Electron microscopy of gametangial interaction and oospore development in Phytophthora capsici

Gametangial development and oospore formation were studied, with emphasis on cell wall morphogenesis, on mated cultures (A¹xA²) of Phytophthora capsici. In this species, the oogonial and antheridial hyphae interact to produce a typical amphigynous antheridium. The following developmental steps were recognized: 1) contact between oogonial and antheridial initials; 2) penetration of the antheridial initial by the oogonial initial; 3) reemergence of the oogonial initial; 4) oogonial expansion; 5) gametangial delimitation and oogonial wall thickening; 6) penetration of the oogonium by the antheridial fertilization tube; 7) oosphere formation; 8) periplasm degeneration and outer oospore wall formation; and 9) inner oospore wall formation. Electron micrographs were obtained of steps 3–9. Steps 1 and 2 were reconstructed from subsequent events. Steps 3–6 are stages of active wall formation with clear indication of intensive dictyosome activity leading to the formation of numerous wall-destined vesicles of two different sizes and electron densities. No vesicles were seen associated with the development of the inner oospore wall; however, by this stage of development the oosphere cytoplasm exhibited an overall intense electron density that obscured fine detail. Cytoplasmic appearance changed enormously during differentiation, from a developing oogonium rich in mitochondria, ribosomes, rough endoplasmic reticulum, dictyosomes and their vesicles, through an oosphere filled with large finger-print vacuoles and lipid-like bodies, to a mature oospore with a large central vacuole (ooplast) surrounded by a cortex of numerous lipid-like bodies; other organelles are confined to the interstitial space between these storage bodies.     

Induction of abnormal male gametes and androgenesis in the aquatic PhycomyceteAllomyces

Summary Induction of cleavage of the cytoplasm in the gametangium of a predominantly male strain of the aquatic PhycomyceteAllomyces under conditions of oxygen starvation, in the presence of dilute lactic acid, or dilute phosphate buffer, pH 7.0, resulted in multinucleate-multi-flagellate gametes. Under certain conditions the frequency for such abnormal gametes approached 70%. Phosphate buffer pH 7.0, at a concentration of 5 × 10^–3-1×10^–2 M and an incubation time of 90 minutes was found to be most effective in inducing multi-flagellate-multinucleate gametes. Nuclear fusion and multiple nuclear fusions were observed in these abnormal gametes as they developed into sporophytic thalli on dilute nutrient agar (YpSS/10). Multinuclear gamete development and nuclear fusion was analysed by electron microscopy. The reported observations reveal the occurrence of androgenesis from multinuclear male gametes inAllomyces.     

Hypoxia and sulphide influence gamete production in Ulva sp.

Gamete production after exposure to hypoxia or sulphide was studied in the marine macroalga Ulva sp. collected in the Sacca di Goro, Italy. Experiments were carried out on discs (12 mm diameter) of thalli cultured in artificial sea water in laboratory at 20 ± 1 °C, 152 μmol m⁻² s⁻¹, 16 h photoperiod and 30‰ salinity. Dehydration of thallus was used as inducer of gametogenesis and growth and gamete release during recovery after 10, 20, 30 or 40 min dehydration (20 ± 1 °C, 25% humidity) were analysed. Unlike non-dehydrated thalli the dehydrated ones produced gametes. Thallus discs, non-dehydrated or subjected to 30 min dehydration, were exposed to hypoxia (1.78–4.02 μmol O₂ L⁻¹) or sulphide (1 mM) for 3, 5, or 7 days at 20 °C in the dark. Non-dehydrated and dehydrated thalli maintained in normoxic conditions in the dark were the controls. Gamete density was checked by counting at the end of the incubation period and during the subsequent 7 days of recovery under 16 h photoperiod in normoxic conditions. Non-dehydrated thalli maintained in normoxic conditions in the dark released gametes when returned to light suggesting that dark constitutes a stimulus to gamete production. The presence of gametes at the end of 3 days incubation of dehydrated thalli in normoxia demonstrated that gametogenesis can occur even in the dark. However, gametes were not present at the end of incubation in hypoxic and sulphidic conditions. Actually, during hypoxic incubation oxygen consumption in D-thalli was very low, only 0.117 × 10⁻³ μmol O₂ mg⁻¹ h⁻¹ compared to 5.93 × 10⁻³ μmol O₂ mg⁻¹ h⁻¹ in normoxia, denoting a reduction of the metabolic rate that could not sustain gametogenesis. During recovery after incubation in normoxic, hypoxic or sulphidic conditions densities of gametes from dehydrated thalli showed significant differences and resulted after hypoxia > after normoxia > after sulphide. Differences in non-dehydrated thalli were not significant. Dehydrated thalli, still green at the end of the incubation period, underwent blanching in the course of recovery in parallel to gamete production, while non-dehydrated thalli maintained their green colour even after exposure to sulphide. Our findings suggest that macroalga Ulva sp. can survive exposure to darkness, severe hypoxia and high sulphide levels and can maintain gamete production even when the exposure to these stress conditions is joined to dehydration.     

