Ultrastructure of the nematode pathogen, Bacillus penetrans

The ultrastructure and development of Bacillus penetrans in root-knot nematodes, Meloidogyne spp., was studied with a transmission electron microscope. Host infection was by a germ tube from the cup-shaped sporangium containing the endospore. The prokaryotic vegetative cells contained septa formed by an ingrowth of the inner layer of the trilaminate cell wall and were associated with mesosomes. Structure of the endospore was similar to other bacteria with a spore protoplast enclosed within two cortical layers and three spore coats. An exosporium which may function in attachment and host specificity surrounded the endospore. Ultrastructural changes accompanying sporulation were similar to those reported for other endospore-forming bacteria but with some parasite specialization. The filamentous vegetative growth was characteristic of some Actinomycetales. Endospore development at the apices of dichotomously branched filaments of the thallus resembled the genus Actinobifida.     

Cytokinesis of the binucleate zoosporangia of Allomyces macrogynus

Allomyces macrogynus, a true fungus, produces zoosporangia which discharge uninucleate zoospores after cytoplasmic cleavage. Binucleate zoosporangia of A. macrogynus were induced and examined to understand the basic principles of cytokinesis associated with the multinucleate zoosporangia. Development of cleavage membranes was visualized by constructing three dimensional models based on electron micrographs and confocal images. Cleavage membranes on the cleavage plane showed asymmetric ingression from the cortex, but cleavage of cytoplasm was completed by the fusion of cleavage membranes with plasma membrane. Also, the position of the cleavage plane was continuously rotated until settled at the last stage. These studies suggest that the positions of the numerous cleavage planes within a multinucleate zoosporangium are continuously adjusted during development of cleavage membranes. The final settlement of cleavage planes would define the exact boundary of cleavage planes and the expansion of cleavage membranes toward the boundary could complete the cleavage of cytoplasm.     

Phagotrophy and Two New Structures in the Malaria Parasite Plasmodium berghei

Blood collected from rats infected with Plasmodium berghei was centrifuged and the pellet was fixed for 1 hour in 1 per cent buffered OsO₄ with 4.9 per cent sucrose. The material was embedded in n-butyl methacrylate and the resulting blocks sectioned for electron microscopy. The parasites were found to contain, in almost all sections, oval bodies of the same density and structure as the host cytoplasm. Continuity between these bodies and the host cytoplasm was found in a number of electron micrographs, showing that the bodies are formed by invagination of the double plasma membrane of the parasite. In this way the host cell is incorporated by phagotrophy into food vacuoles within the parasite. Hematin, the residue of hemoglobin digestion, was never observed inside the food vacuole but in small vesicles lying around it and sometimes connected with it. The vesicles are pinched off from the food vacuole proper and are the site of hemoglobin digestion. The active double limiting membrane is responsible not only for the formation of food vacuoles but also for the presence of two new structures. One is composed of two to six concentric double wavy membranes originating from the plasma membrane. Since no typical mitochondria were found in P. berghei, it is assumed that the concentric structure performs mitochondrial functions. The other structure appears as a sausage-shaped vacuole surrounded by two membranes of the same thickness, density, and spacing as the limiting membrane of the body. The cytoplasm of the parasite is rich in vesicles of endoplasmic reticulum and Palade's small particles. Its nucleus is of low density and encased in a double membrane. The host cells (reticulocytes) have mitochondria with numerous cristae mitochondriales. In many infected and intact reticulocytes ferritin was found in vacuoles, mitochondria, canaliculi, or scattered in the cytoplasm.     

Epiphytism in a subtidal natural bed of Gracilaria gracilis of southwestern Atlantic coast (Chubut, Argentina)

The diversity of epiphytes, their temporal abundance variation and the anatomical structure of host–epiphyte interfaces were studied in the agarophyte Gracilaria gracilis from a natural bed in Bahía Bustamante, Chubut Province, Argentina. Twenty-nine algal species were recorded as epiphytes during 2 years of monthly sampling (March 2006–February 2008). Total epiphyte density ranged between 0.037 ind. cm^?2 (November 2006) and 39.37 ind. cm^?2 (April 2007), with higher density values throughout the second sampling year. Ceramiales species were the most abundant epiphytes. The density of Ceramium rubrum ranged from 0.09 ind. cm^?2 (52 % of the total amount) in September 2006 to 17.4 ind. cm^?2 (44.18 % of the total amount) in April 2007. Epiphyte infection was more dependent on spore recruitment and sporeling development, especially on thalli derived from fragmentation, than on seasonal environmental variations. The different infections were analysed, taking into account the epiphytic attachment strength and invasiveness and the degree of damage inflicted on the host. Calothrix confervicola was one of the most abundant species. This epiphyte, weakly attached to the host surface, generated no host tissue damage. In contrast, C. rubrum, Polysiphonia abscissa and other Ceramiales were the species that caused more damage to the host because their rhizoids penetrated the cortical portion of the host thallus, sometimes reaching the medullary tissue. Some generalisations and characterisations of the different epiphyte groups in relation to their consequences to Gracilaria spp. are presented.     

