MULTILAYERED STRUCTURES (MLSs) IN TWO DINOFLAGELLATES,KATODINIUM CAMPYLOPS AND WOLOSZYNSKIA PASCHERI1

The multilayered structure (MLS), best-known from the flagellar apparatus of charophycean green algae and land plant motile cells, is reported for the first time in members of the Dinophyceae. The MLSs in two dinoflagellates, Katodinium campylops (Harris) Fott and Woloszynskia pascheri (Suchlandt) von Stosch, are similar to other MLSs in possessing the microtubular spline and lamellar strip. Also, as in the majority of MLS-containing organisms, 1) the MLS of each dinoflagellate is closely associated with basal bodies; 2) the spline microtubules possess “keel-like” extensions and 3) extend beyond the MLS, forming a microtubular rootlet that runs beneath the cell surface in a posterior direction; and 4) a mitochondrion is associated with the MLS (K. campylops only) The size, location, and general construction of the MLSs of K. campylops and W. pascheri suggest that they may be homologous to previously described MLSs.     

THE OCCURRENCE AND PHYLOGENETIC SIGNIFICANCE OF A MULTILAYERED STRUCTURE IN COLEOCHAETE SPERMATOZOIDS

Spermatozoid-forming cells of Coleochaete scutata were found in packets of four arranged in concentric internal bands. Spermatozoids, which occur singly in antheridial cells, are spherical to ovoid, approximately 7 μm long by about 3.9 μm wide. As compared to relatively unspecialized zoospores, male gametes undergo a number of specialized cellular changes during development. The spherical nuclei and cytoplasm of mature spermatozoids are increased in density. Posterior plastids are reduced and contain large starch grains. Many small mitochondria are clustered near the cell anterior. The plasmalemma is covered with a layer of flattened, diamond-shaped scales, while body scales of zoospores are pyramidal. The two flagella of both zoospores and spermatozoids are covered with flattened, diamond-shaped scales and hairs. The spermatozoids contain an anterior multilayered structure (MLS) structurally similar to, though smaller than, the MLS observed in zoospores. An asymmetrical cytoskeleton consisting of a band of 30–45 microtubules extends from the MLS down one side of the spermatozoid close to the plasmalemma. An immature MLS was observed in an early stage of spermatozoid development. The finding of an MLS and asymmetrical cytoskeleton in specialized male gametes as well as relatively unspecialized zoospores of Coleochaete strengthens assumptions of homology between MLSs of green algal reproductive cells and those found in flagellated spermatozoids of archegoniate plants. The structure of the spermatozoid of Coleochaete supports the hypothesis that this alga may be relatively close to the phylogenetic line which led directly to archegoniates.     

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.     

SPERMATOGENESIS IN COLEOCHAETE PULVINATA (CHAROPHYCEAE): EARLY BLEPHAROPLAST DEVELOPMENT

Transmission electron microscopy of serial thin sections was used to reconstruct several early developmental stages of the blepharoplast in Coleochaete pulvinata spermatids. These were compared to published studies of blepharoplast development in Charales and the closest relatives of charophycean green algae among embryophytes, i.e., hornworts and liverworts. Bicentriolar centrosomes such as occur in bryophytes and fern allies were not observed in Coleochaete. Centriole replication in C. pulvinata was orthogonal as in Charales. The resulting two daughter centrioles were oriented perpendicularly and joined proximally by electron-dense material. Their orthogonal relationship was maintained throughout blepharoplast development by a massive, banded connective which appeared early. In spermatids of hornworts and liverworts, a multilayered structure (MLS) develops in association with two centrioles destined to become flagellar basal bodies. When the MLS of these lower land plants is sectioned at right angles to the long axis of the microtubular layer, the MLS is observed to lie beneath cross sections of both centrioles. In contrast, when developing MLSs of C. pulvinata and Charales are similarly sectioned, they occur beside a cross section of just one of the two centrioles. In C. pulvinata (as in other charophytes), MLS lamellae are oriented at a 90-degree angle to the long axis of the S₁ microtubules from the beginning. This contrasts with the 40–45 degree angle between the MLS lamellae and S₁ microtubules universally reported for archegoniates. In early C. pulvinata spermatids, spline microtubules are closely associated with an anterior mitochondrion having a low stromal density and few cristae. An anterior mitochondrion is typically associated with blepharoplast development in hornworts and liverworts, but has not previously been reported to occur in Coleochaete or any other charophycean alga. In Coleochaete, as in hornworts and liverworts, but unlike Charales, structure of mature blepharoplasts reflects early blepharoplast ontogeny. Very little change in positional relationships among blepharoplast components (flagella, connective, MLS) occurs during development. These character-state differences are of importance in cladistic analyses of charophycean algae and lower land plants.     

