Rapid and slow mechanisms for loss of cell adhesiveness during fertilization in Chlamydomonas

Although vegetative cells, gametes, and zygotes of the biflagellated alga Chlamydomonas bear flagella, only the flagella of mt+ and mt- gametes are adhesive. The molecules responsible for adhesiveness, mt+ and mt- agglutinins, are long rod-shaped glycoproteins displayed on the flagellar membrane. These flagellar agglutinins, which gametes use both as adhesion and signaling molecules during the early events of fertilization, are lost from the flagella during adhesion. Flagellar adhesiveness can be maintained, however, by recruitment and activation of preexisting, inactive agglutinins from the plasma membrane of the cell body (Hunnicutt et al, 1990, J. Cell Biol. 111, 1605-1616) unless the gametes of opposite mating types fuse to form zygotes. Upon cell fusion, flagellar adhesiveness is lost. In the studies presented here, we have employed an in vitro bioassay to measure agglutinins in both cell bodies and flagella at various times during gametogenesis, during fertilization, and after zygote-formation. By use of the bioassay, which can detect agglutinins that are functionally inactive in vivo, we found that vegetative cells are devoid of agglutinins. These adhesion molecules appear only after gametogenesis is underway with the cell body agglutinins appearing first and then the flagellar agglutinins. Surprisingly, 30 min after zygote formation, when the zygotes' flagella are no longer adhesive, the flagellar agglutinin activity detectable with the bioassay remains high. One interpretation of these results is that zygotes continue to recruit agglutinins from the cell body to the flagella, but cell fusion abrogates activation of the agglutinins. Within 45-90 min after fusion both the cell body and flagellar agglutinins are lost and can be detected in the medium. These mechanisms, which render the zygotes nonadhesive to other zygotes and unmated gametes, contribute to the Chlamydomonas equivalent of a block to polyspermy.     

ELECTRON AND PHASE-CONTRAST MICROSCOPY OF SEXUAL REPRODUCTION IN CHLAMYDOMONAS MOEWUSII1

The sexual process of C. moewusii from gametic activation through germination of the zygote has been studied with phase-contrast and electron microscopy. Long strands emerging from the gametic flagellar tips are the site of early flagellar attraction which is followed by union of compatible flagella within common flagellar sheaths. The gametic connecting strand is formed by coordinated elongation of the plasma papillae of a gametic pair and the penetration of the former through their wall papillae while the flagella are in intimate association. After the free-swimming period, the gametic pairs aggregate in a second period of clumping. The connecting strand is abscised and extruded during plas-mogamy as are the flagellar basal bodies. Evidence is presented which suggests union of the gametic plastids, and stages in karyogamy are illustrated. Formation of the wall layers, accumulation of starch and lipids, and changes in plastid organization in the maturing and germinating zygote are described as is the formation of the gonal walls.     

Sexual agglutination in Chlamydomonas eugametos before and after cell fusion

A new study of sexual agglutination between Chlamydomonas eugametos gametes and between vis-à-vis pairs has been made using techniques that allow one to distinguish between the flagella or cell bodies of individual mating types (mt⁺ or mt^-). It is shown that before mt⁺ and mt^- gametes fuse in pairs, their flagella, which adhere over their whole length, are maintained in a particular conformation around the mt^- cell body. In clumps of agglutinating gametes the cells are asymmetrically distributed with the mt⁺ gametes constituting the outer surface of the clumps with the mt^- gametes on the inside. The flagella are then all directed towards the middle of the clump. This orientation of the flagella is maintained for approx. 8 min after cell fusion before the vis-à-vis pair becomes motile. At this stage, all the flagellar tips are activated. The original mt⁺ flagellar tips then deactivate and swimming is resumed. The original mt^- flagella remain immotile and activated after cell fusion and eventually shorten by a third, but only 30 min or more after fusion. Motile vis-à-vis pairs eventually settle to the substrate when the gamete bodies fuse completely to form a zygote. Settling vis-à-vis pairs are attracted to those that have already settled, to glutaraldehyde-fixed pairs and to flagella isolated from mt^- gametes. They are not chemotactically attracted, rather they are weakly agglutinated. Living vis-à-vis pairs can be shown to aggregate in rows with the cell bodies lying side by side. It is argued that the flagellar agglutination sites involved in gamete recognition are also involved in vis-à-vis pair aggregationAbbreviations mt^+/- mating type plus or minus - FTA flagellar tip activation     