TWO NEW SPECIES OF OEDOCLADIUM1

Two new gynandrosporous species of Oedocladium from the United States were isolated into unialgal culture and comparative studies were made. The new taxa described, O. carolinianum sp. nov. and O. cir-ratum sp. nov., are morphologically very similar. Both species have an opercufate oogonium with inferior division, oospore walls with the middle layer ungulate, and hyaline suffultory cells containing a small amount of vacuolated cytoplasm. Conditions for sexuality in culture zuere determined for both species. Zoospores and akinetes serve as means for asexual reproduction.     

Crossing test among floating Ulva thalli forming `green tide' in Japan

Crossing tests were made to determine the relationship between the identified Ulva pertusa, which commonly grows in Japan as an attached form on exposed rocks, and the floating Ulva forming "green tide" inside calm bays. The floating Ulva thalli were collected from five major green tide sites in Japan (Yokohama, Mikawa, Miyajima, Kochi and Hakata). Reproductive maturation was induced in U. pertusa and the floating thalli from each site. Mating between induced gametes was observed. It is therefore believed that the floating thalli from Yokohama, Mikawa and Miyajima were mainly U. pertusa, while those from Kochi and Hakata were of a different species (Ulva sp.1). Furthermore, the Ulva species found in Mikawa is also a species (Ulva sp.2) different from both U. pertusa and Ulva sp.1.     

Effect of Calcium Chloride Concentration on Growth and Sporulation of Saprolegnia terrestris Cookson ex Seymour

As CaCl₂ concentration in the growth medium was reduced from27.4 to 0.107 µM the range of oospore number per oogoniumin Saprolegnia terrestris increased from 1 to 8 to 1 to 25 ormore, oospore size decreased and the precision of correlationbetween oospore number per oogonium and oogonial diameter decreased,but colony d. wt was not affected. The threshold CaCl₂concentrationrequired to give the lower range of oospore number per oogoniumwas independent of the concentration of other mineral nutrientsin the growth medium. The number of oogonia formed per unitarea of colony was affected by the concentration both of CaCl₂and of other mineral nutrients. Oospores formed in 0.107 µMCaCl₂ medium were often multinucleate but otherwise of normalappearance. The oospore abortion rate was increased from 3.09per cent at 27.4 µM CaCl₂ to 8.2 per cent at 0.107 µMCaCl₂. calcium, oogonia, oospores, Saprolegnia terrestris Cookson ex Seymour     

Morphological changes of giant mitochondria in the unicellular to multicellular phase during parthenogenesis of Ulva partita (Ulvophyceae) revealed by expression of mitochondrial targeting GFP and PEG transformation