The flagellar apparatus ofMesostigma viride (Prasinophyceae): Multilayered structures in a scaly green flagellate

Mesostigma viride Lauterborn (Prasinophyceae) is the first green flagellate found to have multilayered structures (MLS) in its flagellar apparatus. MLS's were previously known from green algae only in charophycean swarmers, linking theCharophyceae to the origin of land plants, whose male gametes (when flagellated) also possess an MLS.M. viride is, therefore, probably more closely related to the origin of theCharophyceae than any other green flagellate that has been thoroughly studied so far. The occurrence of MLS's in green flagellates and apparently in other algae and protozoans suggests that an MLS occurred in an ancient group of flagellates and has survived in various protistan lines, including the line of green algae related to land plants. The occurrence of a synistosome inM. viride and other of its characteristics suggest that it is more closely related toPyramimonas than to other genera of scaly green flagellates.This work was supported by National Science Foundation Grant DEB-78-03554.     

The flagellar apparatus structure inMicrospora (Chlorophyceae) confirms a close evolutionary relationship with unicellular green algae

The spatial configuration of the flagellar apparatus of the biflagellate zoospores of the green algal genusMicrospora is reconstructed by serial sectioning analysis using transmission electron microscopy. Along with the unequal length of the flagella, the most remarkable characteristics of the flagellar apparatus are: (1) the subapical emergence of the flagella (especially apparent with scanning electron microscopy); (2) the parallel orientation of the two basal bodies which are interconnected by a prominent one-piece distal connecting fiber; (3) the unique ultrastructure of the distal connecting fiber composed of a central tubular region which is bordered on both sides by a striated zone; (4) the different origin of the d-rootlets from their relative basal bodies; (5) the asymmetry of the papillar region which together with the subapical position of the basal bodies apparently cause the different paths of corresponding rootlets in the zoospore anterior; (6) the presence of single-membered d-rootlets and multi-membered s-rootlets resulting in a 7-1-7-1 cruciate microtubular root system which, through the different rootlet origin, does not exhibit a strict 180° rotational symmetry. It is speculated that the different basal body origin of the d-rootlets is correlated with the subapical implant of flagella. It is further hypothesized that in the course of evolution the ancestors ofMicrospora had a flagellar papilla that has migrated from a strictly apical position towards a subapical position. Simultaneously, ancestral shift of flagella along the apical cell body periphery has taken place as can be concluded from the presence of an upper flagellum overlying a lower flagellum in the flagellar apparatus ofMicrospora. The basic features of the flagellar apparatus of theMicrospora zoospore resemble those of the coccoid green algal generaDictyochloris andBracteacoccus and also those of the flagellate green algal genusHeterochlamydomonas. This strengthens the general supposition thatMicrospora is evolutionarily closely related to taxa which were formerly classified in the traditionalChlorococcales.     

Fine structure of the zoospore ofUlothrix belkae with emphasis on the flagellar apparatus

Summary The anterior end of the zoospore ofUlothrix belkae has been examined in detail and is compared toStigeoclonium and other filamentous green algae. The nature of the symmetry of green algal motile cells is discussed and the term, 180° rotational symmetry, is proposed to describe the type of arrangement of anterior end components seen inU. belkae, including the four basal bodies, rootlets and striated fibers. The four microtubular rootlets are cruciately arranged. A striated microtubule-associated component (SMAC) has a periodicity of 6.4 nm and extends with each 2-membered rootlet posteriorly into the cell. One 5-membered rootlet passes very near the eyespot. Phylogeny in green algal motile cells is discussed.     

THE ULTRASTRUCTURE OF MULTILAYERED STRUCTURES ASSOCIATED WITH FLAGELLAR BASES IN MOTILE CELLS OF TRENTEPOHLIA AUREA1

An ultrastructural study of motile cell development in the green alga Trentepohlia aurea has revealed the presence of multilayered structures (MLS) associated With flagellar bases. These MLS are ultrastructurally similar to MLS described in pteridophyte and bryophyte sperm and in the zoospore of the green algae Coleochaete and Klebsormidium. However, 2 MLS are found in each biflagellate motile cell of T. aurea, while other previously described MLS occur singly in biflagellate motile cells. In addition, the MLS of T. aurea consist of fewer microtubules and are structurally simpler than most other MLS described. The MLS of Trentepohlia may represent a stage in the evolutionary development of the MLS of land plants. The presence or absence of the MLS in motile cells of green algae may be a useful character in phylogenetic studies.     