Regulated targeting of a protein kinase into an intact flagellum. An aurora/Ipl1p-like protein kinase translocates from the cell body into the flagella during gamete activation in chlamydomonas

In the green alga Chlamydomonas reinhardtii flagellar adhesion between gametes of opposite mating types leads to rapid cellular changes, events collectively termed gamete activation, that prepare the gametes for cell-cell fusion. As is true for gametes of most organisms, the cellular and molecular mechanisms that underlie gamete activation are poorly understood. Here we report on the regulated movement of a newly identified protein kinase, Chlamydomonas aurora/Ipl1p-like protein kinase (CALK), from the cell body to the flagella during gamete activation. CALK encodes a protein of 769 amino acids and is the newest member of the aurora/Ipl1p protein kinase family. Immunoblotting with an anti-CALK antibody showed that CALK was present as a 78/80-kDa doublet in vegetative cells and unactivated gametes of both mating types and was localized primarily in cell bodies. In cells undergoing fertilization, the 78-kDa CALK was rapidly targeted to the flagella, and within 5 min after mixing gametes of opposite mating types, the level of CALK in the flagella began to approach levels normally found in the cell body. Protein synthesis was not required for targeting, indicating that the translocated CALK and the cellular molecules required for its movement are present in unactivated gametes. CALK was also translocated to the flagella during flagellar adhesion of nonfusing mutant gametes, demonstrating that cell fusion was not required for movement. Finally, the requirement for flagellar adhesion could be bypassed; incubation of cells of a single mating type in dibutyryl cAMP led to CALK translocation to flagella in gametes but not vegetative cells. These experiments document a new event in gamete activation in Chlamydomonas and reveal the existence of a mechanism for regulated translocation of molecules into an intact flagellum.     

Mitosis in the Hemoflagellate Trypanosoma cyclops*

SYNOPSIS. The ultrastructure of interphase and mitotic nuclei of the epimastigote form of Trypanosoma cyclops Weinman is described. In the interphase nucleus the nucleolus is located centrally while at the periphery of the nucleus condensed chromatin is in contact with the nuclear envelope. The nucleolus fragments at the onset of mitosis, but granular material of presumptive nucleolar origin is often recognizable in the mitotic nucleus. Peripheral chromatin is in contact with the nuclear envelope throughout mitosis, and it seems reasonable to assume that the nuclear envelope is involved in its segregation to the daughter nuclei. Spindle microtubules extend between the poles of the dividing nucleus and terminate close to the nuclear envelope. The basal body and kinetoplast divide before the onset of mitosis and do not appear to have any morphologic involvement in that process. Spindle pole bodies, kinetochores, and chromosomal microtubules have not been observed.     

Experimental dissection of flagellar surface motility in chlamydomonas

Experiments have explored the possible relationships between the flagellar surface motility of chlamydomonas, visualized as translocation of polystyrene beads by paralyzed (pf) mutants (Bloodgood, 1977, J. Cell Biol. 15:983-989), and the capacity of gametic flagella to participate in the mating reaction. While vegetative and gametic flagella bind beads with equal efficiencies and are capable of transporting them along entire flagellar lengths, beads on vegetative flagella are primarily associated with the proximal half of the flagella whereas those of gametic flagella exhibit no such preference. This difference may relate to the "tipping" response of gametes during sexual flagellar agglutination (Goodenough and Jurivich, 1978, J. Cell Biol. 79:680-693). Colchicine, vinblastine, chymotrypsin, cytochalasins B and D, and anti-β-tubulin antiserum are all able to inhibit the binding of beads to the flagellar suface. Trysin digestion and an antiserum directed against whole chlamydomonas flagella have no effect on the ability of flagella to bind beads, but the beads remain immobile. These results suggest that at least two flagellar activities participate in surface motility: (a) bead binding, which may involve a tubulin-like component at the flagellar surface; and (b) bead translocation, which may depend on a second component (e.g. an ATPase) of the flagellar surface. Surface motility is shown to be distinct from gametic adhesiveness per se, but it may participate in concentrating dispersed agglutinins, in driving them toward the flagellar tips, and/or in generating a signal-to-fuse from the flagellar tips to the cell body. Directly supporting these concepts is the observation that bound beads remain immobilized at the flagellar tips during the "tip-locking" stage of pf x pf matings, and the observation that bound ligands such as antibody fail to be tipped by trypsinized flagella.     