A giant mitochondrion that branches and connects as a single mitochondrion in a cell has been observed during specific phases of the cell cycle of unicellular green algae, but has not been observed in multicellular algae. The genus Ulva is a green macroalga in which the haploid and diploid phases are isomorphic and its gametes develop parthenogenetically. The existence or absence of the giant mitochondrion, and its behavior in Ulva partita, were investigated using a parthenogenesis system. To observe the parthenogenesis of gametes and the dynamics of mitochondria by fluorescence microscopy, we developed an experimental system for culturing and observing U. partita on cover slips: gametes were suspended in 6‐well plates filled with artificial seawater, and cover slips were placed on the well bottoms. The gametes settled on the cover slips as spherical cells (1‐cell S phase). These cells grew into larger cells, losing their eyespot (1‐cell L phase), and developed into multicellular thalli. Gene introduction using the polyethylene glycol (PEG) method is available with transformation efficiencies of 9.0–15.1%. Transformation was performed using a plasmid encoding green fluorescent protein (GFP) fused to the mitochondrial targeting sequence, and mitochondria were labeled by GFP fluorescence. This revealed a string‐shaped giant mitochondrion in a cell of the 1‐cell S phase. In the 1‐cell L phase, a reticular mitochondrion was observed. After the initiation of cell division, the reticular mitochondrion was fragmented, and small oval mitochondria were observed in the 5‐cell phase.     

A Histochemical Study of Cytoplasmic Changes During Wall Layer Formation in the Oospore of Albugo candida

The young multinucleate oogonium in Albugo is double-walled with an outer layer exhibiting a negative staining reaction for insoluble polysaccharides and an inner layer which is strongly PAS-positive. The oogonial nuclei exhibit an unusual staining behaviour with aniline blue showing an outer dark blue sheath of proteins surrounding a central hyaline nuclear core. Various histochemical localizations were performed for tracing the chronological sequence of development of the wall layers of the oospore. The first wall of the fertilized oospore was laid at the interphase of the periplasm and the ooplasm. Subsequent wall layers were formed both on the inner and outer side of the first oosporic wall. The second oosporic wall was formed just internal to the first one and exhibited faint PAS positivity. The third wall of the oospore was formed external to the first one and the PAS-negative material for this was apparently contributed by the periplasm. This wall layer at later stages acquired a ridged appearance and these ridges in a mature oospore appear as distinct “pegs”. The last wall to be formed is the innermost one and it completely surrounds the central ooplasm. This wall layer is callosic in nature.     

Protoplast isolation optimization and regeneration of cell wall in Gracilaria gracilis (Gracilariales, Rhodophyta)

This paper reports the first successful isolation and cell wall regeneration of Gracilaria gracilis (Stackhouse) Steentoft, Irvine et Farnham protoplasts. These results form an important foundation for the development of a successful tissue culture system for G. gracilis. Initially, an isolation protocol was optimized by investigation of the effects of the enzyme constituents and concentrations, the pre-treatment of thalli, the incubation period and temperature, and the pH of the enzymatic medium on protoplast yields. A pre-treatment of G. gracilis thalli with 1 % (w/v) papain for 30 min followed by a 3-h enzymatic digestion of thalli with an enzymatic mixture containing 2 % (w/v) cellulase Onozuka R-10, 1 % (w/v) macerozyme R-10, and 10 U mL^?1 agarase at pH 6.15 was found to produce the highest yield of protoplasts at 22 °C. Reliably high yields (20–30?×?10⁵ protoplasts g^?1 f.wt) of protoplasts could be obtained from G. gracilis thalli when this optimized protocol was used. Cell wall re-synthesis by G. gracilis protoplasts, which constitutes the first step towards whole plant regeneration, was followed using calcoflour staining and scanning electron microscopy. Protoplasts were shown to complete the initial stages of cell wall re-synthesis within the first 24 h of culturing.     

