NOVEL PELLICLE SURFACE PATTERNS ON EUGLENA OBTUSA (EUGLENOPHYTA) FROM THE MARINE BENTHIC ENVIRONMENT: IMPLICATIONS FOR PELLICLE DEVELOPMENT AND EVOLUTION1

Euglena obtusa F. Schmitz possesses novel pellicle surface patterns, including the greatest number of strips (120) and the most posterior subwhorls of strip reduction in any euglenid described so far. Although the subwhorls form a mathematically linear pattern of strip reduction, the pattern observed here differs from the linear pattern described for Euglena mutabilis F. Schmitz in that it contains seven linear subwhorls, rather than three, and is developmentally equivalent to three whorls of exponential reduction, rather than two. These properties imply that the seven‐subwhorled linear pattern observed in E. obtusa is evolutionarily derived from an ancestral bilinear pattern, rather than from a linear pattern, of strip reduction. Furthermore, analysis of the relative lateral positions of the strips forming the subwhorls in E. obtusa indicates that (1) the identity (relative length, lateral position, and maturity) of each strip in any mother cell specifies that strip’s identity in one of the daughter cells following pellicle duplication and cell division, (2) the relative length of any given pellicle strip regulates the length of the nascent strip it will produce during pellicle duplication, and (3) pellicle pores develop within the heels of the most mature pellicle strips. These observations suggest that continued research on pellicle development could eventually establish an ideal system for understanding mechanisms associated with the morphogenesis and evolution of related eukaryotic cells.     

THE FLAGELLAR APPARATUS AND RESERVOIR/CANAL CYTOSKELETON OF CRYPTOGLENA PIGRA (EUGLENOPHYCEAE)1

The flagellar apparatus and reservoir cytoskeleton of Cryptoglena pigra Ehrenberg are described. Three flagellar roots are associated with the two basal bodies. The four-membered dorsal root arises from the dorsal basal body and extends anteriorly following the reservoir membrane. At the base of the reservoir the dorsal root nucleates a large microtubular group termed the dorsal band. The dorsal band continues anteriorlhy between the reservoir and eyespot and is continuous with the microtubules of the canal and ultimately the pellicle. The ventral basal body is associated with two roots. The four-membered intermediate root proceeds anteriorly and extends the length of the reservoir. The seven-to eight-membered ventral root projects anteriorly along the reservoir membrane and bends away from the reservoir. At this point, the microtubules of the ventral root line a cytoplasmic pocket and are termed the MTR (reinforcing microtubules). The canal region is composed of longitudinal microtubules surrounded by two semicircles of microtubles. Ultimately, the fifteen ridges of the canal give rise to the pellicular ridges.     

ULTRASTRUCTURE OF THE BASAL BODY COMPLEX AND PUTATIVE VESTIGIAL FEEDING APPARATUS IN PHACUS PLEURONECTES (EUGLENOPHYCEAE)

Phacus pleuronectes (O. F. Müller) Dujardin is a phototrophic euglenoid with small discoid chloroplasts, a flat rigid body, and longitudinally arranged pellicular strips. The flagellar apparatus consisted of two basal bodies and three flagellar roots typical of many phototrophic euglenoids but also had a large striated fiber that connected the two basal bodies and associated with the ventral root. The three roots, in combination with the dorsal microtubular band, extended anteriorly and formed the major cytoskeletal elements supporting the reservoir membrane and ultimately the pellicle. A cytoplasmic pocket arose in the reservoir/canal transition region. It was supported by the ventral root and a C-shaped band of electron-opaque material that lined the cytoplasmic side of the pocket. A large striated fiber extended from this C-shaped band toward the reservoir membrane. The striated fibers in the basal apparatus and associated with the microtubule-reinforced pocket in P. pleuronecte s appear to be similar to those of the phagotrophic euglenoids.     

Immunological and structural evidence for patterned intussusceptive surface growth in a unicellular organism. A postulated role for submembranous proteins and microtubules

The surface complex of Euglena has been examined intact and after isolation and purification by the use of mild sonication to disrupt cells. In intact cells the surface complex (pellicle complex) is oriented in a series of parallel ridges and grooves, and possesses among other components a characteristic group of four to seven microtubules. Isolated pellicles retain the ridge and groove pattern but no microtubules are present. Isolates yielded at least three major polypeptides on SDS acrylamide gels; one or more of the polypeptides are postulated to be identical with a submembrane layer present in both intact and isolated pellicles; one polypeptide appears to be in or on the surface membrane. Antibodies directed against the isolated pellicles were conjugated directly or indirectly to fluorescein, latex spheres, or ferritin. In appropriate experiments with these antibody conjugates, it has been found that antigenic sites are immobile and that new antigenic sites (daughter strips) are inserted between parental strips in replicating cells. These results together with direct observation of daughter strips by transmission electron microscopy suggest that surface growth in Euglena occurs by intussusception. Microtubules associated with the pellicle complex are postulated to play a role in the development of new daughter strips, and possibly also in cell movements.     