Giant flagellar bundles ofVibrio alginolyticus (NCMB 1803)

Summary Following swarming ofVibrio alginolyticus on solid medium a large number of giant flagellar bundles appear behind the growth front. The suggested sequence of events leading to bundle formation is as follows. After inoculation from liquid to solid media the short rods with a single polar sheathed flagellum develop peritrichous nonsheathed flagella and elongate into long filamentous swarmers. After division into short rods, some of the cells become spherical in shape with many peritrichous flagella concentrated at one pole in close association with the sheathed polar flagellum. These tufted spherical bodies form the template upon which masses of loose peritrichous flagella spontaneously aggregate.Flagellar bundles formed when bacteria are grown at pH 8.5 are longer than those formed at pH 7.2 and shorter when grown at pH 6.5. In distilled water the flagellar bundles disintegrate into masses of flagellar fragments.     

ULTRASTRUCTURE OF FERTILIZATION IN A CRYPTOMONAD1

By means of various electron microscopic techniques, the ultrastructure of fusing gametes in a cryptomonad is described for the first time. The isolate used in this study is bisexual, and vegetative cells may act as isogametes. Plasmogamy usually is initiated at the posterior end of one gamete and the mild-ventral region of the other gamete. A posterior, pointed protuberance may be a specialized mating structure which initiates the fusion process. Fusion proceeds toward the anterior end, forming a quadriflagellate cell which becomes spherical and settles to the bottom of the culture flask. The quadriflagellate, spherical cell contains two nuclear-nucleomorph-chloroplast complexes which remain intact throughout karyogamy. During karyogamy the nuclei are positioned close to each other and become lobed on the sides where fusion takes place. At the points where the lobes touch, the nuclear membranes break down and direct karyogamy is initiated. Nuclear fusion continues and eventually a single zygotic nucleus is formed. The zygote nucleus and the two nucleomorphs and chloroplasts become enclosed in a common periplastidial compartment. The nucleomorphs, however, remain apart and do not fuse. Meiosis presumably is zygotic, but the stages of post-karyogamy remain to be elucidated.     

Acetylcholine receptors in the central nervous system of Drosophila melanogaster.

Mating between gametes of the biflagellated unicellular green alga Chlamydomonas reinhardi consists of several events culminating in zygote formation. Initially, the cells agglutinate by their flagellar tips. This is followed by pairing, cell wall loss, and cell fusion. Here we report on the relationship between the length of the flagellum, and the cells' ability to agglutinate, undergo cell wall loss (as measured by medium carbohydrate accumulation), and to form zygotes. We found that deflagellated gametes regained the potential for sexual agglutination when the flagella had regenerated to less than 3 μm (compared to the full length flagella of approx. 11 μm), while medium carbohydrate appeared only after the flagella had reached an average length greater than 5 μm. By inhibiting flagellar regeneration with cycloheximide or colchicine, we determined that carbohydrate release is related to the length of the flagellum and not to the time after deflagellation. A flagellar length dependence similar to that of carbohydrate release was also observed when we measured the relationship between the gametes' ability to fuse and flagellar length.     

