Preliminary Observations on the Fine Structure of the Pansporoblast of Thelohania bracteata (Strickland, 1913) (Microsporida, Nosematidae) as Revealed by Freeze-Etching Electron Microscopy

SYNOPSIS. The ultrastructure of a microsporidan pansporoblast was observed with freeze-etching electron microscopy. The cross-fractured face of ovoid mature spores, with the upper part of the spore coat fractured off, revealed the spore membrane; the convex face had many small depressions and the concave face bore fine particles. In cross-section the spore-coat was highly laminated and about 0.5 μ in diameter.
In the cytoplasm of the pansporoblast, fluid-filled and finger-print-life profiles of vesicles were observed. The vesicles were approximately 180 nm in diameter and laminated, each lamella being about 15–18 nm thick. In addition to these vesicles, a population of elevations, each with an average diameter of 40 nm, was evenly distributed in the pansporoblast among the spores. No other cytoplasmic organelles were observed within the pansporoblast. The pansporoblast wall was about 15–19 nm thick with particles 15–18 nm in diameter on its outer surface.     

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).     

Fertilization in a crab. II. Cytological aspects of the cortical reaction and fertilization envelope elaboration

At fertilization, the egg of Carcinus maenas undergoes cortical vesicle exocytosis, in response to the first contacts between the spermatozoon and the egg plasma membrane. This process was observed in vitro and may be connected with a cortical reaction. Carcinus maenas eggs display two populations of cortical vesicles which, during the reaction, successively release two different exudates: a fine granular material and a mass of ring-shaped granules. During the first steps of exocytosis, the two superimposed vitelline envelopes are detached from the egg surface, and the inner one gradually changes. Thus a new coating, derived from the coalescence of the secreted ring-shaped granules, is progressively elaborated under the vitelline envelopes. These events occur over a 7–8 hr period. The morphological uniqueness of the cortical vesicle exudates and the complexity of the related events are discussed in terms of the cortical reaction and of the formation of the fertilization envelope in Carcinus maenas.     

Lesions induced by complement in vitro on the protoscoleces of Echinococcus multilocularis: a study by electron microscopy.

Changes in the ultrastructure of the tegument of Echinococcus multilocularis protoscoleces during complement-mediated lysis in vitro was studied using transmission and scanning electron microscopy. It was found that the total disintegration of protoscoleces by complement proceeds through formation of ‘tegumental bubbles’ and disruption of the external plasma membrane. This sequence of events was evident in the appearance of numerous loose membrane fragments and vesicles, the lifting of the external unit membrane of the microtriches and the release of organelles from the distal cytoplasm. Subsequent events, such as the appearance of a ‘fuzzy’ coat and disruption of the basement membrane, were probably due to autolysis.     

A Light and Electron Microscopical Study of Trichoduboscqia epeori Léger (Microspora: Duboscqiidae)1

Trichoduboscqia epeori Léger was found to parasitize nymphs of the mayfly Rhithrogena iridina Kolenati in southwest Germany for a new host record. It was studied by light and electron microscopy. The pansporoblast membrane is evaginated at several points, usually four, to produce long needle-like appendages >20 μm in length with a resilient inner core superficially resembling collagen, which is thought to maintain their orientation. It is suggested that the pansporoblast appendages may play a role in host infection. The structure and ultrastructure of developmental stages are recorded for the first time. Apart from the pansporoblast appendages, the ultrastructure of T. epeori conforms to the general pattern seen in many other pansporoblastic Microspora. Typically 16 spores are produced per pansporoblast but 32-spore pansporoblasts were also found, and the taxonomic significance of this is discussed.     

Light and electron microscope study of Thelohania solenopsae n. sp. (Microsporida: Protozoa) in the red imported fire ant,Solenopsis invicta

A new species of Microsporida, Thelohania solenopsae, is described from the red imported fire ant, Solenopsis invicta. The Thelohania infections are localized in the fat body of workers. Meronts causing infections of progeny are found in the ovaries of queens. Spores occur only in adult ants and only vegetative stages are present in larvae and pupae. Both uninucleate octospores (eight spores within a pansporoblast membrane) and binucleate free spores (spores developing in isolation) are formed.     

