Ultrastructure and isolation of glyoxysomes (microbodies) in zoospores of the fungusentophlyctis sp.

Summary A correlative approach, involving light and electron microscopic, cytochemical, and biochemical techniques, was used to study the structure and function of microbodies in zoospores ofEntophlyctis sp. The same population of microbodies already existing in the zoosporangium appeared to be segregated into zoospore initials during cytoplasmic cleavage. Microbodies laid at the anterior end of zoospores and were part of an organized assemblage of organelles, the microbody-lipid globule complex. In the microbody-lipid globule complex, endoplasmic reticulum occurred on the surface of the lipid globules toward the zoospore's exterior, and the microbody, subtended by mitochondria, was appressed to the opposite surface of the lipid globule. The organization of the microbody-lipid globule complex changed as the zoospore swam and encysted. As lipid globules coalesced, the microbody-lipid globule complex became disorganized. After lipid globule coalescence was completed, the microbody-lipid globule complex regained its order, and several microbodies were clustered adjacent to a single lipid globule. The microbodies persisted even in the encysted zoospore, but they were found on all sides of the lipid globule.Microbodies isolated from zoospores contained catalase as well as malate synthase and isocitrate lyase, two enzymes of the glyoxylate cycle. When zoospores encysted greater activities of these glyoxylate cycle enzymes could be detected. The presence of glyoxylate cycle enzymes and the close association between the microbody and lipid globule suggest that microbodies function as glyoxysomes in zoospores and encysted zoospores. The functional significance of the morphological organization of the microbody-lipid complex is discussed in terms of energy production and the conversion of storage lipid into structural components of the cell.     

ZOOSPORE STRUCTURE OF THE MYCOPARASITIC CHYTRID CAULOCHYTRIUM PROTOSTELIOIDES OLIVE

The subcellular organization of zoospores released from sessile, parasitic sporangia of Caulochytrium protostelioides was studied with light and electron microscopy. A single flagellum is posteriorly directed but laterally inserted into the cylindrical motile zoospore. A striated rhizoplast attaches the proximal end of the kinetosome to a specialized region of the nuclear envelope. A system of rough endoplasmic reticulum, smooth endoplasmic reticulum, dictyosomes and bristle-coated vesicles are associated with the one to several pulsating vacuoles typically located near the flagellar apparatus. The microbody-lipid globule complex (MLC) comprises one to many lipid globules. An extensive microbody branches around each lipid globule and encloses a portion of the rhizoplast. A reticulum of smooth surfaced cisternae interdigitates among the branches of the complex microbody, and cisternae are opposed to the surface of lipid globules opposite the microbodies. Mitochondria with predominantly circular profiles are scattered throughout the zoospore body, but several are always adjacent to the microbody, and hence, are also part of the MLC. Ribosomes are uniformly distributed throughout the zoospore, and one to several cisternae of rough endoplasmic reticulum are adjacent to the nuclear envelope. Zoospores of C. protostelioides are similar to several other chytrid zoospores, which also have the same type of microbody-lipid globule complex, but yet are structurally distinct from any other chytrid zoospore.     

THALASSOCHYTRIUM GRACILARIOPSIDIS (CHYTRIDIOMYCOTA), GEN. ET SP. NOV., ENDOSYMBIOTIC IN GRACILARIOPSIS SP. (RHODOPHYCEAE)

Thalassochytrium gracilariopsidis gen. et sp. nov. is an endosymbiotic, polycentric, zoosporic fungus that infected cultures of the red alga Gracilariopsis sp. Based on the posteriorly uniflagellate zoospore and the platelike cristae of the mitochondria, the fungus is placed in the Chytridiomycota. Ultrastructurally, the fungal zoospore is distinguished by the anterior position of the kinetosome, a unique microbody-lipid globule complex, an electron-opaque helix associated with the kinetosome and lipid globules, and a beaked nucleus. Zoospores are positively phototactic, and the unusual helix might constitute part of the photosensory apparatus. Zoospores lack certain taxonomically important structures, such as a rumposome, props, a nonflagellated kinetosome, and flagellar roots. The organism does not fit into any described genus, and the features of its zoospore differ from those of any described order. The fungal thallus is polycentric with multinucleate, septate hyphae. Haustoria form within the algal cells. The fungus does not appear to cause major harm to its host and seems to be host specific. However, during intense sporulation of the fungus, degradation of host chloroplasts was observed in medullary cells.     