Observation on the Sexual Reproduction of Peridinium bipes Stein (Dinophyceae)

Sexual reproduction of the dinoflagellate Peridinium bipes Stein was observed. At the late growth season (from late March to early April), the small, unarmored motile cells i.e. gametes are produced by division of the cate cell. Isogamy occurs in this species. Two gametes in fusion are morphologically indistinguishable, but their behavior are different. Before fusion, two gametes are connected by a transparent granular structure and move quickly for about ten minutes. When the plasmogamy almost completes, the fusing cells stop moving for a while and the transverse flagellum of one gamete is cast off. By staining with modified carbol fuchsin, it was proved that the karyogamy takes place soon after plasmogamy and the change of chromosomes in this period was also observed. The zygote keeps motile for about 14 days before casts off its two flagella and becomes aplanozygote. During this period i.e. planozygote stage, the zygote enlarges from 55 × 50 μm to 75 ×70 μm, intercolary bands connecting thecai plates widen, lots of oil droplets are produced as storage granules. After sinking to the bottom of flask, aplano- zygote continues changing: exospore wall is cast off, mesospore wall and endospore wall are thickened, oil droplets turn to starch grains, protoplast contracts and becomes spherical, a large red lipid granule, perhaps eye-spot, appears. Afterward, aplanozygote has become hypnozygote i.e. resting cyst. The type of sexual reproduction, the amphiesma of the zygote, the resistance of hypnozygotic wall to acid and alkali, the relationship between fossil dinoflagellate remain and the wall of hypnozygote were discussed. It was also considered that the three formas of P. bipes named by Huber-Pestalozzi were the different stages of zygote development.     

Tipping and mating-structure activation induced in Chlamydomonas gametes by flagellar membrane antisera

Antisera raised against vegetative and gametic flagella of Chlamydomonas reinhardi have been used to probe dynamic properties of the flagellar membranes. The antisera, which agglutinate cells via their flagella, associate with antigens that are present on both vegetative and gametic membranes and on membranes of both mating types (mt+ and mt-). Gametic cells respond to antibody presentation very differently from vegetative cells, mobilizing even high concentrations of antibody towards the flagellar tips; the possibility is discussed that such "tipping" ability reflects a differentiated gametic property relevant to sexual agglutinability. Gametic cells also respond to antibody agglutination by activating their mating structures, the mt+ reaction involving a rapid polymerization of microfilaments. Several impotent mt+ mutant strains that fail to agglutinate sexually are also activated by the antisera and procede to form zygotes with normal mt- gametes. Fusion does not occur between activated cells of like mating type. Monovalent (Fab) preparations of the antibody fail to activate mt+ gametes, suggesting that the cross-linking properties of the antisera are essential for their ability to mimic, or bypass, sexual agglutination.     

Agglutination factor in the cell body of Chlamydomonas eugametos

Chlamydomonas eugametos gametes agglutinate via the surfaces of their flagella. The mating-type minus (mt ^-) agglutination factor is a high-molecular-weight glycoprotein called PAS-1.2, present on the exterior surface of the flagellar membrane. During flagellar regeneration, mt ^- gametes were able to agglutinate as soon as the flagella protruded as short stumps. This was also observed when protein synthesis was blocked, indicating that gametes possess a pool of PAS-1.2. When the exterior surface of flagella-less gametes was extracted and the proteins were subjected to gel electrophoresis, large quantities of PAS-1.2 were detected. Using anti-PAS-1.2 serum, the presence of PAS-1.2-like material was visualized on the plasma membrane of mt ^- gamete cell bodies. By assaying the biological activity of extracts of the cell bodies and of isolated flagella, it was calculated that the plasma membrane of the cell bodies contains 25 times the activity present in the flagella and could, therefore, represent a large pool of mt ^- agglutination factor.     