Reconstruction of the flagellar apparatus in Ploeotia costata (Euglenozoa) and its relationship to other euglenoid flagellar apparatuses

The flagellar apparatus of Ploeotia costata Farmer and Triemer was reconstructed using serial sectioning and TEM. The flagellar apparatus is similar to other euglenoids having two flagella arising from basal bodies connected by a striated fiber, and three asymmetrically arranged roots. The flagella emerge subapically from between the two ventral pellicle strips. The dorsal flagellum is 1/2 the body length and actively pulls the cell, while the ventral flagellum is twice the body length and drags along the substrate surface. The ventral and dorsal roots are on the opposite sides of their respective basal bodies, while the intermediate root is associated with the ventral flagellum on the side closest to the dorsal basal body. The dorsal root lines the dorsal side of the reservoir and after giving rise to the dorsal band lines the right side of the reservoir/canal. The ventral and intermediate roots join at the reservoir forming the intermediate-ventral root, which lines the left and ventral sides of the reservoir/canal. There was no evidence of a microtubule-reinforced pocket in P. costata. Comparisons with Ploeotia vilrea, Lentomonas applanatum, and related flagellar apparatuses led to the conclusion that the basic euglenoid flagellar structure is symplesiomorphic but with enough variation to be taxonomically diagnostic.     

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     

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.     

A model for the morphogenesis of strip reduction patterns in phototrophic euglenids: evidence for heterochrony in pellicle evolution

We propose a general developmental model that explains the evolutionary origin, diversification, and inheritance of pellicle strip patterns in phototrophic euglenids. Dividing cells of Euglena gracilis, E. viridis, and Phacus similis were observed with scanning electron microscopy in order to study the morphogenesis of posterior whorls of strip reduction. We found evidence that constant whorl numbers are maintained through cell division because of organized strip growth before and during cytokinesis. Alternating nascent strips form a new whorl of strip reduction at each of the anterior and posterior ends of daughter cells. Strips that terminated to form posterior whorls in the mother cell change in length during the development of daughter cells. In the mother cells of E. gracilis, the strips forming whorls I and II grow to become whorls II and III, respectively, in the daughter cells; the strips forming whorl III in the mother cell lengthen and meet with other strips already present at the posterior tip of daughter cells. This process of whorl morphogenesis during asexual reproduction is consistent with known variation in pellicle strip patterns and suggests that heterochrony played a major role in the ultrastructural evolution of phototrophic euglenids.     

Cell division of Giardia intestinalis: flagellar developmental cycle involves transformation and exchange of flagella between mastigonts of a diplomonad cell

Giardia intestinalis is a binucleated diplomonad possessing four pairs of flagella of distinct location and function. Its pathogenic potential depends on the integrity of a complex microtubular cytoskeleton that undergoes a profound but poorly understood reorganization during cell division. We examined the cell division of G. intestinalis with the aid of light and electron microscopy and immunofluorescence methods and present here new observations on the reorganization of the flagellar apparatus in the dividing Giardia. Our results demonstrated the presence of a flagellar maturation process during which the flagella migrate, assume different position, and transform to different flagellar types in progeny until their maturation is completed. For each newly assembled flagellum it takes three cell cycles to become mature. The mature flagellum of Giardia is the caudal one that possesses a privileged basal body at which the microtubules of the adhesive disk nucleate. In contrast to generally accepted assumption that each of the two diplomonad mastigonts develops separately, we found that they are developmentally linked, exchanging their cytoskeletal components at the early phase of mitosis. The presence of the flagellar maturation process in a metamonad protist Giardia suggests that the basal body or centriole maturation is a universal phenomenon that may represent one of the core processes in a eukaryotic cell.     

