperm has approximately 70 flagella that emerge from a sinistrally-coiled flagellar apparatus, and swims forward by ciliary beat of these flagella. Backward swimming was not observed even after sperm collided with obstacles. Video microscopy showed that the flagella of the swimming sperm are oriented laterally and oblique-anteriorly. TEM and SEM observations revealed that the basal bodies of these flagella are arranged in at least two rows and oriented in the same directions as observed by video microscopy. These basal bodies (flagella) are categorized into two types according to their orientation: group I (laterally directed) and group II (oblique-anteriorly directed). The directionality of the basal bodies appears to be fixed by electron-dense material around their base. The outer dynein arms of the flagellar axoneme are entirely absent. These morphological characteristics of basal bodies (flagella) may relate to the lack of backward swimming behavior of the sperm. Based on these results, the evolution of swimming behavior in the archegoniates is discussed in connection with lack of backward swimming in a distantly related green alga, Mesostigma viride, and the Streptophyta.     

FLAGELLAR MOTION AND FINE STRUCTURE OF THE FLAGELLAR APPARATUS IN CHLAMYDOMONAS

The biflagellate alga Chlamydomonas reinhardi was studied with the light and electron microscopes to determine the behavior of flagella in the living cell and the structure of the basal apparatus of the flagella. During normal forward swimming the flagella beat synchronously in the same plane, as in the human swimmer's breast stroke. The form of beat is like that of cilia. Occasionally cells swim backward with the flagella undulating and trailing the cell. Thus the same flagellar apparatus produces two types of motion. The central pair of fibers of both flagella appear to lie in the same plane, which coincides with the plane of beat. The two basal bodies lie in a V configuration and are joined at the top by a striated fiber and at the bottom by two smaller fibers. From the area between the basal bodies four bands of microtubules, each containing four tubules, radiate in an X-shaped pattern, diverge, and pass under the cell membrane. Details of the complex arrangement of tubules near the basal bodies are described. It seems probable that the connecting fibers and the microtubules play structural roles and thereby maintain the alignment of the flagellar apparatus. The relation of striated fibers and microtubules to cilia and flagella is reviewed, particularly in phytoflagellates and protozoa. Structures observed in the transitional region between the basal body and flagellar shaft are described and their occurrence is reviewed. Details of structure of the flagellar shaft and flagellar tip are described, and the latter is reviewed in detail.     

X-ray equatorial diffraction studies on the cross-sectional structure of Salmonella flagella

X-ray diffraction studies have been made on the cross-sectional structure of the normal Salmonella flagella. Two approaches have been made: one based upon small-angle equatorial scatterings (2θ 3°) and the other upon moderate-angle angle equatorial diffractions (3° 2θ 10°).Analysis of small-angle scattering data gives the radius of gyration of the flagella as 68 Å. Cylindrically averaged electron density of the cross-section of the flagella is obtained by means of the Fourier-Bessel transformation method. The average radius of the flagella is about 65 Å.In the investigation of the moderate-angle diffraction pattern, validity is examined of the model that a flagellum consits annularly arranged strands, of which each has a cylindrically symmetric structure. Features of the pattern observed in the range of 3° < 2θ < 10° can be interpreted fairly well by this model. Average radii of the flagella obtained for the 11 and 13 strands models are close to that obtained by the analysis of the small-angle scattering data.     

Phylogenetic analysis of ITS2 sequences suggests the taxonomic re‐structuring of Dunaliella viridis (Chlorophyceae,Dunaliellales)

We analyzed the ITS2 primary and secondary structure (including Compensatory Base Changes (CBCs)) of 17 new Dunaliella strains (11 D. viridis, two D. tertiolecta, and four Dunaliella sp.), and compared these with other Dunaliella sequences available from the ITS2 database to circumscribe their taxonomic position. The ITS2 primary and secondary structure analysis positioned the majority of D. viridis strains in four main clades, showing that D. viridis is polyphyletic. The detection of at least one CBC among these clades strongly suggests that they could correspond to different biological species. Unexpectedly, while D. viridis var. euchlora (CCAP19/21) was positioned within the subgenus Dunaliella, D. viridis var. palmelloides (CCAP11/34) was positioned clearly outside this subgenus, suggesting that this taxon may not be properly placed in Dunaliella. Furthermore, the detection of at least three compensatory base changes (CBCs) between D. viridis var. palmelloides (CCAP11/34) and the other strains analyzed, confirm that this strain is a different species. For these reasons we propose re‐naming D. viridis var. palmelloides (CCAP11/34) to incertae sedis, and D. viridis var. euchlora (CCAP19/21) to Dunaliella sp. Therefore, the ITS2 primary and secondary structure data suggest a taxonomic re‐structuring of D. viridis.     