A monoclonal antibody that blocks adhesion of Chlamydomonas mt+ gametes

During the mating reaction (fertilization) in the biflagellated alga, Chlamydomonas reinhardtii, mt+ and mt- gametes adhere to each other via their flagella and subsequently fuse to form quadriflagellated zygotes. In the studies reported here, we describe a monoclonal antibody directed against an mt+ flagellar surface molecule. The antibody blocks the adhesiveness of mt+ gametes, isolated mt+ flagella, and detergent extracts thereof. It has no effect on mt- gametes. Cyanogen bromide- activated Sepharose beads derivatized with the antibody bind only mt+ gametes; mt- gametes and mt+ and mt- vegetative cells are unreactive with the derivatized beads. The interaction of mt+ gametes with the beads is dynamic and cells continuously bind, detach, and rebind to the beads. Surprisingly, antibody-derivatized beads that have been incubated with mt+ gametes acquire the ability to bind mt- gametes. Moreover, extraction of the preincubated beads with detergents releases active mt+ adhesion molecules. The evidence suggests that binding of the antibody to the flagellar surface adhesion molecules causes their release from the flagellar surface, possibly mimicking the normal mechanism of flagellar de-adhesion.     

Flagellar adhesion between mating type plus and mating type minus gametes activates a flagellar protein-tyrosine kinase during fertilization in Chlamydomonas

When Chlamydomonas gametes of opposite mating type are mixed together, flagellar adhesion through sex-specific adhesion molecules triggers a transient elevation of intracellular cAMP, leading to gamete activation in preparation for cell-cell fusion and zygote formation. Here, we have identified a protein-tyrosine kinase (PTK) activity that is stimulated by flagellar adhesion. We determined that the protein-tyrosine kinase inhibitor genistein inhibited fertilization, and that fertilization was rescued by dibutyryl cAMP, indicating that the genistein-sensitive step was upstream of the increase in cAMP. Incubation with ATP of flagella isolated from non-adhering and adhering gametes followed by SDS-PAGE and immunoblotting with anti-phosphotyrosine antibodies showed that adhesion activated a flagellar PTK that phosphorylated a 105-kDa flagellar protein. Assays using an exogenous protein-tyrosine kinase substrate confirmed that the activated PTK could be detected only in flagella isolated from adhering gametes. Our results indicate that stimulation of the PTK is a very early event during fertilization. Activation of the PTK was blocked when gametes underwent flagellar adhesion in the presence of the protein kinase inhibitor staurosporine, but not in the presence of the cyclic nucleotide-dependent protein kinase inhibitor, H8, which (unlike staurosporine) does not block the increases in cAMP. In addition, incubation of gametes of a single mating type in dibutyryl cAMP failed to activate the PTK. Finally, flagella adhesion between plus and minus fla10-1 gametes, which have a temperature-sensitive lesion in the microtubule motor protein kinesin-II, failed to activate the PTK at elevated temperatures. Our results show that kinesin-II is essential for coupling flagellar adhesion to activation of a flagellar PTK and cAMP generation during fertilization in Chlamydomonas.     

Flagellar adhesion-dependent regulation of Chlamydomonas adenylyl cyclase in vitro: a possible role for protein kinases in sexual signaling

Interactions between adhesion molecules, agglutinins, on the surfaces of the flagella of mt+ and mt- gametes in Chlamydomonas rapidly generate a sexual signal, mediated by cAMP, that prepares the cells for fusion to form a zygote. The mechanism that couples agglutinin interactions to increased cellular levels of cAMP is unknown. In previous studies on the adenylyl cyclase in flagella of a single mating type (i.e., non-adhering flagella) we presented evidence that the gametic form of the enzyme, but not the vegetative form, was regulated by phosphorylation and dephosphorylation (Zhang, Y., E. M. Ross, and W. J. Snell. 1991. J. Biol. Chem. 266:22954-22959; Zhang, Y., and W. J. Snell. 1993. J. Biol. Chem. 268:1786-1791). In the present report we describe studies on regulation of flagellar adenylyl cyclase during adhesion in a cell-free system. The results show that the activity of gametic flagellar adenylyl cyclase is regulated by adhesion in vitro between flagella isolated from mt+ and mt- gametes. After mixing mt+ and mt- flagella together for 15 s in vitro, adenylyl cyclase activity was increased two- to threefold compared to that of the non-mixed (non- adhering), control flagella. This indicates that the regulation of gametic flagellar adenylyl cyclase during the early steps of fertilization is not mediated by signals from the cell body, but is a direct and primary response to interactions between mt+ and mt- agglutinins. By use of this in vitro assay, we discovered that 50 nM staurosporine (a protein kinase inhibitor) blocked adhesion-induced activation of adenylyl cyclase in vitro, while it had no effect on adenylyl cyclase activity of non-adhering gametic flagella. This same low concentration of staurosporine also inhibited adhesion-induced increases in vivo in cellular cAMP and blocked subsequent cellular responses to adhesion. Taken together, our results indicate that flagellar adenylyl cyclase in Chlamydomonas gametes is coupled to interactions between mt+ and mt- agglutinins by a staurosporine- sensitive activity, probably a protein kinase.     