The zoospore ofOlpidium brassicae

Summary The ultrastructure of the zoospore ofOlpidium brassicae is described and compared with observations made of other zoospores of the uniflagellatePhycomycetes. The zoospore ofO. brassicae is characterized by an extensive, cone-shaped rhizoplast and a lack of a nuclear cap, as well as a side-body complex or a rumposome. Vacuoles which contain osmiophilic material are termed gamma-like particles. Three-dimensional reconstructions based on serial sectioning were made of the organelles in the region of the nucleus, showing that the zoospore ofO. brassicae contains one or at most two elaborately branched mitochondria. Microbodies have a high degree of interconnection and are in intimate association with the mitochondrion, lipid drops, and the nuclear envelope.     

禾谷多粘菌游动孢子超微结构观察

本文描述了寄生在大麦根部的禾谷多粘菌Polymyxa graminis Led.的次生游动孢子的超微结构,包括核、内质网、高尔基体、线粒体、脂质粒、排泄泡、小囊、具膜小囊、核糖体以及鞭毛基体(Kinetosome)和鞭毛杆等细胞器。游动孢子中未见微体。同时也在电镜下观察了游动孢子接触和穿透根细胞时所形成的管腔(Rohr)和棘杆(Stachel)以及游动孢子穿透细胞壁的详细过程。     

The zoospore of Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew

The zoospore of Pseudosporonospora cubensis is typical of the secondary zoospore of the Peronosporales. The reniform zoospore contains a central nucleus with a prominent beak-like extension to the kinetosomes on the lateral side of the spore in the groove region. "Fuzzy" vesicles derived from dictyosomes surround and fuse with the contractile vacuole. Mitochondria and microbodies are located in the peripheral cytoplasm of the zoospore but the latter are confined to the groove region of the spore. The microbodies usually contain a laminate inclusion and the microbodies are not in a fixed position in relation to the peripheral cisternae. Neither a microbody-lipid body complex nor a "U-body" were observed.
The kinetosomes of the spore are almost perpendicular to each other at the distal end of the beak-like extension of the nucleus. A complex system of cytoplasmic microtu-bules flare out from the kinetosomes to surround the nucleus and bundles of cytoplasmic microtubules extend under the plasmalemma of the spore. The zoospore contain numerous vesicles with osmiophilic inclusions which are finely striated; these are the so-called finger-print vesicles.     

Phylogenetic implications of the microbody-lipid globule complex in zoosporic fungi

Chytridiomycetous fungal zoospores contain a unique and intricate association of organelles, the 'microbody-lipid globule complex' (MLC). The spatial arrangement of organelles in the MLC appears important in the utilization of lipid globules for energy, but in addition, the structural association of organelles in the MLC reveals phylogenetic trends within this diverse group of organisms. Variations in the structure of the MLC correlate well with current phylogenetic concepts of aquatic fungi, yet suggest new relationships among these posteriorly uniflagellate zoospores. Based upon the organization of organelles in the MLC, 4 basic patterns of MLCs can be recognized, and these correspond to the 4 orders of Chytridiomycetes. The MLC in its simplest form consists of a microbody appressed to the edge of a lipid globule. In more highly organized MLCs, mitochondria subtend the microbody and a cisterna surmounts one side of the lipid globule. The organization and structure is still more complex in other MLCs where ER is elaborated into a tubular network of membranes or where small microbodies or mitochondria fuse into 'giant' organelles. The structural organization of the MLC provides an additional criterion by which the phylogeny of awuatic fungi can be evaluated.     