Ormiersia carcini gen. n., sp. n., microsporidian of the Mediterranean crab, Carcinus mediterraneus Czerniavsky, 1844: developmental cycle and ultrastructural study]

A microsporidan parasite, Ormieresia carcini gen. n., sp. n., was found in the crab, Carcinus mediterraneus Czerniavsky. Its development and fine structure are the subject are the subject of the present study. The life cycle begins with a schizont surrounded by a unit membrane and containing a diplokaryon. The entire process of sporogony takes place in the host musculature. The sporogonic stages are enclosed in the pansporoblastic membrane. In each pansporoblast, sporogony gives rise to 8 sporoblasts; the octonucleate sporogonial plasmodium is lacking. In the course of schizogonic and sporogonic divisions, each kinetic center consists of 2 plaques, one located within and the other outside the nuclear envelope. The dividing sporonts and sporoblasts sevrets "metabolic" substances (granules, tubules) which are depostied in the pansporoblast. The uninucleate spore is long and cylindrical, measuring 19.1 X 2.4 micronm. A rectilinear manubrium traverses the spore. Its posterior end attenuates abruptly into a polar filament with 4 or 5 coils; its anterior end is attached to the polar cap, which is compressed by a double polar ring. The anterior part of the manubrium is surrounded by a polaroplast consisting of a "spongy" (vesicular) and a lamellar zone.     

Fertilization in a crab: I. Early events in the ovary,and cytological aspects of the acrosome reaction and gamete contacts

Early events in fertilization were studied in Carcinus maenas by in vitro experiments and ultrastructural analysis; some were found to occur in the lumen of ripe ovaries. The acrosome reaction generally conformed to the usual Reptantia Decapoda pattern. However, a prominent membrane system continuous with the nuclear envelope and located close to the base of the acrosome tubule characterized the type of spermatozoon observed in Carcinus maenas. Such complex anatomical connections linking the three parts of the reacted spermatozoon (acrosome tubule, membrane system and nucleus envelope) may be significant in relation to the membrane system's contribution to the acrosome reaction. The outer layer of the everted acrosomal vesicle was found to comprise tubular elements ending in bell-shaped corpuscles, deeply interdigitated with the oolemma microvilli during the establishment of the initial contacts between the reacted spermatozoon and the egg plasma membrane. At the site of contact, the oolemma formed a minute fertilization cone, locally depressed by the acrosome tubule. During these early fertilization events, the nucleus, like the other spermatozoon components, was seen to penetrate the egg coatings first, and later to be located near the oolemma.     

A novel complete reconstitution system for membrane integration of the simplest membrane protein

A complete reconstitution system for membrane integration of the simplest protein was developed by means of defined factors. A mutant version of Pf3 coat protein, 3L-Pf3 coat, requires neither signal recognition particle/Sec factors nor a membrane potential for its integration into the cytoplasmic membrane of Escherichia coli. Although 3L-Pf3 coat is spontaneously integrated into liposomes composed of phospholipids, diacylglycerol completely blocks such spontaneous integrations at a physiological level. Under the conditions where spontaneous integration does not occur, 3L-Pf3 coat integration was absolutely dependent on a novel integration-stimulating factor. Combination of the PURE system, an in vitro translation system composed of the purified factors involved in translation in E. coli, with liposomes containing the highly purified integration-stimulating factor revealed multiple cycles of 3L-Pf3 coat integration, achieving the complete reconstitution of membrane integration. Based on the function of the factor, we propose that the factor is named MPIase (Membrane Protein Integrase).     