Mating in Chlamydomonas: a system for the study of specific cell adhesion. I. Ultrastructural and electrophoretic analyses of flagellar surface components involved in adhesion

To determine the ultrastructural and biochemical bases for flagellar adhesiveness in the mating reaction in Chlamydomonas, gametic and vegetative flagella and flagellar membranes were studied by use of electron microscope and electrophoretic procedures. Negative staining with uranyl acetate revealed no differences in gametic and vegetative flagellar surfaces; both had flagellar membranes, flagellar sheaths, and similar numbers and distributions of mastigonemes. Freezecleave procedures suggested that there may be a greater density of intramembranous particles on the B faces of gametic flagellar membranes than on the B faces of vegetative flagellar membranes. Gamone, the adhesive material that gametes release into their medium, was demonstrated, on the basis of ultrastructural and biochemical analyses, to be composed of flagellar surface components, i.e., membrane vesicles and mastigonemes. Comparison of vegetative (nonadhesive) and gametic (adhesive) "gamones" by use of SDS polyacrylamide gel electrophoresis showed both preparations to be composed of membrane, mastigoneme, and some microtubule proteins, as well as several unidentified protein and carbohydrate-staining components. However, there was an additional protein of approximately 70,000 mol wt in gametic gamone which was not present in vegetative gamone. When gametic gamone was separated into a membrane and a mastigoneme fraction on CSCl gradients, only the membrane fraction had isoagglutinating activity; the mastigoneme fraction was inactive, suggesting that mastigonemes are not involved in adhesion.     

Behavior of flagella and flagellar root systems in the planozygotes and settled zygotes of the green alga Bryopsis maxima Okamura (Ulvophyceae,Chlorophyta) with reference to spatial arrangement of eyespot and cell fusion site

Behaviors of male and female gametes, planozygotes and their microtubular cytoskeletons of a marine green alga Bryopsis maxima Okamura were studied using field emission scanning electron microscopy, high‐speed video microscopy, and anti‐tubulin immunofluorescence microscopy. After fusion of the biflagellate male and female gametes, two sets of basal bodies lay side by side in the planozygote. Four long female microtubular roots extended from the basal bodies to the cell posterior. Four short male roots extended to nearly half the distance to the posterior end. Two flagella, one each from the male and female gametes, become a pair. Specifically, the no. 2 flagellum of the female gamete and one male flagellum point to the right side of the eyespot of the female gamete, which is located at the cell posterior and which is associated with 2s and 2d roots of the female gamete. This spatial relationship of the flagella, microtubular roots, and the eyespot in the planozygote is retained until settlement. During forward swimming, the planozygote swings the flagella backward and moves by flagellar beating. The male and female flagella in the pair usually beat synchronously. The cell withdraws the flagella and becomes round when the planozygote settles to the substratum 20 min after mixing. The axoneme and microtubular roots depolymerize, except for the proximal part and the basal bodies. Subsequently, distinct arrays of cortical microtubules develop in zygotes until 30 min after mixing. These results are discussed with respect to the functional significance of the spatial relationships of flagellar apparatus‐eyespot‐cell fusion sites in the mating gametes and planozygote of green algae.     