Ultrastructural characterization of the locomotory cytoskeletal system of the spermatozoid inGinkgo biloba

Summary The development of the locomotory cytoskeletal system of sperm is carefully coordinated with the development of the sperm inGinkgo biloba. Here we report further ultrastructural characterization of the locomotory cytoskeletal system in the developing spermatid and mature spermatozoid, particularly with respect to the initiation and early development of the flagellar apparatus. A multilayered structure (MLS) assembles from an electron-dense matrix that self-organizes after blepharoplast breakup and then further elongates. At the tail of the assembling MLS, the spline microtubules connect to an anterior beak of the nuclear envelope. Nuclear-pore complexes are found on the nuclear envelope close to this beak. The mitochondria which elongate and line up one behind the other are tightly associated with the MLS. The MLS ofG. biloba is composed of an upper layer of parallel spline microtubules and a lower layer consisting of a fibrous lamellar strip composed of paralled fibers about 9 nm in diameter. Higher-magnification images show that the fully assembled fibers of the lamellar strip consist of subunits which suggest that protofilaments are involved in the assembly processes. A unique cytoskeletal system of the spermatozoid inG. biloba is given by the anterior bundle of microtubules. This bundle, in which microtubules are arranged parallel to each other, forms between the plasmalemma and the MLS and is about 214–392 nm in cross section. These microtubules expand spirally along the MLS band. Other details of cellular fine structure of the mature spermatozoid are described.     

Dynamic changes in microtubule organization during division of the primitive dinoflagellate Oxyrrhis marina

The marine dinoflagellate Oxyrrhis marina has three major microtubular systems: the flagellar apparatus made of one transverse and one longitudinal flagella and their appendages, cortical microtubules, and intranuclear microtubules. We investigated the dynamic changes of these microtubular systems during cell division by transmission and scanning electron microscopy, and confocal fluorescent laser microscopy. During prophase, basal bodies, both flagella and their appendages were duplicated. In the round nucleus situated in the cell centre, intranuclear microtubules appeared radiating toward the centre of the nucleus from densities located in some nuclear pores. During metaphase, both daughter flagellar apparatus separated and moved apart along the main cell axis. Microtubules of ventral cortex were also duplicated and moved with the flagellar apparatus. The nucleus flattened in the longitudinal direction and became discoid-shaped close to the equatorial plane. Many bundles of microtubules ran parallel to the short axis of the nucleus (cell long axis), between which chromosomes were arranged in the same direction. During ana-telophase, the nucleus elongated along the longitudinal axis and took a dumbbell shape. At this stage a contractile ring containing actin was clearly observed in the equatorial cortex. The cortical microtubule network seemed to be cut into two halves at the position of the actin bundle. Shortly after, the nucleus divided into two nuclei, then the cell body was constricted at its equator and divided into one anterior and one posterior halves which were soon rebuilt to produce two cells with two full sets of cortical microtubules. From our observations, several mechanisms for the duplication of the microtubule networks during mitosis in O. marina are discussed.     

ULTRASTRUCTURE OF THE BASAL APPARATUS AND PUTATIVE VESTIGIAL FEEDING APPARATUSES IN A QUADRIFLAGELLATE EUGLENOID (EUGLENOPHYTA)1

The flagellar apparatus and presumptive vestigial feeding apparatuses of a cold-water, photosynthetic, quadriflagellate euglenoid is described. The organism possesses two similar sets of flagella each consisting of one short and one long flagellum. Each pair of flagella is associated with three microtubular roots for a total of six roots in the basal apparatus. At the level of the ventral basal bodies, each intermediate root is nine-membered, while the ventral roots are composed of eight to nine microtubules. Only one of the ventral roots lines the single microtubule reinforced pocket. A four-membered dorsal root attaches to each dorsal basal body, and at the level of the reservoir each gives rise to a dorsal band. An additional bundle of microtubules, not arising from the microtubular roots of the basal apparatus, begins posterior to the basal apparatus as a small group of a few microtubules and extends anteriorly on the right ventral side of the reservoir ending at the canal. At the level of the stigma, the microtubules are organized into a multi-layered bundle that continues to increase in size and eventually splits to form two bundles at the level of the canal. We postulate that these bundles may represent the remnants of a rod-and-vane-type feeding apparatus like that found in many phagotrophic euglenoids.     

Transformation and development of the flagellar apparatus ofCryptomonas ovata (Cryptophyceae) during cell division

Summary InCryptomonas ovata, long, dorsal flagella are produced which transform during the following cell division into short, ventral flagella. At division there is a reorientation in cell polarity, and the parental basal apparatus, which comprises the basal bodies and associated roots, is distributed to the daughter cells via a complex sequence of events. Flagellar apparatus development includes the transformation of a four-stranded microtubular root into a mature root of different structure and function. Each newly formed basal body nucleates new microtubular roots, but receives a striated fibrous root from a parental basal body. The striated roots are originally produced on the transforming basal body and are transferred to the new basal bodies at each successive division. The development of the asymmetric flagellar apparatus throughout the cell cycle is described.     