Viral Genomic Single-Stranded RNA Directs the Pathway Toward a T = 3 Capsid

The molecular mechanisms controlling genome packaging by single-stranded RNA viruses are still largely unknown. It is necessary in most cases for the protein to adopt different conformations at different positions on the capsid lattice in order to form a viral capsid from multiple copies of a single protein. We showed previously that such quasi-equivalent conformers of RNA bacteriophage MS2 coat protein dimers (CP₂) can be switched by sequence-specific interaction with a short RNA stem-loop (TR) that occurs only once in the wild-type phage genome. In principle, multiple switching events are required to generate the phage T = 3 capsid. We have therefore investigated the sequence dependency of this event using two RNA aptamer sequences selected to bind the phage coat protein and an analogous packaging signal from phage Qβ known to be discriminated against by MS2 coat protein both in vivo and in vitro. All three non-cognate stem-loops support T = 3 shell formation, but none shows the kinetic-trapping effect seen when TR is mixed with equimolar CP₂. We show that this reflects the fact that they are poor ligands compared with TR, failing to saturate the coat protein under the assay conditions, ensuring that sufficient amounts of both types of dimer required for efficient assembly are present in these reactions. Increasing the non-cognate RNA concentration restores the kinetic trap, confirming this interpretation. We have also assessed the effects of extending the TR stem-loop at the 5′ or 3′ end with short genomic sequences. These longer RNAs all show evidence of the kinetic trap, reflecting the fact that they all contain the TR sequence and are more efficient at promoting capsid formation than TR. Mass spectrometry has shown that at least two pathways toward the T = 3 shell occur in TR-induced assembly reactions: one via formation of a 3-fold axis and another that creates an extended 5-fold complex. The longer genomic RNAs suppress the 5-fold pathway, presumably as a consequence of steric clashes between multiply bound RNAs. Reversing the orientation of the extension sequences with respect to the TR stem-loop produces RNAs that are poor assembly initiators. The data support the idea that RNA-induced protein conformer switching occurs throughout assembly of the T = 3 shell and show that both positional and sequence-specific effects outside the TR stem-loop can have significant impacts on the precise assembly pathway followed.     

Le fer dans la branchie des crustacés décapodes Carcinus maenas (L.) et Homarus americanus Milne Edw.: étude quantitative et histochimique

Carcinus maenas (L.) gills accumulate iron. This iron occurs in a ferric and inorganic state. Up to 99.9 % of this metal is included in the chitinous-cellular debris fraction as isolated by centrifugation. The remaining iron belongs to the soluble protein and mitochondrial fractions. At the site of the iron polymucosaccharic acids and polysaccharides are also observed. The iron content of Homarus americanus Milne Edw. gills is smaller than that of Carcinus maenas gills. In Homarus americanus the iron is accumulated as scattered ‘tablets’ along the branchial tubes, while in Carcinus maenas it coats the branchial lamellae. It seems that the accumulation of iron in Carcinus maenas is related to the rôle of filtration or adsorption by the chitin against the particulate iron of the sea water. The differences observed between Carcinus maenas and Homarus americanus are probably related to morphological and structural differences.     

Plasmodium falciparum: merozoite invasion in vitro in the presence of chloroquine.

The fine structure of invasion of human erythrocytes by merozoites of the malaria parasite Plasmodium falciparum was observed in vitro. The invasion process is similar to that described for P. knowlesi. Merozoites enter apical end first by invagination of the erythrocyte membrane. At the rim of the invagination, where merozoite and erythrocyte are in closest contact, the erythrocyte membrane is thickened. The brushy cell coat of the P. falciparum merozoite appears to be lost at this attachment zone. The part of the merozoite within the erythrocyte invagination has no visible coat. The coat on the portion outside is unaltered. Merozoites can successfully invade erythrocytes after 3 hr in the presence of a concentration of chloroquine harmful to feeding stages.     

Sauertylenchus labiodiscus n. gen., n. sp., from Australia (Nematoda:Tylenchorhynchinae)

Sauertylenchus labiodiscus n. gem, n. sp. is described and illustrated from soil around Rhagodia sp. in Australia. It can be distinguished from the most closely related genus Tylenchorhynchus Cobb, 1913 by the distinctly set-off, rounded, lip region with a conspicuous labial disc, and long thin stylet. The face view and spicules of Sauertylenchus labiodiscus are illustrated with scanning electron micrographs. The subfamilies Tylenchorhynchinae and Merlininae ale discussed.     

The Cell Surface of Trypanosoma musculi Bloodstream Forms. I. Fine Structure and Cytochemistry*†