Gametangial interaction and oogenesis in the phycomycete Ciliomyces spectabilis (Fungi,Lagenidiales)

Gametangial interaction and oospore formation were studied in Ciliomyces spectabilis, a Lagenidiaceous fungus which is parasitic on ciliate cysts. Electron dense and granular vesicles of the antheridium are engaged in formation of the copulation porus between adjacent thalli. The oosphere is delimited by Golgi-derived cisternae which give rise to the membranes of the oosphere and the periplasm. The contents of the antheridium and the periplasm degenerate. The outer oospore wall is formed by wall vesicles originating from the endoplasmic reticulum. No vesicles are involved in the development of the thick inner oospore wall. Vacuoles with electron dense spherical contents fuse and form the central reserve globule. Lipid bodies aggregate first and disintegrate later into numerous small ones. The number of cytoplasmic organelles decreases. The possibility of wall formation via secretion of soluble wall material is discussed.     

Characterization of sexual reproductive processes in Chara braunii (Charales,Charophyceae)

Chara braunii is distributed worldwide and is the most common charalean species in Japan. This species is monoecious and produces numerous sets of sex organs, each of which consists of one antheridium and one oogonium, under laboratory culture conditions. In this study, we report that light intensity strongly affected the vegetative phase and sexual reproductive phase of this species. Under high‐light conditions (70.0 μmol photons m^?2 s^?1), thalli grew but did not form reproductive organs. Under a low‐light intensity (10.0 μmol photons m^?2 s^?1), algal bodies formed many reproductive organs. In addition, antheridia without the corresponding oogonia (lone antheridia) were observed under low‐light conditions. The absence of oogonium primordia adjacent to the lone antheridium was confirmed by several microscopic approaches. The addition of liquid fertilizer increased the total number of sex organs and growth; however, the number of lone antheridia decreased with increasing fertilizer concentrations. Exogenously applied gibberellin did not affect the number of lone antheridia. These results suggest that regulatory mechanisms for the appropriate allocation of resources exist in this alga, similar to those reported in some land plants.     

Antheridial dehiscence in ferns

We investigated the mechanism of antheridial dehiscence in ferns for the first time using fluorescence microscopy as well as scanning and transmission electron microscopy. The mechanism leading to antheridial dehiscence in Polystichum setiferum, Asplenium trichomanes and A. onopteris was found to depend on the different cellulose contents of the inner and outer walls of the ring cells detected with calcofluor white stain and the Thiéry test. The extremely low cellulose content of the ring cell walls facing spermatozoids made them less mechanically resilient than external wall cells. When the ring cells absorbed water they expanded only into the antheridial cavity, pushing the gametes against the cap cell, which detached from the ring cell below and enabled spermatozoid release. The newly released spermatozoids were spherical bodies covered in cellulose fibrils. The significance of cellulose fibrils could be to isolate the gametes from each other, to reinforce the electron transparent material and to protect the gamete from pressure created by the ring cells during release.     

Eyespot behavior during the fertilization of gametes in Ulva arasakii Chihara (Ulvophyceae, Chlorophyta)

Behavior of the eyespots during the fertilization of Ulva arasakii Chihara was studied using field emission scanning electron microscopy (FE‐SEM). FE‐SEM enabled the visualization of the eyespot of biflagellate male and female gametes. The smaller male gamete has one protruded smaller (1.3 ± 0.15 μm× 1.0 ± 0.29 μm) eyespot and the larger female gamete has a larger (1.6 ± 0.2 μm× 1.1 ± 0.13 μm) one on a posterior position of the cell. The cell membrane over the eyespot region is relatively smooth compared to other parts of the cell body and exhibits hexagonal arranged lipid globules. Because the size of the cell and the morphology of the eyespot are different between male and female gametes, we could follow the fate of the eyespots during the fertilization. The initial cytoplasmic contact and fusion of the gametes takes place at their anterior end, slightly posterior to the flagellar base. The morphology of the fusing gametes followed two clearly distinguishable patterns. About half the gamete pairs lie side‐by‐side with their longitudinal axes nearly parallel, while the rest are oriented anti‐parallel to each other. In all cases, the larger female gamete fused along the same side as the eyespot, while the smaller male gamete fused along the side away from its eyespot. As fusion proceeds, the gamete pair is transformed into the quadriflagellate planozygote, in which the eyespots are positioned side‐by‐side on the region of cell fusion. These observations indicated that the opposite positioning of the eyespot relative to the cell fusion site in male and female gametes is important for the proper arrangement of the eyespots in the planozygote. The significance of this feature in advanced green algae is briefly discussed.     