Taxonomy, molecular phylogeny, and ultrastructural morphology of Olpidiopsis porphyrae sp. nov. (Oomycetes, straminipiles), a unicellular obligate endoparasite of Bangia and Porphyra spp. (Bangiales, Rhodophyta)

Olpidiopsis porphyrae sp. nov., a marine oomycete endoparasite that infects the commercially cultivated red alga Porphyra yezoensis, is described and its phylogenetic position based on molecular data and ultrastructural morphology is discussed. O. porphyrae infects the host Porphyra by means of encysted zoospores. Spherical-shaped holocarpic thalli develop within the cytoplasm of its algal host, which produce monoplanetic, subapically biflagellate zoospores. The characteristic features of this isolate are the ellipsoidal, unicellular thallus and simple holocarpic zoosporangial development, which show morphological similarity with the genus Olpidiopsis. Laboratory infection experiments with a wide range of green, brown, and red algae revealed that O. porphyrae infects several stages of the bangialean red algae (the genera Bangia and Porphyra). Molecular phylogenetic analyses inferred from both SSU rRNA and cox2 genes showed O. porphyrae branched before the main saprolegnian and peronosporalean lineages within the monophyletic oomycete clade, indicating its phylogenetic separation from them. A single or double K-body-like organelle, which contains tubular inclusions, is found located to one side of the zoospore nucleus and shows similarities to homologous organelles previously described in O. saprolegniae. The ultrastructural morphology of O. porphyrae with zoospore initials containing K-bodies and tubular mitochondrial cristae is characteristic of oomycetes. Group I intron-like multiple insertions were found in the SSU rRNA gene of O. porphyrae. This is the first report of SSU group I introns in the class Oomycetes.     

A new oomycete species parasitic in nematodes, Chlamydomyzium dictyuchoides sp. nov.: Developmental biology and phylogenetic studies

The genus Chlamydomyzium is a little studied holocarpic oomycete parasite of nematodes of uncertain phylogenetic and taxonomic position. A new holocarpic species, Chlamydomyzium dictyuchoides, is described which has usually refractile cytoplasm and a dictyuchoid pattern of spore release. This new species infects bacteriotrophic rhabditid nematodes and was isolated from diverse geographical locations. Infection was initiated by zoospore encystment on the host surface and direct penetration of the cuticle. A sparsely branched, constricted, refractile thallus was formed which eventually occupied almost the entire host body cavity, often accompanied by complete dissolution of the host cuticle. Walled primary cysts formed throughout the thallus and each cyst released a single zoospore via an individual exit papillum, leaving a characteristic dictyuchoid wall net behind. At later stages of infection some thalli formed thick-walled stellate resting spores in uniseriate rows. Resting spore formation appeared to be parthenogenetic and was not accompanied by the formation of antheridial compartments. These spores had ooplast-like vacuoles and thick multi-layered walls, both of which suggest they were oospores. The maximum likelihood tree of sequences of the small ribosomal subunit (SSU) gene placed this new isolate in a clade before the main saprolegnialean and peronosporalean lines diverge. A second undescribed Chlamydomyzium sp., which has direct spore release forms a paraphyletic clade, close to C. dictyuchoides and Sapromyces. The fine structure of other documented Chlamydomyzium species was compared, including an undescribed (but sequenced) isolate, SL02, from Japan, Chlamydomyzium anomalum and Chlamydomyzium oviparasiticum. Chlamydomyzium as currently constituted is a paraphyletic genus that is part of a group of phylogenetically problematic early diverging clades that lie close to both the Leptomitales and Rhipidiales.     

Host-parasite interface of Veronica persica and Sorosphaera veronicae (Plasmodiophoromycetes)

The host-parasite interface between host, Veronica persica Poir., and parasite, Sorosphaera veronicae Schroeter, is observed to consist of a single, unit membrane, presumably the plasma membrane of the fungal parasite.     