SYNOPSIS. An extracellular surface coat was observed at the fine-structural level on the outer lamina of the pellicular and flagellar membranes of intact Trypanosoma musculi bloodstream forms. The surface coat had a mean width of 9.2 nm, and was composed of a somewhat electron dense, uneven, fibrillar-like matrix. Brief trypsin treatment of living blood forms completely removed the cell surface coat. Several cytochemical methods applicable to electron microscopy were used to detect the presence and distribution of carbohydrates in the trypanosome's surface coat and pellicular membrane. The polycationic dye compounds employed were: ruthenium red, ruthenium violet, Alcian blue chloride, and lanthanum nitrate. Electron-dense stain reaction products, indicative of polysaccharides, were evident in the surface coat of cells treated with these dyes, which also agglutinated both living and glutaraldehyde fixed cells. Like the surface coat, the pellicular membrane of trypsinized cells gave strong positive staining reactions with the several dyes, indicating the presence of membrane bounded carbohydrates, and living and glutaraldehyde-fixed trypsinized cells were agglutinated with the polycationic stains. Bloodstream forms were treated with the enzymes, α-amylase, dextranase, and neuraminidase. No obvious morphologic difference, however, was apparent between the surface coat of untreated cells and those subjected to treatment with any of the various glycoside hydrolase enzymes. Further, these enzymes had no apparent gross effect on the staining affinity of the surface coat for the several polycationic dyes. Cationized ferritin was used to visualize the negative cell surface charge of T. musculi bloodstream forms. Large quantities of cationized ferritin were bound in the surface coat matrix. Glycoside hydrolase enzyme treatments had no apparent effect on the amount of ferritin bound in the surface coat. Cationized ferritin was bound also to the outer lamina of the pellicular membrane in trypsinized cells, which had quantitatively less ferritin bound per surface unit area than bloodstream forms untreated by the enzyme. Living and glutaraldehyde-fixed cells were agglutinated with cationized ferritin. The results obtained in the various experiments indicated that polyanionic polysaccharides were constituent terminal ligands of the surface coat matrix and pellicular membrane in T. musculi bloodstream forms.     

Synthesis of intracellular and extracellular gold nanoparticles with a green machine and its antifungal activity

Green synthesis method is being increasingly used in the development of safe, stable, and eco-friendly nanostructures with biological resources. In this study, extracellular and intracellular synthesis of gold nanoparticles (AuNPs) was carried out using green algae Chlorella sorokiniana Shihira & R.W. Fresh algae were isolated and identified from Musaözü Pond located in the province of Eskişehir and then extraction process were performed. Optimization studies were studied using pH value, metal salt concentration, and time parameters for extracellular synthesis and using only time parameter for intrasellular synthesis. Since more controlled and optimum conditions can be achieved in the production of AuNPs by extracellular synthesis, these nanoparticles (NPs) were used for characterization and antifungal activity studies. Optical, physical, and chemical properties of synthesized NPs were characterized by UV visible spectrophotometer (UV-Vis), dynamic light scattering (DLS), Zetasizer, X-Ray diffraction (XRD), Fourier transform ınfrared spectroscopy (FTIR), field emission scanning electron microscope (FE-SEM), ınductively coupled plasma mass spectrometer (ICP-MS) and transmission electron microscope (TEM) analysis. The optimum conditions for AuNPs synthesis were determined as 1 mM for HauCl₄ concentration, 6 for pH value, and 60th min for time. AuNPs obtained from extracellular synthesis from C. sorokiniana extract are 5–15 nm in size and spherical shape. TEM images of extracellular synthesis show noticeable cell wall and membrane damages, cytoplasma dissolutions, and irregularities. AuNPs obtained by intracellular synthesis are in 20–40 nm size and localized in the cell wall and cytoplasm. These NPs exhibited significant antifungal activity against C. tropicalis, C. glabrata, and C. albicans isolates. AuNPs obtained by algae-mediated green synthesis have a significant potential for medical and industrial use, and this eco-friendly synthesis method can be easily scaled for future studies.     

Fertilization in crabs: IV. The fertilization potential consists of a sustained egg membrane hyperpolarization

The electrophysiological properties of immature and mature oocytes of two crabs were analyzed. Growing immature oocytes of Carcinus maenas and fully grown immature oocytes of Maia squinado had essentially K⁺ dependent resting potentials, Em, of ?61 ? 1 mV, n=19, and ?67.3 ± 0.5 mV, n=68, respectively. Fully grown immature oocytes of Carcinus maenas showed an Em of ?40 ± 1.5 mV, n=19, that was k⁺ and Cl^? dependent. In mature oocytes of both species, the plasma membrane became exclusively permeable to cl^? and the Em attained–41 ± 1 mV, n=49 and ?34 ± 1.5 mV, n=27 for Carcinus maenas and Maia squinado, respectively. After in vitro insemination, a dramatic increase in egg membrane permeability to K⁺ was observed. This instantaneously caused a sustained hyperpolarization constituting, for these crabs, the fertilization potential. We observed that concurrently with this electrical response to fertilization, sperm reinitiated the oocyte meiotic maturation previously arrested at the first metaphase. The triggering mechanism of the fertilization potential as well as the possible occurrence of a physiological polyspermy are discussed.     