LIFE HISTORY OF COLPOMENIA SINUOSA (SYCTOSIPHONACEAE,PHAEOPHYCEAE) IN THE AZORES1

Colpomenia sinuosa (Mertens ex Roth) Derbès and Solier (Scytosiphonaceae, Phaeophyceae) is a common species on the rocky intertidal shores of the Azores, where reproductive gametophytes occur throughout the year. Life‐history studies of this species were carried out in culture, and both sexual and asexual reproduction were observed. Anisogamous gametes fused to form zygotes. The zygotes gave rise to a filamentous prostrate sporophyte generation bearing unilocular sporangia, under both short‐day and long‐day conditions at 15 and 22°C, and to both unilocular and plurilocular sporangia, under the lower temperature condition. Unispores developed into gametophytes, and plurispores gave rise to filamentous sporophytes. Asexual reproduction was carried out by unfused female gametes and asexual plurispores produced from the same gametophyte. Unfused gametes developed into filamentous prostrate sporophytes producing unilocular sporangia in both culture conditions, and unispores released from the sporangia gave rise to gametophytes. Asexual plurispores from field gametophytes, under both culture conditions, developed directly into new gametophytes. The species exhibited three types of life history: a heteromorphic, diplohaplontic; a heteromorphic, monophasic (both with alternation between the erect and filamentous prostrate thalli); and a monomorphic, monophasic.     

腐霉属的一个新种和二个新记录

本文报告作者1982—1983年在北京和广东一些地区土壤中用已知方法(余永年,1975)分离到的几种腐霉,其中1个新种(Pythium borealis sp.nov.)、2个国内未报道的种(P.oligandrum和P.myriotylum)和1个已知种的变异株(P.ultimum variant)。文中对以上各菌进行了形态学描述。     

Developmental pattern of crust into upright thalli of Grateloupia asiatica (Halymeniaceae,Rhodophyta)

Grateloupia asiatica is an edible seaweed and source of carrageenan in Korea. Considering the economic importance of this edible seaweed, mass culturing methods from spores have been conducted. Moreover, it helps prevent natural population depletion. Spores germinate to form crusts, filaments and spherical structures that subsequently differentiate and develop into thalli. The objective of our research was to study the developmental pattern of crust into upright thalli. Vegetative cells of crust divided circularly to expand and coalescence with other crusts, while those cells that divide vertically increase the thickness of the crust. The specific growth rate of the crusts was optimal at 20 °C (8.6 % day^?1) and 42 μmol photons m^?2 s^?1 (10.8 % day^?1). At optimum condition, they grew in regularity that forming pattern of zonation and circular mounds onto the crusts subsequently produce upright thalli. Thalli would be generated if the crusts, generally composed of six cells in thickness, will start to produce cortical and medulla cells to support and generate upright thalli. In the tank culture, the upright thalli grew rapidly during July to October and reached a maximum length of around ±5.2 cm. The thalli matured and contained many carposporangia in the branches after 150 culture days.     

Transmission of sex cells of one species through the body of a second species in the genus Xenopus. II. Interspecific matings

Hybrids between X. mulleri and X. laevis have been prepared by two methods. By one method the hybridization has been effected either by a natural coupling following hormonal stimulation or by means of an artificial fertilization. By the other method, the primordial germ cells of mulleri have been introduced into laevis at the neurula stage and, when the host laevis had reached sexual maturity, the mulleri gametes produced have been brought into contact with laevis gametes through a natural coupling.