ULTRASTRUCTURAL FEATURES OF MITOSIS IN PLOEOTIA COSTATA (HETERONEMATALES,EUGLENOPHYTA)1

The ultrastructural features of mitosis in the colorless phagotrophic euglenoid, Ploeotia costata (Farmer and Triemer 1988bn; syn: Serpenomonas costata, Triemer 1986) are described. During interphase the nucleus is rounded and lies adjacent to the reservoir and the four basal bodies, two of which bear flagella. At the onset of mitosis, two additional flagella are generated from the accessory basal bodies such that four basal bodies with flagella now lie at one pole of the prophase nucleus. Microtubules develop in the nucleus prior to migration of one of the basal body pairs to the opposite pole of the nucleus. By metaphase, chromosomes with layered kinetochores are aligned on the equator of the spindle, and a dumbbellshaped nucleolus stretches from pole to pole. Continued elongation of the nucleus results in the separation of the chromosomal masses at anaphase. The distance between the nuclear poles from metaphase to anaphase changes little although the overall length of the nucleus nearly doubles. By telophase a large interzonal spindle develops between the forming daughter nuclei. The extended interzonal spindle breaks near the center prior to cell cleavage.     

Rearrangement of the flagellar apparatuses and eyespots of isogametes during the fertilization of the marine green alga,Monostroma nitidum (Ulvophyceae,Chlorophyta)

Behaviors of the flagellar apparatuses (flagella, basal bodies, microtubular roots, etc.), mating structures and eyespots of gametes during the fertilization of Monostroma nitidum were studied using field emission scanning electron microscopy and transmission electron microscopy. The biflagellate isogamete (mt⁺ and mt^?) mating structure has a position that is converse between mt⁺ and mt^? gametes relative to the flagellar beat plane and the eyespot. After the adhesion of mt⁺ and mt^? gametes, gamete fusion occurred between the two mating structures. The cell fusion plane expanded to the cell surface as circumscribed by 1s–2d roots in mt⁺ gamete and 1d–2s roots in the mt^? gamete. Two sets of flagellar apparatuses lay side by side in the planozygote and soon become mutually close. The no. 1 basal body of mt⁺ gamete and the no. 2 basal body of mt^? gamete rotated in a counterclockwise direction, as viewed from the cell anterior. Then, the no. 2 basal body of mt⁺ gamete and the no. 1 basal body of mt^? gamete slid into a face to face position. Finally, four flagella and basal bodies exhibited a cruciate arrangement. The basal bodies of the opposing pair (no. 1 and no. 2) were offset in a counterclockwise orientation by the basal body diameter. The 1s and 2d roots of the mt⁺ gamete lay nearly parallel to the 1d and 2s roots of the mt^? gamete, respectively, at the cell fusion plane. Because of the asymmetric localization of the mating structure, association, and subsequent rearrangement of basal bodies and microtubular roots, two eyespots lay on the same side of the planozygote. After the settlement of the planozygote, the flagellar apparatus started to disintegrate in the zygote cytoplasm.     

SEXUAL PROCESSES IN THE LIFE CYCLE OF GYRODINIUM UNCATENUM (DINOPHYCEAE): A MORPHOGENETIC OVERVIEW1

Sexual processes in the life cycle of the dinoflagellate Gyrodinium uncatenum Hulburt were investigated in isolated field populations. Morphological and morphogenetic aspects of gamete production, planozygote formation, encystment, excystment, and planomeiocyte division are described from observations of living specimens, Protargol silver impregnated material and scanning electron microscope preparations. The sexual cycle was initiated by gamete formation which involved two asexual divisions of the vegetative organism. Gametes were fully differentiated following the second division and immediately capable of forming pairs. Either isogamous or anisogamous pairs were formed by the mid-ventral union of gametes. Gametes invariably joined with flagellar bases in close juxtaposition. Complete fusion of gametes required ca. 1 h, involved plasmogamy followed by karyogamy and resulted in a quadriflagellated planozygote. Planozygotes encysted in 24–48 h to yield a hypnozygote capable of overwintering in estuarine sediments. Hypnozygotes collected from sediment in late winter readily excysted upon exposure to temperatures above 15°C. A single quadriflagellated planomeiocyte emerged from the cyst and under culture conditions divided one to two days later. The four flagella were not evenly distributed at the first division and both bi- and tri-flagellated daughter cells were formed.