Isolation and Properties of Flagella of Trypanosomatids*

SYNOPSIS. A procedure is described for the isolation of flagella of Crithidia fasciculata. Herpetomonas samuelpessoai and Leishmania tarentolae in a highly purified state and giving reasonably good yield. The 3 types of flagella give a similar electrophoretic pattern of proteins. It is shown that H. samuelpessoai and, to a lesser extent, C. fasciculata flagella confer protection against Trypanosoma cruzi infection.     

Swimming patterns of the quadriflagellate Tetraflagellochloris mauritanica (Chlamydomonadales,Chlorophyceae)

Chlamydomonadales are elective subjects for the investigation of the problems related to locomotion and transport in biological fluid dynamics, whose resolution could enhance searching efficiency and assist in the avoidance of dangerous environments. In this paper, we elucidate the swimming behavior of Tetraflagellochloris mauritanica, a unicellular–multicellular alga belonging to the order Chlamydomonadales. This quadriflagellate alga has a complex swimming motion consisting of alternating swimming phases connected by in‐place random reorientations and resting phases. It is capable of both forward and backward swimming, both being normal modes of swimming. The complex swimming behavior resembles the run‐and‐tumble motion of peritrichous bacteria, with in‐place reorientation taking the place of tumbles. In the forward swimming, T. mauritanica shows a very efficient flagellar beat, with undulatory retrograde waves that run along the flagella to their tip. In the backward swimming, the flagella show a nonstereotypical synchronization mode, with a pattern that does not fit any of the modes present in the other Chlamydomonadales so far investigated.     

HYPERSALINE SOIL SUPPORTS A DIVERSE COMMUNITY OF DUNALIELLA (CHLOROPHYCEAE)1

Numerous isolates of the green halophile Dunaliella were studied as part of a survey of microbial diversity at the Great Salt Plains (GSP) in Oklahoma, USA. The GSP is a large (～65 km²) salt flat with extreme temporal and spatial fluctuations in salinity and temperature. Although the flagellate halophile Dunaliella is common worldwide, nearly all cultured isolates are from saline habitats that are primarily aquatic rather than primarily terrestrial. The diverse GSP Dunaliella strains exhibit three morphotypes: a predominantly motile form, a motile form with a prominent palmelloid phase (nonmotile, mucilage rich), and a palmelloid form with a weakly motile phase. All had broad salinity optima well below typical in situ salinities at the GSP, and two of the palmelloid isolates grew as well in freshwater as in highly saline media. Molecular phylogenetic and evolutionary analyses revealed that Dunaliella from the GSP (and two similar habitats in the Great Basin, USA) are allied with D. viridis Teodor. but possess phylogenetic diversity in excess of existing global isolates from aquatic habitats. In addition, isolates from primarily terrestrial habitats exhibit statistically higher rates of nucleotide substitution than the phylogenetically homogeneous set of primarily aquatic Dunaliella taxa. We hypothesize that dynamically extreme saline soil habitats may select for different and more diverse Dunaliella lineages than more stable saline aquatic habitats. We also propose Dunaliella as a tractable microbial model for in situ testing of evolutionary and phylogeographic hypotheses.     

THE LOCOMOTOR BEHAVIOR OF LYGODIUM AND MARSILEA SPERM

Flagella of living sperm of the ferns, Lygodium japonicum (Thunb.) Sw. and Marsilea vestita Hook, and Grev., beat three dimensionally with a continuous traveling helical wave. The wave is propagated from base to tip of the flagellum. Flagella of Lygodium and Marsilea complete 65 and 30 beat cycles per sec, respectively. Each flagellum circumscribes an open conicoid oriented in a latero-posterior direction. Only dead sperm have anteriorly directed flagella as illustrated in plant morphology textbooks.     