An attachment tip and pili-like structures in insect- and plant-pathogenic spiroplasmas of the class Mollicutes

Ultrastructural studies using scanning electron microscopy (SEM), negative-staining transmission electron microscopy (TEM), and thin-sectioning TEM on four species of Spiroplasma, in vitro and/or in vivo, indicated that their helices commonly possess one tapered end (tip structure) and one blunt or round end. These tip structures appeared morphologically different from the rest of the helix, exhibiting an electron-dense conical or rod-shaped core. In thin sections of the midgut of the leafhopper Dalbulus elimatus, the tip structures of Spiroplasma kunkelii in the midgut lumen were mostly aligned between microvilli, perpendicular to the apical plasma membrane of epithelial cells. These tip structures appeared frequently attached or closely apposed to the plasma membrane, in which cup-shaped invaginations close to the tips were observed. Pleomorphic forms of spiroplasma, enclosed in membranous vesicles, were found in the cytoplasm of the midgut epithelial cells. These findings suggest that the tip structure may be involved in the orientation and attachment of spiroplasma helices in relation to their host cells, and thus may be functionally comparable to the attachment organelle of mycoplasmas. Additionally, pili-like structures were observed by negative-staining TEM on the surface of Spiroplasma melliferum, and in thin sections of S. kunkelii infecting the leafhopper vector Dalbulus gelbus. Abbreviations CSS Corn stunt spiroplasma - SEM Scanning electron microscopy - TBS Tris-buffered saline - TEM Transmission electron microscopy     

Microbiology in the 1990s: reflections about open questions and trends

The optimal conditions for protoplast formation ofCandida apicola were by using an enzyme fromArthrobacter sp. in combination with 2-mercaptoethanol. The kinetic data support the two-layered structure model of cell wall for this yeast but the structure of the cell wall depended on the age of cells and culture conditions. To regenerate the protoplasts, the type of osmotic stabilizer was important: sorbitol gave 16 to 30% regeneration. Electron microscopy revealed the presence of vesicles in the sections of protoplasts and whole cells ofCandida apicola grown in production medium and producing glycolipids. In sections of whole cells, vesicle-like structures are located in the periplasmic space and in protoplasts they can either be attached to, or released from, the cell surface. These vesicles are thought to be involved in the transport of the surface-active glycolipids and in the protection of the cell against denaturing effects.     

The flagellar root system of zoospores of the green algaChlorosarcinopsis (Chlorosarcinales) as compared withChlamydomonas (Volvocales)

The flagellar root system of zoospores in two species ofChlorosarcinopsis (C. minuta andC. spec.) has been studied in detail. The biflagellate zoospores show a cruciate root system, two of the four microtubular roots containing two microtubules, the other two four microtubules. The flagellar apparatus is otherwise identical with that ofChlamydomonas reinhardi as described byRingo (1967). Evidence is presented that the genusChlamydomonas is characterized by a bilateral symmetric root system (4-2-4-2) rather than a system with four equally numbered roots (i.e. 4-4-4-4). It is suggested that a root system with four identical cruciate roots is not present in any biflagellate algal cell. The taxonomic significance of cruciate root systems in green algae is discussed refering to the identical root systems ofChlorosarcinopsis andChlamydomonas.     

Osservazioni Sulle Infrastrutture di Anabaena Cycadinae E delle Cellule dei Tubercoli Radicali di Cycas Revoluta

Abstract

Observations on the infrastructures of ANABAENA CYCADINAE and of root-nodules cells of CYCAS REVOLUTA. — Light and electron microscope observations carried on the fine and ultrafine sections of root-nodules of Cycas revoluta, brought in evidence differences structural and infrastructural of cells. These differences concern mainly the modifications presented by reticulum endoplasmic which in middle-aged cells takes a fragmentary and vescicular aspect, and the mitochondria which, in the same cells, show short cristae starting by outside wall of the organul.

About the plastyds, I could bring in evidence that there is plenty of leucoplasts but the proplastyds are still numerous, as the cloroplastyds showing some development of the lamellar system and of grana. Plastyds green are present either in while nodules, either in the root-tops.