Induction of gel-phase lipid in plasma membrane of chick intestinal cells after coccidial infection

When chickens are infected with the coccidial parasite Eimeria necatrix, the plasma membrane of intestinal cells harbouring second-generation schizonts becomes refractory to mechanical shearing, hypotonic shock and ultrasonication. Plasma membrane from these infected cells was isolated to high purity as judged by enriched levels of ouabain-sensitive (Na⁺ + K⁺)-stimulated Mg²⁺-dependent ATPase activity and sialic acid content, the lack of detectable cytochrome oxidase and glucose-6-phosphatase activities and electron microscopic analysis of the final preparation. Wide-angle X-ray diffraction patterns recorded from the isolated membranes revealed that during the later stages of parasite maturation the host cell plasma membrane acquires increasing proportions of gel-phase lipid. By contrast, purified membrane from isolated parasites is in a liquid-crystalline state. The transition temperature of host cell plasmalemma at 100 h postinfection is 61°C, about 20°C above physiological temperature. By contrast, liposomes of plasma membranes from infected cells undergo a thermal transition at about 28°C. The accumulation of gel-phase lipid in the host cell plasma membrane is not attributable either to an increase in the constituent ratio of saturated to unsaturated fatty acids or to a significant change in the cholesterol to phospholipid ratio. During the late stages of infection, the cells become stainable with trypan blue which suggests that the acquisition of crystalline phase lipid disrupts the permeability of the host cell plasmalemma.     

Fine structure of the microsporidan Abelspora portucalensis gen.n., sp.n. (Microsporida) parasite of the hepatopancreas of Carcinus maenas (Crustacea,Decapoda)

A new species of a microsporidan, Abelspora portucalensis, was found in the hepatopancreas of Carcinus maenas, forming white xenomas. Each xenoma seems to consist of an aggregate of hypertrophic host cells in which the parasite develops and proliferates. This cytozoic microsporidan being characterized by one uninucleate schizont giving rise to two sporonts, each originating two sporoblasts, resulting in two spores within a persistent sporophorous vacuole (pansporoblast) should be included in a new family Abelsporidae. In fresh smears most spores were 3.1–3.2 μm long and 1.2–1.4 μm wide. Fixed, stained, and observed in SUS mature spores measured 3.1 ± 0.08 × 1.3 ± 0.06 μm (n = 25 measurements). Spore cytoplasm was dense and granular, polyribosomes were arranged in helicoidal tape form. The polar filament was anisofilar and consisted of a single coil with 5–6 turns. The anchoring disc and and the anterior zone of the filament are surrounded by the polaroplast composed of two usual zones. In the anterior zone, the membrane of the polar filament is in continuity with the membranes of the polaroplast. The appearance of a microsporidan with described nuclear divisions in life cycle, spores shape and size, polaroplast and polar filament morphology and identity of the host suggests that we may erect a new genus Abelspora and a new species A. portucalensis (Portugal = Portucalem).     

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.     

The effects of infection by Tetracapsuloides bryosalmonae (Myxozoa) and temperature on Fredericella sultana (Bryozoa)

The myxozoan, Tetracapsuloides bryosalmonae, exploits freshwater bryozoans as definitive hosts, occurring as cryptic stages in bryozoan colonies during covert infections and as spore-forming sacs during overt infections. Spores released from sacs are infective to salmonid fish, causing the devastating Proliferative Kidney Disease (PKD). We undertook laboratory studies using mesocosm systems running at 10, 14 and 20 °C to determine how infection by T. bryosalmonae and water temperature influence fitness of one of its most important bryozoan hosts, Fredericella sultana, over a period of 4 weeks. The effects of infection were context-dependent and often undetectable. Covert infections appear to pose very low energetic costs. Thus, we found that growth of covertly infected F. sultana colonies was similar to that of uninfected colonies regardless of temperature, as was the propensity to produce dormant resting stages (statoblasts). Production of statoblasts, however, was associated with decreased growth. Overt infections imposed greater effects on correlates of host fitness by: (i) reducing growth rates at the two higher temperatures; (ii) increasing mortality rates at the highest temperature; (iii) inhibiting statoblast production. Our results indicate that parasitism should have a relatively small effect on host fitness in the field as the negative effects of infection were mainly expressed in environmentally extreme conditions (20 °C for 4 weeks). The generally low virulence of T. bryosalmonae is similar to that recently demonstrated for another myxozoan endoparasite of freshwater bryozoans. The unique opportunity for extensive vertical transmission in these colonial invertebrate hosts couples the reproductive interests of host and parasite and may well give rise to the low virulence that characterises these systems. Our study implies that climate change can be expected to exacerbate PKD outbreaks and increase the geographic range of PKD as a result of the combined responses of T. bryosalmonae and its bryozoan hosts to higher temperatures.     