Ultrastructure of the flagellar apparatus inPyramimonas octopus (Prasinophyceae)

Summary While green algae usually lack one of the outer dynein arms in the axoneme, flagella of the octoflagellated prasinophytePyramimonas octopus possess dynein arms on all peripheral doublets. The outer dynein arm on doublet no. 1 is modified, and additional structures are associated with doublets no. 2 and 6. The flagellar scales are asymmetrically arranged. Thus the two rows of thick flagellar hairscales are displaced towards doublet no. 6,i.e., in the direction of the effective stroke of each flagellum. The underlayer of small scales includes two nearly opposite double rows scales, arranged in the longitudinal direction of the flagellum. The hairscales emerge from these rows. The double rows are separated on one side by 9, on the other by 11 rows of helically arranged scales. The central pair of microtubules twists, but the axoneme itself (represented by the 9 peripheral doublets), does not seem to rotate. The flagella are arranged in two groups, showing modified 180° rotational symmetry. The effective strokes of the two central flagella are exactly opposite, while the other flagella beat in six intermediate directions.     

Phylogenetic analysis of Dunaliella (Chlorophyta) emphasizing new benthic and supralittoral isolates from Great Salt Lake

Dunaliella, a commercially important chlorophyte, is globally distributed in saline habitats. Morphological species have not been definitively reconciled with phylogenetic analyses. Considerable genetic diversity continues to be discovered in new isolates, especially from soil and benthic habitats. Twenty‐nine new isolates from Great Salt Lake, Utah, many from benthic or supralittoral habitats, were phylogenetically analyzed using ITS1+5.8S+ITS2 in comparison to a broad sampling of available sequences. A few new isolates align in one branch of a bifurcated monophyletic Dunaliella salina clade and several cluster within monophyletic D. viridis. Several others align with relatively few unnamed strains from other locations, comprising a diverse clade that may represent two or more new species. The overall Dunaliella clade is relatively robust, but the nearest outgroups are ambiguously placed with extremely long branches. About half of the isolates, all from benthic or supralittoral habitats, have been persistently sarcinoid in liquid media since isolation. This trait is spread across the Dunaliella phylogeny. The morphology of two sarcinoid strains was documented with light microscopy, revealing an extensive glycocalyx. Clumping behavior of unicellular and sarcinoid strains was unaffected by presence or absence of Mg²⁺ or Ca²⁺, addition of lectin‐inhibiting monosaccharides, or water‐soluble factors from morphologically opposite strains. Results from this investigation have significantly expanded our current understanding of Dunaliella diversity, but it seems likely that much remains to be discovered with additional sampling.     

A backward swimming mutant of Chlamydomonas reinhardii

A backward swimming mutant (RL-10) was isolated from Chlamydomonas reinhardii. In contrast to the wild-type flagellum which usually displays a ciliary type beating pattern, the flagella in the RL-10 cells always propagated such undulating waves as found in sperm flagella. This abnormal beating pattern was maintained after the cell was demembranated by a non-ionic detergent (Nonidet P40) and reactivated with ATP. Reactivated axonemes (demembranated flagella) of the wild-type cells changed the beating pattern from the ciliary type to the flagellar type when the Ca²⁺ concentration was increased from 10⁻⁷ to 10⁻⁶ M. However, the RL-10 axonemes did not show such a Ca-dependent change in the beating pattern. Hence the RL-10 flagella might carry defects in the controlling mechanisms of flagellar beating pattern, at sites other than the membrane.     

MORPHOLOGICAL AND GENETIC CHARACTERIZATION OF PHAEOCYSTIS CORDATA AND P. JAHNII (PRYMNESIOPHYCEAE), TWO NEW SPECIES FROM THE MEDITERRANEAN SEA

Two new Phaeocystis species recently discovered in the Mediterranean Sea are described using light and electron microscopy, and their systematic position is discussed on the basis of an analysis of their nuclear-encoded small-subunit ribosomal RNA gene (SSU rRNA) sequences. Phaeocystis cordata Zingone et Chrétiennot-Dinet was observed only as flagellated unicells. Cells are heart shaped, with two flagella of slightly unequal length and a short haptonema. The cell body is covered with two layers of thin scales. The outermost layer scales are oval, with a faint radiating pattern, a raised rim, and a modest central knob. The inner-layer scales are smaller and have a faint radiate pattern and an inflexed rim. Cells swim with their flagella close together, obscuring the haptonema, pushing the cell, and causing it to rotate about its longitudinal axis while moving forward. Phaeocystis jahnii Zingone was isolated as a nonmotile colony. It forms loose aggregates of cells embedded in a mucilaginous, presumably polysaccharide matrix without a definite shape or visible external envelope. The flagellated stage has the features typical of other Phaeocystis species. Cells are rounded in shape and slightly larger than P. cordata. The cell body is covered with extremely thin scales of two different sizes with a very faint radiating pattern toward their margin. Swimming behavior is similar to that of P. cordata, with the flagella in a posterior position as the cells swim. The SSU rRNA sequence analysis indicated that both species are distinct from other cultivated Phaeocystis species sequenced to date. Regions previously identified as specific for the genus Phaeocystis are not found in P. jahnii, and new genus-specific regions have been identified. P. cordata is more closely related to the colonial species P. globosa, P. antarctica, and P. pouchetii and has branched prior to the divergence of the warm-water P. globosa species complex from the cold-water species P. antarctica and P. pouchetii. These results are discussed within a framework ofthe available data on the evolution of the world’s oceans.     