In no nodules it has been possible to point out the presence of bacteria while it has been possible to verify that Algae have some polymorphism of structure because the chromatoplasma may result essentially of tree different aspects, by me, indicated as reticular type (fig. 12), lamellar type (fig. 10) and type at mixed structure (fig. 11).     

Flagellar apparatus ultrastructure inMesostigma viride (Prasinophyceae)

The ultrastructure of the flagellar apparatus ofMesostigma viride Lauterborn (Prasinophyceae) has been studied in detail with particular reference to absolute configurations, numbering of basal bodies, basal body triplets and flagellar roots. The two basal bodies are interconnected by three connecting fibers (one distal fiber = synistosome, and two proximal fibers). The flagellar apparatus shows 180° rotational symmetry; four microtubular flagellar roots and two system II fibers are present. The microtubular roots represent a 4-6-4-6-system. The left roots (1s, 2s) consist of 4 microtubules, each with the usual 3 over 1 root tubule pattern. Each right root (1d, 2d) is proximally associated with a small, but typical multi-layered structure (MLS). The latter displays several layers corresponding to the S₁ (the spline microtubules: 5–7), and presumably the S₂—S₄ (the lamellate layers) of the MLS of theCharophyceae. At its proximal origin (near the basal bodies) each right root originates with only two microtubules, the other spline microtubules being added more distally. The structural and positional information obtained in this study strongly suggest that one of the right roots (1d) ofMesostigma is homologous to the MLS-root of theCharophyceae and sperm cells of archegoniate land plants. Thus the typical cruciate flagellar root system of the green algae and the unilateral flagellar root system of theCharophyceae and archegoniates share a common ancestry. Some functional and phylogenetic aspects of MLS-roots are discussed.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.     

Comparative cytology and taxonomy of theUlvaphyceae II. Ulvalean characteristics of the stephanokont flagellar apparatus ofDerbesia tenuissima

Summary The stephanokont flagellar apparatus of the zoospores ofDerbesia tenuissima (De Not.) Crouan is examined and compared to the flagellar apparatuses of other green algae. The flagella ofDerbesia are attached to two of three bands which lie at the junction of the body and papilla. Serial longitudinal and cross sections reveal that the basal bodies are attached to the bands along their sides and at their proximal ends. The bands are not striated in any plane. The lack of striation in the bands and the partial covering of the proximal end of the basal bodies by one of the bands closely resemble the type of flagellar connection system described as the Bryopsis-type byMelkonian (1980). Zoospores of ulvalean green algae also possess these features, suggesting that green siphons are phylogenetically related to theUlvales. It is proposed that green siphons be tentatively classified in theUlvaphyceae rather than in theChlorophyceae orCharophyceae.This work supported by NSF Grant DEB 78-03554.     

Microalgal biotechnology: Carotenoid production by the green algae<Emphasis Type="Italic">Dunaliella salina</Emphasis>

Unicellular green algae of the genusDunaliella thrive in extreme environmental conditions such as high salinity, low pH, high irradiance and subzero temperatures. Species ofDunaliella are well known in the alga biotechnological industry and are employed widely for the production of valuable biochemicals, such as carotenoids. Some strains ofDunaliella are cultivated commercially in large outdoor ponds and are harvested to produce dry algal meals, such as polyunsaturated fatty acids and oils for the health food industry, and coloring agents for the food and cosmetic industries. During the past decade, the advances in molecular biology and biochemistry of microalgae, along with the advances in biotechnology of microalgal mass cultivation, enabled this microalga to become a staple of commercial exploitation. In particular, the advent of molecular biology and mutagenesis inDunaliella has permitted enhancements in the carotenoids content of this green alga, making it more attractive for biotechnological applications. Accordingly, the present review summarizes the recent developments and advances in biotechnology of carotenoid production inDunaliella.     

An algal medium based on fertilizers and its evaluation in mariculture

Five agricultural fertilizers were tested as potential nutrient enrichments for the mass culture ofTetraselmis suecica. Maximum algal growth was observed for the trade fertilizer Igromurtonik^R (Murphy Ltd, England) adjusted for a nitrate to phosphate ratio of 24:1. The gross biochemical composition ofT. suecica grown in the enriched fertilizer was compared to the composition of the alga grown in control medium. The nutritional value of the algal material was then tested on the rotiferBrachionus plicatilis. The medium based on the fertilizer is as an inexpensive substitute for mass algal culture ofTetraselmis suecica, a food source for the rotiferBrachionus plicatilis.     