Ultrastructure of the gametes ofCoelomomyces dodgei Couch (Blastocladiales,Chytridiomycetes)

Summary As part of an investigation on the developmental biology ofCoelomomyces dodgei Couch (Blastocladiales, Chytridiomycetes), the ultrastructure of the male and female gametes was studied. The nucleus is central and conical in shape except for a basal spur that curves back towards the large plate-like mitochondrion. A nuclear cap of ribosomes sits on the flat anterior end of the nucleus. Approximately seven lipid globules are partially embedded in the mitochondrion and are interconnected by membrane cisternae. The lipid globules are covered by a single fenestrated microbody and a backing membrane lies between the microbody and the gamete plasma membrane. The kinetosome is at the base of the nucleus and is connected to a single, posterior, whiplash flagellum. A nonkinetosomal centriole is absent. In the peripheral cytoplasm of both mating types there is a paracrystalline body of unknown composition and function. No significant ultrastructural differences were found between the male and female gametes.     

WHAT ARE THE CHROMIDIA OF POLYPHAGUS EUGLENAE?

Zoospores, prosporangia, and asexual sporangia were studied with electron microscopy to determine the ultrastructural identification of “chromidia,” granular masses surrounding nuclei that classical mycologists believed to be extruded chromatin used for lipid synthesis. In the zoospore the nucleus was enclosed by an aggregation of ribosomes. In other developmental stages the behavior of microbodies was identical to that described for “chromidia.” A microbody network with interspersed ER surrounded nuclei in young prosporangia. As the prosporangium matured, lipid globules became associated with the microbodies. When the single, large nucleus migrated into the elongate asexual sporangium, microbodies still surrounded the nucleus; but after the nucleus divided and a multinucleate sporangium formed, microbodies were scattered throughout the cytoplasm. When incubated in the diaminobenzidine medium for the cytochemical detection of catalase, reaction product was found in these microbodylike structures, confirming that “chromidia” described in prosporangia and asexual sporangia by classical mycologists are really microbodies. Rather than giving rise to lipid, these microbodies are probably involved in the metabolism of the lipid globules with which they are associated. The “chromidia” in zoospores are not extruded chromatin as suggested earlier, but correspond in their location around the nucleus to an aggregation of ribosomes.     

Production and modifications of extracellular structures during development of chytridiomycetes

Summary In development of the primitive fungi, chytridiomycetes, unwalled zoospores bearing single, posterior flagella are transformed into walled, round-cells which elaborate the thallus. Production, structural modification, or release of extracellular material are involved with each transition of developmental stage. This article reviews the variety and developmental changes of extracellular materials found at the cell surface of chytridiomycetes. A cell coat, produced from Golgi-derived vesicles during zoosporogenesis, is visible around free swimming zoospores of some chytridiomycetes. How the zoospore surface receives and transduces signals is not widely explored, but it is known that fenestrated cisternae and simple cisternae, which are integrated into the microbody-lipid globule complex, are spatially and structurally associated with the plasma membrane and flagellar apparatus. This spatial association, as well as the cytochemical localization of calcium in fenestrated cisternae, suggest a mechanism for signal transduction and for regulation of zoospore motility. Zoospores become encased in a new layer of extracellular material as the zoospore encysts. Among some chytrids the source of this material is preexisting vesicles which fuse with the plasma membrane. Among other zoospores, a readily identifiable population of encystment vesicles is not apparent, demonstrating that there is no single pattern or mechanism for zoospore encystment in chytridiomycetes. Encysted zoospores developing into thalli, typically produce cell walls with a microfibrillar substructure. Ultrastructural analysis of walls reveals distinctive architecture and remarkable sculpturing which have been used in systematics of some members of chytridiomycetes. Nothing is known as to underlying controls of cytoskeletal elements and plasma membrane enzyme complexes in wall biogenesis. Many changes in cell surface structures accompany thallus maturation. Septa, many traversed with plasmodesmata, are produced in most chytrid thallus types. As sporangia and resting spores prepare for the production and release of zoospores, additional extracellular layers of material are frequently produced. Polarized deposits of extracellular material become discharge plugs, discharge vesicles, or endoopercula. Interstitial material is also released into cleavage furrows. Circumscissile or localized digestion of walls produce operculate or inoperculate exit ports for zoospore release. Cryofixation preserves more extensive extracellular material than does conventional chemical fixation, and broader application of cryofixation may radically alter our current view of cell surface structure. Thus chytridiomycetes exhibit a range in patterns for the occurrence and subsequent modifications of extracellular materials, even for members within the same order. The most universally recognized role for these extracellular materials is protection. Although there is a reasonable view of the types of extracellular material involved in chytridiomycete development, we have only limited understandings of their biogenesis or roles in regulation and communication, areas awaiting more investigations.Abbreviations DIC Nomarski-differential contrast optics - TEM transmission electron microscopy     