The biotechnology of cultivating Dunaliella for production of β-carotene rich algae

Dunaliella accumulates massive amounts of β-carotene when cultivated under high light intensity and growth-limiting conditions. The pathway for biosynthesis of β-carotene was elucidated by analysis of the effect of selected inhibitors. The presence of the inhibitors elicited the accumulation of the following intermediates: β-zeacarotene, lycopene, ζ-carotene, phytofluene, phytoene and a few unidentified long-chain isoprenoids. Each of the accumulated intermediates was composed of about equal amounts of two stereisomers, as is the case for β-carotene in the untreated algae. It is deduced, therefore, that the isomerization reaction occurs early in the pathway of β-carotene biosynthesis, at or before phytoene.The unique caratenogenesis properties of Dunaliella led to the development of a new biotechnological process for mass-cultivation of the alga. Commercial production facilities for β-carotene rich Dunaliella exist today in Israel, USA, Australia, Spain and China. Recent developments, which indicate that the stereoisometric mixture of β-carotene present in Dunaliella is preferentially absorbed in animal tissues, coupled with new evidence for the efficacy of β-carotene in reducing the incidence of cancer, have opened new vistas of potential markets for the high β-carotene algae.     

High-speed video observation of swimming behavior and flagellar motility ofProrocentrum minimum (Dinophyceae)

Summary To understand the functions of the longitudinal and transverse flagella of dinoflagellates, the flagellar waveform and frequency of each flagellum were observed by high-speed video-recording. The longitudinal flagellum emerged from the anterior end of the cell and beat with a planar undulating wave whose plane was perpendicular to the valval sutural plane. The transverse flagellum curved around the anterior end of the cell and beat with a helical wave, with different alternating half pitches. The half pitch corresponding to the parts farther from the cellular antero-posterior axis was shorter than that of the parts closer to the axis. This pattern is described by the ratio of the outer-parts half pitch to the pitch of the whole period of the helix and seems to be characteristic of the dinoflagellates' transverse flagellum.Abbreviations p _in half pitch corresponding to the inner parts of the transverse flagellum - p _out half pitch corresponding to the outer parts of the transverse flagellum - P _p pitch of helical swimming trajectory - R _p radius of helical swimming trajectory - _c rotational frequency of the cell     

Ribosomes ofDunaliella

Summary The high molecular weight ribonucleic acids from the green algaDunaliella were isolated from wholeDunaliella cells and fromDunaliella ribosomes and analysed by the technique of sucrose density centrifugation. Ribonucleic acids from whole cells and fromDunaliella ribosomes showed the same sedimentation profile only when ribosomes were prepared in the presence of the RNase inhibitor polyvinyl sulfate. Otherwise ribonucleic acids fromDunaliella ribosomes were degraded to some extent, as compared with those from whole cells, although the ribosomes were still physically intact. The ribonucleic acids fromDunaliella were resolved by sucrose density centrifugation into three high molecular weight components sedimenting with 26, 23 and 17.5s. The 80s ribosomal fraction contained mainly a 26 and 17.5 s RNA, whereas the 50 s ribosomal fraction contained a 23 s RNA. The 26 s RNA and the 23 s RNA may represent the heavy ribonucleic acids from the cytosol and the chloroplast of the cell respectively, whereas the 17.5 s RNA may be a mixture of the two light RNA's from the two cell compartments.The experiments described in this paper were submitted by H. J.Rahmsdorf to the Fachbereich Biologie der Freien UniversitÄt Berlin in partial fulfillment of the requirements for a doctor's degree.