Invasion of Babesia microti into Epithelial Cells of the Tick Gut1

During feeding a peritrophic membrane (PM) is formed in the gut of the tick Ixodes dammini, dividing the lumen of the gut into an ecto- and endoperitrophic space. Babesia and all food particles ingested with the blood meal by the tick are retained in the endoperitrophic space, the lumen proper. Only Babesia equipped with a highly specialized organelle, the arrowhead, are able to pass the PM and enter the ectoperitrophic compartment. During the crossing of the PM the arrowhead loses its density, suggesting that enzymes released from it dissolve the polymers in the PM, making passage of the parasite through this barrier possible. In the ectoperitrophic space the arrowhead of Babesia touches the epithelial cell. At the point of contact the membrane of the host cell starts to invaginate, and simultaneously the arrowhead's fine structure loses its highly organized pattern. The growing host membrane encircles the parasite and the arrowhead diminishes progressively in size. When the piroplasm is inside the host cell, the arrowhead can no longer be found. During invasion the host membrane often touches the parasite's plasma membrane at the site of a coiled structure, and the host membrane becomes ruptured and the nearby host cytoplasm appears to be lysed. Babesia inside the host cell is covered solely by its own plasma membrane; the invaginated host membrane is missing. It is postulated that the latter disintegrates during invasion by the parasite through the action of enzymes from the coiled structure. The parasite is surrounded by a halo of homogeneous material deriving most probably from the lysed host cytoplasm.     

Host age determines parasite load of Laboulbeniales fungi infecting ants: Implications for host-parasite relationship and fungal life history

Arthropod-parasitic fungi of the order Laboulbeniales are known to exhibit specialization to individual host taxa in most cases. Some species exhibit ecological specificity to multiple, often unrelated hosts in certain microhabitats; and often position specificity to different host body parts. The myrmecophilous Rickia wasmannii (Ascomycota: Laboulbeniales) infects Myrmica species (Hymenoptera: Formicidae) (host specificity), and occasionally other arthropod inquilines inside the ant nest (ecological specificity). An effect of the position of infection on the thallus densities has also been reported. Another determinative factor that may also exist in the Rickia-Myrmica host-parasite system, the chronological age of ant worker hosts, has also been linked to parasite load. Comprehensive studies on the age-related infection intensity, however, are still lacking. Here we investigated whether the level of infection correlates with the age of the M. scabrinodis host consistently. We found that older hosts exhibited higher parasite load, even though the infection level of the different colonies varied widely. The results highlight that the level of R. wasmannii infections are strongly influenced by host individual and host colony factors.     

Development and ultrastructure of the marine,parasitic oomycete,Lagenisma coscinodisci Drebes (Lagenidiales)

The thalli of Lagenisma coscinodisci are coenocytic. In young thalli each nucleus is associated with two procentrioles and four dictyosomes. Before nuclear division two pairs of procentrioles are found on two opposite poles each lying in a pocket of the nuclear membranes. The spindle microtubules radiate from these poles; the nuclear membrane remains completely closed in all divisional stages. The thallus transforms holocarpically into a zoosporangium. The differentiation begins after the breakdown of the host with the formation of a cell wall and with the development of a discharge tube. During these processes peripheral dilated tubules are observed which are in close, local contact with small infoldings of the plasmalemma. The discharge tube develops independent of the point of entry of the infection tube and enlarges by tip growth. There are several light and electron microscopical observations which suggest that the last nuclear divisions in zoosporangia of old cultures are meiotic.They give rise to sexually determined zoomeiospores.     

THE HOST-PARASITE INTERFACE OF ALBUGO CANDIDA ON RAPHANUS SATIVUS

The fine structure of the intercellular hyphae of the obligate parasite Albugo candida infecting radish does not differ markedly from that described previously for cells of Peronospora manshurica. The stalked, capitate haustoria do not contain nuclei and are packed with mitochondria and lomasomes. The fungal plasma membrane and cell wall are continuous from the intercellular hypha throughout the haustorium except that there is no evidence of fungal cell wall around a portion of the haustorial stalk proximal to the haustorial head. Within the vacuolate host mesophyll cell, the haustorium is always surrounded by host plasma membrane and with at least a thin layer of host cytoplasm. The host cell wall invaginates at the point of haustorial penetration to form a short sheath around the region of penetration, but normally there is no host cell wall around the balance of the haustorium. About 1% of the haustoria observed were necrotic, and these were invariably walled-off completely from host cytoplasm by host cell wall. An amorphous, moderately electron-dense encapsulation lies between the haustorium proper and the host plasma membrane and extends into the penetration region between the sheath and the fungal cell wall. Invaded host cells contain more ribosomal-rich ground cytoplasm than uninfected cells. Glandular-like systems of tubules and connecting vesicles are often numerous in host cytoplasm in the vicinity of haustorial heads. These tubules open into the encapsulation, their limiting unit membranes being continuous with the host plasma membrane. We suggest that these represent a secretory mechanism of the host specifically induced by the parasite.     