The secondary zoospore of Aphanomyces astaci and A. laevis (Oomycetes, Saprolegniales)

The ultrastructure of the secondary zoospores of Aphanomyces astaci and A. laevis was compared. The general appearance of the organelles and their compartmental–ization is the same, but some subtle differences were found. A. laevis has a less distinct distribution of fuzzy vesicles around the border of the water expulsion apparatus than A. astaci. The content of the U–body of the A. astaci zoospore is paracrystalline but the A. laevis U–body has a tubular content. In the peripheral cytoplasm of both species are seen vesicles with a granular content. Flattened cisternae are found in a narrow zone just below the plasmalemma. These two structures are confined to the zoospore stage of the fungi. The lipid–microbody complex of A. laevis cysts is not present in the zoospore. The ultrastructural organization of uniflagellate and bi–flagellate zoospores is compared.     

Localization of antimonate-mediated precipitates of cations in zoospores ofChytriomyces hyalinus

The osmium tetroxide-potassium pyroantimonate technique was used to detect cations in zoospores ofChytriomyces hyalinus. Electron-opaque precipitates were located on the fenestrated and closed cisternal portions of rumposomes in zoospores. Precipitates also appeared in the fenestrae of rumposomes, in mitochondria, in lipid globules, and on the plasma membrane. Ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid, which selectively chelates calcium ions, removed most of the precipitates from sections of rumposomes, mitochondria, and lipid globules, indicating the electron-opaque material was antimonate precipitates of calcium ions concentrated in these organelles. The localization of calcium in therumposome and the close association of the rumposome with the flagellar apparatus suggest a role for the rumposome in the regulation of flagellar activities.     

The zoospore and meiospore of the aquatic phycomyceteCatenaria anguillulae

Summary The zoospore and meiospore of the aquatic phycomyceteCatenaria anguillulae (Phycomycetes, Blastocladiales, Catenariaceae) have a nuclear cap enclosing the cellular ribosomes within a double membrane, and a side body complex which is very similar to that observed in zoospores ofBlastocladiella andCoelomomyces and is structurally related to the side body complex observed in spores ofAllomyces. The structural organization of the side body complex and striated rootlet is analyzed from serial sections.The meiospore also contains an array of flattened cisternae which are in direct contact with, and appear to be derived from, the outer nuclear membrane and the backing membrane of the side body complex.The structural organization of the zoospore and meiospore ofC. anguillulae is compared to and contrasted with the structural organization observed in spores of members of theChytridiales, Blastocladiales, Monoblepharidales, andHarpochytriales. It is concluded that the structural organization of the spores of theBlastocladiales, Monoblepharidales, andHarpochytriales is similar, and affinities in spore organization can be found in some members of theChytridiales.     