ELECTRON MICROSCOPE OBSERVATIONS ON THE FLAGELLAR APPARATUS OF BRYOPSIS MAXIMA (CHLOROPHYCEAE)1

The flagellar apparatus in male gametes of the siphonaceous green alga, Bryopsis maxima Okamura, was studied and compared with that of other green biflagellate cells. The proximal portions of two basal bodies are connected by a single striated proximal band, unique among the biflagellate reproductive cells of green algae studied. Anterior to the flagellar bases is a pair of distal bands different from the single structure in other biflagellate cells. These bands which arise from the distal portion of each basal body, extend upward in the papilla and curve down toward the lower edges of the basal bodies. They seem to have no direct association with each other. Two pairs of distinct flagellar roots, one consisting of 3–5 microtubules and the other of a partially striated fiber of undetermined numbers of microtubules, diverge from the basal body region and extend towards the cell posterior. Their component microtubules are disorganized into single or smaller groups midway over the cell length. The uniqueness of the flagellar apparatus is briefly discussed.     

Cell shape and microtubules in zoospores of the green algaChlorosarcinopsis gelatinosa (Chlorosarcinales): Effects of low temperature

Summary The effect of low temperature (2 °C) on cell shape and microtubules in zoospores of the green algaChlorosarcinopsis gelatinosa has been investigated. The zoospores are 4–6 times longer than wide with a mean length of 12,5 m and can be kept in the dark for several hours without changes in cell shape. Cell shape changes have been evaluated quantitatively by measuring changes in cell length. Low temperature induces a decrease in cell length which exhibits a two-step kinetic: during the first 30 minutes a rapid rate of decrease in cell length was measured, while during the next 4 hours a slow rate of decrease in cell length was observed. Complete regeneration of zoospore length occurs when cold-treated cells are subjected to the original zoospore induction temperature (30 °C) for two hours. Observation of numbers, disposition and types of microtubules in the zoospore during decrease in cell length has shown that within 30 minutes after cold application the secondary cytoskeletal microtubules (scmt) disappear, while flagellar root microtubules are unaffected. During this period most cells develop a prominent posterior appendage (tail). Sections demonstrate the presence of several microtubules in these tails. Flagellar root microtubules probably extend into the tails and disappearance of scmt starts at the posterior pole of the cell. Regeneration of zoospores to original cell length is coupled with reappearance of scmt starting at the anterior pole of the cell. It is concluded that secondary cytoskeletal microtubules constitute the main cytoskeleton inChlorosarcinopsis zoospores and that flagellar root microtubules contribute to only a minor extent to the cytoskeleton, because they cannot retain the cell shape. The results are discussed with respect to the functional significance of flagellar root microtubules in green algae.     

Flagellar apparatus duplication and partition, flagellar transformation during division in Entosiphon sulcatum.

Electron microscopic examination of serial sections of developmental stages of the flagellar apparatus during the cell cycle indicates that the basal bodies replicate in a semi-conservative manner and that there is a flagellar transformation over two cell cycles in euglenoids as in other algal flagellate groups. Two new pairs of basal bodies are formed, each pair comprising one parental and one newly developed basal body. There is a transformation of the parental dorsal flagellum containing a thin paraxonemal rod into a ventral flagellum bearing a large paraxonemal rod. Observation of the roots associated with the basal bodies shows that the dorsal root transforms into an intermediate root over two cell cycles following the transformation of the dorsal basal body/flagellum to a ventral one. Also the two ventral roots are newly formed in relation to the formation of two new phagotrophic apparatuses during the division. After the breakage of the connection between the parental basal bodies the two new pairs move apart and are guided/drawn by transverse microfibrillar bundles which connect them to opposite sides of the pellicle. The axis of the separation/migration of the pairs of basal bodies is parallel to the axis of elongation of the dividing nucleus.     