Ultrastructural study of spore wall morphogenesis inOphioglossum thermale kom. var.nipponicum (Miyabe et Kudo) nishida

Spore wall morphogenesis ofOphioglossum thermale var.nipponicum was examined by transmission electron microscopy. The spore wall of this species consists of three layers: endospore, exospore, and perispore. The spore wall development begins at the tetrad stage. At first, the outer undulating lamellar layer of the exospore (Lo) is formed on the spore plasma membrane in advance of the inner accumulating lamellar layer (Li) of the exospore. Next, the homogeneous layer of the exospore (H) is deposited on the outer lamellar layer. Both lamellar layers may be derived from spore cytoplasm; and the homogeneous layer, from the tapetum. Then the endospore (EN) is formed. It may be derived from spore cytoplasm. The membranous perispore (PE), derived from the tapetum, covers the exospore surface as the final layer. Though the ornamentation of this species differs distinctly from that ofO. vulgatum, the results mentioned above are fundamentally in accordance with the data obtained fromO. vulgatum (Lugardon, 1971). Therefore, the pattern of spore wall morphogenesis appears to be very stable in the genusOphioglossum.     

Grazing on green algae by the periwinkleLittorina littorea in the Wadden Sea

On sedimentary tidal flats in the Wadden Sea near the Island of Sylt, the periwinkleLittorina littorea occurred preferentially on clusters and beds of mussels and on shell beds (100 to 350 m⁻²), achieved moderate densities on green algal patches or mats (20 to 50 m⁻²), and remained rare on bare sediments (<5 m⁻²). Green algae covering>10% of sediment surface appeared in summer on approximately one third of the tidal zone, mainly in the upper and sheltered parts and almost never on mussel and shell beds. In feeding experiments,L. littorea ingested more of the dominant alge,Enteromorpha, than ofUlva, irrespective of whether or not algae were fresh or decaying. The tough thalli ofChaetomorpha were hardly consumed. Snails feeding onEnteromorpha produced fecal pellets from which new growth ofEnteromorpha started. In the absence of periwinkles,Enteromorpha developed on mussels and the attached fucoids. Experimentally increased snail densities on sediments prevented green algal development, but the snails were unable to graze down established algal mats. It is concluded that natural densities ofL. littorea hardly affect the ephemeral mass development of green algae on sediments. However, where the snails occur at high densities, i.e. on mussel beds, green algal development may be prevented.     

Ultrastructure and phylogeny of the spermatozoid ofChara vulgaris (Charophyceae)

At maturity, spermatozoids of the green algaChara vulgaris are biflagellated, contain little cytoplasm, and coil for approximately 2 1/2 gyres within the mother cell wall. The anterior of the cell contains an ovoid headpiece anchoring two slightly staggered basal bodies that are positioned above and directly in front of approximately 30 linearly arranged mitochondria. An elongated stellate pattern occupies the transition zone between the BBs and axonemes. Flagella emerge from the cell just in front of the nucleus and encircle the full length of the spermatozoid. The spline comprises a maximum of 38 microtubules surrounding the anterior mitochondria and gradually decreases posteriorly to a minimum of 11. The dense nucleus is narrow, cylindrical, and occupies the central revolution of the cell. Six starch-laden plastids and associated mitochondria are linearly arranged at the cell posterior. Phylogenetic analyses of charalean taxa and archegoniates based on spermatogenesis strongly support the orderCharales, withNitella as the sister group toChara. Diagnostic features ofChara spermatozoids include absence of a lamellar strip and axonemes embedded in the cell for almost the entire length of the anterior mitochondria. Potential relationships amongCharales, Coleochaetales and archegoniates are evaluated in regards to the probable course of evolution of streamlined biflagellated gametes.     

Presence of myelin-like structures in tanniferous vascular parenchyma cells ofSalix dasyclados Wim.

Summary In the cambial region ofSalix dasyclados plants, extensive spherical formations of myelin-like lamellar configurations were found during the transit period between winter dormancy and reactivation of growth in early spring. The lamellar structures occurred in parenchyma cells containing tannins and were associated with spherosomes. Cytomembranes in tannin-containing cells appeared with negative contrast on electron micrographs, indicating the presence of tanniferous substances in the cytoplasm.Abbreviation R_G relative growth rate (% fresh weight increase per day)