Lipid turnover during morphogenesis in the water mold Blastocladiella emersonii

The lipid content of $\text{Blastocladiella emersonii}$ zoospores is 5 pg/cell or about 13% of dry weight. Within the first few minutes of germination 60–70% of total zoospore lipid is lost, with neutral lipid, glycolipid and phospholipid fractions decreasing to about the same extent. These changes in lipid content precede the breakdown during germination of the complex and extensive membrane system of zoospores. During growth, which immediately follows germination, net phospholipid synthesis resumes so that total lipid is maintained at 6% of dry weight, but net synthesis of neutral and glycolipid does not begin until induction of sporulation. During sporulation the phospholipid level decreases so that the distribution of lipid among the three fractions approaches that found in zoospores. These changes in lipid content suggest that zoospore membranes containing neutral and glycolipids are synthesized $\text{de novo}$ during spore formation.     

The peroxisome (microbody) membrane: effects of detergents and lipid solvents on its ultrastructure and permeability to catalase

Synopsis The effects of detergents, organic lipid solvents, and several adjuvants used in cell fractionation on the ultrastructure of the peroxisomal (microbody) membrane and its permeability to catalase have been investigated. Chopper sections of glutaraldehyde-fixed liver were incubated in the presence of various agents, followed by cytochemical staining for catalase and processed for electron microscopy. Catalase activity was also determined biochemically in the incubation medium. Marked catalase diffusion was found after treatment with 1% or 0.5% Triton X-100 or deoxycholate, as well as with 50% ethanol or acetone or 20% propanol ort-butanol. In contrast, 1% digitonin and lower concentrations of the above agents, as well as sucrose or glycerine caused selective diffusion of catalase from a limited population of peroxisomes. Tieatment with 10% polyvinylpyrrolidone (PVP), which has been used as a protective agent in the isolation of microbodies, did not produce any alteration in the fine structure and cytochemical appearance of peroxisomes. These findings concur with earlier biochemical studies on freshly isolated peroxisomes and demonstrate the susceptibility of microbodies, even in glutaraldehyde-fixed rat liver to the effects of various agents which affect the microbody membrane. A close correlation between the ultrastructural integrity of the microbody membrane and its permeability to catalase has been found. The significance of these observations for the assessment of the permeability characteristics of the microbody membrane is discussed.     

Secretory membranes of the lactating mammary gland

Summary The lactating mammary gland is one of the most highly differentiated and metabolically active organs in the body. Membranes of the lactating mammary cell have important roles in transmitting from one membrane to another of hormonal information and in milk secretion, which is the final event. During milk secretion, the projection of the surface membrane into the alveolar lumen by enveloping intracellular lipid droplets with the apical plasma membrane is one of the most remarkable aspects of biological membrane action throughout nature.This review focuses on current knowledge about membranes in the lactating mammary gland. (1) Advances in the isolation and properties of membranes, especially the plasma membrane and Golgi-derived secretory vesicles, concerned with milk secretion from the lactating mammary gland are described. (2) Milk serum components are secreted by fusing the membranes of secretory vesicles that condense milk secretions with the plasma membrane in the apical regions. This occurs through the formation of a tubular-shaped projection and vesicular depression in a ball-and-socket configuration, as well as by simple fusion. (3) Intracellular lipid droplets are directly extruded from the mammary epithelial cells by progressive envelopment of the plasma membranes in the apical regions. (4) The balance between the surface volume lost in enveloping lipid droplets and that provided by fusion of the secretory vesicle and other vesicles with the apical plasma membrane is discussed. (5) The membrane surrounding a milk fat globule, which is referred to as the milk fat globule membrane (MFGM), is composed of at least the coating membrane of an intracellular lipid droplet, of the apical plasma membrane and secretory vesicle membrane, and of a coat material. Consequently, MFGM is molecularly different from the plasma membrane in composition. (6) MFGM of bovine milk is structurally composed of an inner coating membrane and outer plasma membrane just after segregation. These two membranes are fused and reorganized through a process of vesiculation and fragmentation to stabilize the fat globules. Hypothetical structural models for MFGM from bovine milk fat globules just after secretion and after rearrangement are proposed.Abbrevations MFGM milk fat globule membrane - HEPES N-2-hydroxylpiperazine-N-2-ethanesulfonic acid - INT 2-(p-indophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - Sph sphingomyelin - PC phosphatidyl choline - PE phosphatidyl ethanolamine - PS phosphatidyl serine - PI phosphatidyl inositol - PAS periodic acid-Schiff reagent - CB Coomassie brilliant blue R-250 Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