Norlevinea n. g., a New Genus for Glugea daphniae (Protozoa: Microspore), a Parasite of Daphnia longispina (Crustacea: Phyllopoda)1

ABSTRACT. Norlevinea n. g. is established for microsporidia in which a uninucleate meront changes into a sporont by secreting a thin, membranous, sporontogcnetic and fragile sporophorous vesicle (pansporoblast membrane) in which four uninucleate sporoblasts are formed. In contrast to the genus Gurleya, the sporoblasts and later the spores are permanently joined into doublets, being laterally cemented by an electron-dense substance structurally identical to and continuous with the exospore layer. The polar filament is of the anisofilar type. The type species is Norlevinea daphniae (Weiser, 1947) n. comb., a parasite of the ovaries of Daphnia longispina occurring in several carp ponds in Czechoslovakia.     

Sec23 Homolog Nel1 Is a Novel GTPase-activating Protein for Sar1 but Does Not Function as a Subunit of the Coat Protein Complex II (COPII) Coat

The coat protein complex II (COPII) generates transport carriers from the endoplasmic reticulum (ER) under the control of the small GTPase Sar1. Sec23 is well known as a structural component of the COPII coat and as a GTPase-activating protein (GAP) for Sar1. Here, we showed that Saccharomyces cerevisiae contains a novel Sec23 paralog, Nel1, which appears not to function as a subunit of the COPII coat. Nel1 does not associate with any of the COPII components, but it exhibits strong Sar1 GAP activity. We also demonstrated that the chromosomal deletion of NEL1 leads to a significant growth defect in the temperature-sensitive sar1D32G background, suggesting a possible functional link between these proteins. In contrast to Sec23, which is predominantly localized at ER exit sites on the ER membrane, a major proportion of Nel1 is localized throughout the cytosol. Our findings highlight a possible role of Nel1 as a novel GAP for Sar1.     

Comparative ultrastructural topography of the gut epithelia of the lung fluke Paragonimus (Trematoda: Troglotrematidae)

The gut epithelia in the species of Paragonimus known in Japan, P. westermani, P. ohirai, P. iloktsuenemis, P. miyazakii, and P. sadoensis, were observed with the scanning electron microscope. Transmission electron microscopy was employed to substantiate and clarify the results of these topographic studies. Morphological differences in the cytoplasmic projections of the gut epithelial surface were found to be present among some species of Paragonimus. The cytoplasmic projections in this group are of a sheet-like or broad lamellar type, although fine digitiform type projections have never been described in other trematodes.     

Ultrastructure of the tail region of the second stage preparasitic larva of the root-knot nematode

Studies on the tail of second-stage infective larvae (L₂s) of Meloidogyne javanica, M. incognita and M. hapla from a region anterior to the rectal gland to the tail tip have revealed the presence of a previously undescribed sensory organ, the caudal sensory organ, in the posterior region of the tail.The extreme tip of the tail consists of solid cuticle and the different zones of this structure are described throughout the tail region. Longitudinal sections through the anus and rectum have revealed that the cortical (external cortical, epicuticle) layer gradually decreases in thickness until its outermost layer appears to merge with the plasma membrane of the rectal inflation or gland. This gland appears to be similar in all three species studied.The two phasmidial glands and their canals are described from transverse sections. Somatic muscle is first found in the region of the anus and it extends anteriorly throughout most of the length of the L₂. Depressor ani muscles which insert on the dorsal surface of the rectum are also described.The rectal gland in the dilated state occupies about three quarters of the diameter of the L₂. It contains a matrix which resembles that extruded from the adult female rectal gland cells. The rectal gland cells contain large amounts of rough endoplasmic reticulum and desmosomes are found close to the junction of these cells and the plasma membrane of the gland itself. More anteriorly in the L₂ most of the area is taken up by large lipid droplets which function as an energy reserve.It is suggested that the rectal gland should not be used as a taxonomic criterion for separating the La_2^S of Meloidogyne because it can vary so much in appearance within the same species.