Trafficking of malarial proteins to the host cell cytoplasm and erythrocyte surface membrane involves multiple pathways

During the asexual stage of malaria infection, the intracellular parasite exports membranes into the erythrocyte cytoplasm and lipids and proteins to the host cell membrane, essentially "transforming" the erythrocyte. To investigate lipid and protein trafficking pathways within Plasmodium falciparum-infected erythrocytes, synchronous cultures are temporally analyzed by confocal fluorescence imaging microscopy for the production, location and morphology of exported membranes (vesicles) and parasite proteins. Highly mobile vesicles are observed as early as 4 h postinvasion in the erythrocyte cytoplasm of infected erythrocytes incubated in vitro with C6-NBD-labeled phospholipids. These vesicles are most prevalent in the trophozoite stage. An immunofluorescence technique is developed to simultaneously determine the morphology and distribution of the fluorescent membranes and a number of parasite proteins within a single parasitized erythrocyte. Parasite proteins are visualized with FITC- or Texas red-labeled monoclonal antibodies. Double-label immunofluorescence reveals that of the five parasite antigens examined, only one was predominantly associated with membranes in the erythrocyte cytoplasm. Two other parasite antigens localized only in part to these vesicles, with the majority of the exported antigens present in lipid-free aggregates in the host cell cytoplasm. Another parasite antigen transported into the erythrocyte cytoplasm is localized exclusively in lipid-free aggregates. A parasite plasma membrane (PPM) and/or parasitophorous vacuolar membrane (PVM) antigen which is not exported always colocalizes with fluorescent lipids in the PPM/PVM. Visualization of two parasite proteins simultaneously using FITC- and Texas red-labeled 2 degrees antibodies reveals that some parasite proteins are constitutively transported in the same vesicles, whereas other are segregated before export. Of the four exported antigens, only one appears to cross the barriers of the PPM and PVM through membrane-mediated events, whereas the others are exported across the PPM/PVM to the host cell cytoplasm and surface membrane through lipid (vesicle)-independent pathways.     

Spermatogenesis and sperm structure of Acerella muscorum, (Ionescu, 1930) (Hexapoda, Protura)

The general organization of the male genital system, the spermatogenesis and the sperm structure of the proturan Acerella muscorum have been described. At the apex of testis apical huge cells are present; their cytoplasm contains a conventional centriole, a large amount of dense material and several less electron-dense masses surrounded by mitochondria. Spermatocytes have normal centrioles and are interconnected by cytoplasmic bridges. Such bridges seem to be absent between spermatid cells and justify the lack of synchronization of cell maturation. Spermatids are almost globular cells with a spheroidal nucleus and a large mass of dense material corresponding to the centriole adjunct. Within this mass a centriole is preserved. Mitochondria of normal structure are located between the nucleus and the plasma membrane. The spermatids are surrounded by a thick membrane. No flagellar structure is formed. Sperm have a compact spheroidal nucleus, a large cap of centriole adjunct material within which a centriole is still visible. A layer of mitochondria is located over the nucleus. The cytoplasm is reduced in comparison to spermatids; many dense bodies are interspersed with sperm in the testicular lumen. The sperm are small, immotile cells of about 2.5-3 μm in diameter.     

Electron microscopical observations onPseudaphelidium drebesii Schweikert and Schnepf, a parasite of the centric diatomThalassiosira punctigera

Summary Ultrastructural observations revealed details of the infection process and the fine structure ofPseudaphelidium drebesii Schweikert and Schnepf, a parasite of the marine centric diatomThalassiosira punctigera (Castracane) Hasle. After attachment and encystment of the zoospore an intracellular infection tube is generated. This structure is everted between the overlap of the girdle bands or the gap between the valve and the girdle band of the host diatom. The bulk of thePseudaphelidium protoplast enters the silica shell of the diatom via the infection tube but does not pierce the host plasma membrane. Parts of the host cytoplasm are phagocytized with microfilament involvement.Pseudaphelidium drebesii is characterized by unusual microbodies containing tubular inclusions and by closed mitosis with a perinuclear spindle. The taxonomic position ofP. drebesii is discussed.