Gametogenesis inCoelomomyces dodgei Couch (Blastocladiales,Chytridiomycetes)

Summary The ultrastructure of gametogenesis was studied inCoelomomyces dodgei Couch (Blastocladiales, Chytridiomycetes), an obligate parasite of anopheline mosquito larvae and the copepod,Acanthocyclops vernalis. In infected copepods reared under a 16/8 hours light/dark photoperiod at 25 +2 °C., the gametophyte develops over a period of approximately seven days, and gametogenesis is triggered by the onset of the dark period during the last day of development. The initial step of gametogenesis is the elongation of the centriole to form the kinetosome, and measuring time from the onset of the final dark period (0 hours), this occurs prior to the beginning of the light period (8 hours). Subsequently, small vesicles that appear to originate from elements of the rough endoplasmic reticulum (rER) fuse at the distal end of the kinetosome forming the flagellar vesicle into which the axonemal microtubules elongate to form the flagellum (8–12 hours). Similar small vesicles apparently also derived from rER align in planes and fuse to form cleavage furrows which delineate the gamete initials (12–14 hours). As the gamete initials begin forming, the mitochondria within each initial fuse to form a single mitochondrion that associates with the lipid globules and microbodies forming the microbody-lipid globule complex (12–16 hours). The time elapsed between the formation of the flagellar vesicle to the release of mature gametes from the copepod host is about 8.5 hours. No differences were observed in the processes or timing of gametogenesis in male and female gametophytes.     

Alogomyces tanneri gen. et sp. nov., a chytrid in Lobulomycetales from horse manure

The order Lobulomycetales contains chytrids from soil, freshwater and marine habitats; environmental DNA sampling has indicated that representatives of this order might be found in deep ocean localities. We describe Alogomyces tanneri as the first lobulomycetalean chytrid isolated from horse manure; A. tanneri is also the first species in the order to possess a rumposome in its zoospore. This species widens the range of habitats, ultrastructural variation and thallus morphology for Lobulomycetales.     

ULTRASTRUCTURAL MORPHOLOGY OF MICROTHAMNION ZOOSPORES1

The Ultrastructure of Microthamnion zoospores is described (exclusive of the finer details of the flagellar apparatus). The zoospores have a typical chlorophycean morphology but, in addition, many unique features. The chloroplasts contain starch but no pyrenoid. Thylakoids may run from one edge of the chloroplast to the other and usually anastomose into 2- to 8-membered stacks. The internal morphology is highly polarized and characterized by an intimate proximity and constant spacing between many organelle membranes. All the organelles are asymmetrically distributed within the cell in a precise manner. The anterior region of the zoospore is attenuated into a neck which contains a single, massive mitochondrion. A fibrous rhizoplast lies beside the mitochondrion and appears to connect the flagellar apparatus directly with the outer membrane of the nuclear envelope. In addition, this outer membrane is extended over a distance of several microns to eventually lie in close proximity to the basal bodies. Oil vacuoles and lipid bodies are restricted to the posterior end of the zoospore.     

Endocytosis of cationized ferritin by zoospores of the fungusAphanomyces euteiches

Summary Cationized ferritin, a marker for adsorptive endocytosis, was taken up by zoospores of the fungusAphanomyces euteiches. The probe was endocytosed into the numerous, often coated, vesicles surrounding the contractile vacuole. The vacuole itself contained very little ferritin. It is suggested that the contractile vacuole complex is the main area of membrane recycling in the zoospore. After zoospore encystment some of the ferritin was found in multivesicular bodies and the remnants of the contractile vacuole.