The cytology and ultrastructure of zoospores of Hydrurus foetidus (Chrysophyceae)

The unusual tetrahedral shape of Hydrurus foetidus (Vill.) Trev. zoospores is associated with a complex skeletal system of microtubules extending from a broad flagellar root (up to 19 microtubules) into each of three, pointed anterior processes. The posterior end, also pointed and supported by a separate set of microtubules, contains a single large chloroplast with a prominent posterior furrow containing mitochondrial elements. A large immersed pyrenoid is penetrated by paired thylakoids. There is no eyespot. Numerous large Golgi bodies occur immediately anterior to the nucleus and up to 5–6 contractile vacuoles lie near the cell surface at the anterior end. Two terminally inserted flagella extend from the cell surface, a long one serving for cell locomotion, and the other vestigial with an axonemal pattern of 9+0. The flagellar root system consists of: (1) a thin, striated rhizoplast extending from the basal body of the long flagellum and ramifying over the surface of a conspicuous, anteriorly directed, conical projection of the nucleus; (2) a broad microtubular root which emanates from near the basal body of the long flagellum and appears to function as a MTOC; (3) a compound root, consisting of a striated fiber and two associated microtubules, which runs alongside the basal body of the stubby flagellum before terminating at the cell surface; and (4) a short two-membered microtubular root, also associated with the basal body of the stubby flagellum. Other components of the flagellar apparatus include a large dense body near the proximal end of the basal body of the short flagellum, and a small, dense, core-like structure closely associated with one of its triplet fibers. The flagellar apparatus of H. foetidus is remarkably similar in ultrastructure to that of Chrysonebula holmesii Lund.     

An Ultrastructural Study of Lentomonas applanatum (Preisig) N. G. (Euglenida)

ABSTRACT. Lentomonas applanatum (syn. Entosiphon applanatum Preisig) is a biflagellate, phagotrophic euglenid found in intertidal salt marshes. Lentomonas applanatum bears a superficial similarity to Entosiphon sulcatum , however, an ultrastructural study of L. applanatum revealed many features that are atypical for other described species of the genus Entosiphon . These features include number and organization of pellicular strips, construction of the feeding apparatus, nature of the flagellar transition zone and flagellar apparatus, and point of flagellar emergence. These differences show that L. applanatum is related more closely to phagotrophic genera such as Ploeotia than to E. sulcatum . The construction of the feeding apparatus and pellicle suggest that L. applanatum has retained many of the pleisiomorphic characters that were present in the earliest euglenids. The presence of similar structures in other related protists may provide important clues as to the evolution of the Euglenida.     

MALE GAMETES OF ATRACTOMORPHA ECHINATA HOFFMAN (CHLOROPHYCEAE)1

Many naked gametes are produced in each fusiform, male gametangium of Atractomorpha echinata Hoffman and are liberated through irregularly shaped pores in the gametangial wall. They are typically biflagellate, pyriform or fusiform in shape, 6-11 μm long, and only a few micrometers wide. A mature male gamete is characterized by: (i) a nucleus with condensed chromatin and no nucleoli, (ii) a reduced, starch filled chloroplast occupying a posterior position, and (iii) a cup shaped eyespot consisting of a single layer of plastoglobuli. The flagellar apparatus includes two types of flagellar roots alternating in a cruciate pattern. One type consists of two microtubules, while the other consists of microtubules of varying number, usually eight or nine, but rarely as many as eleven. The paired basal bodies are connected anteriorly by a broad, striated distal fiber; there is no dense apical cap as reported in Sphaeroplea sperm. A unique structure, consisting of three layers of small subunits (6–8 nm diameter) arranged in a paracrystalline array, is positioned beneath each basal body. Based on the structure of its male gametes, Atractomorpha clearly demonstrates affinity with the chlorophycean rather than the ulvaphycean line of evolution. Moreover, if phylogenetic affinities for the Sphaeropleaceae are to be sought among other groups of green algae, the Chlorococcales appears the most promising candidate.     

Pellicle complex ofEuglena gracilis: Characterization by disruptive treatments

Summary Euglena gracilis was treated with 4% colchicine, 0.5% -mercaptoethanol, 0.5% Triton X-100, and 8% tannic acid in attempting to characterize its pellicle complex. Colchicine had no visible effects on the microtubules of the pellicle, canal or reservoir. Colchicine in dimethyl sulfoxide disorganized the reservoir region. Colchicine was shown to enter the cell by its ability to inhibit flagellar regeneration. Mercaptoethanol destroyed most of the organelles in the cell. Only the tripartite plasmalemma of the pellicle complex remained normal. The microtubules of the cell and the protein layer underlying the plasmalemma were disrupted by mercaptoethanol treatment. Triton X-100 caused membranes of internal organelles to round, up become distended and swollen, but had no morphological effect on the plasmalemma. The ridge-groove shape of the pellicle remained intact after every treatment. Mercaptoethanol caused an indentation of the ridges over the position of a disrupted microtubule, suggesting a supporting function of the microtubules. Distension of endoplasmic reticulum by Triton X-100 revealed more clearly its association with the pellicle complex. The ability of the pellicle ofEuglena gracilis to maintain its integrity under a number of disruptive treatments was demonstrated.