Ultrastructure and Cytochemistry of the Mature Spermatozoon of Khawia Armeniaca (Cholodkovsky, 1915) (Caryophyllidea: Lytocestidae), a Parasite of Capoeta Capoeta Sevangi (De Filippi, 1865) (Teleostei, Cyprinidae).

The mature spermatozoon of Khawia armeniaca, a monozoic caryophyllidean parasite of templar fish Capoeta capoeta sevangi (De Filippi, 1865) from the Lake Sevan, Armenia, has been studied using transmission electron microscopy and cytochemical technique of Thiéry (1967) for the first time. The mature spermatozoon of K. armeniaca consists of a single axoneme with the 9+'1' trepaxonematan structure, cortical microtubules and nucleus which are situated parallel to the longitudinal axis of the spermatozoon, and a moderately electrondense cytoplasm with glycogen particles. The cortical microtubules are arranged in one continuous semicircle beneath the plasma membrane in Region II and anterior part of Region III of the mature spermatozoon. The two opposite rows of cortical microtubules are observed in the remaining nuclear and at the beginning of the postnuclear part (Regions III, IV) of the male gamete The number of cortical microtubules is remarkably variable in the spermatozoa of various Khawia species. K. armeniaca exhibits the highest number of cortical microtubules in comparison with K. sinensis and K. rossittensis. Glycogen was detected in the cytoplasm of prenuclear (II), nuclear (III) and postnuclear (IV) regions with different ultrastructural organization of the mature spermatozoon of K. armeniaca. Variations of sperm ultrastructural characters within caryophyllideans and other cestodes are discussed.

Simultaneous detection of multiple targets for ultrastructural immunocytochemistry.

Simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. While fluorescent microscopy allows for simultaneous detection of multiple antigens, the sensitive electron microscopy immunodetection is limited to only two antigens. In order to overcome this limitation, we prepared a set of novel, shape-coded metal nanoparticles readily discernible in transmission electron microscopy which can be conjugated to antibodies or other bioreactive molecules. With the use of novel nanoparticles, various combinations with commercial gold nanoparticles can be made to obtain a set for simultaneous labeling. For the first time in ultrastructural histochemistry, up to five molecular targets can be identified simultaneously. We demonstrate the usefulness of the method by mapping of the localization of nuclear lipid phosphatidylinositol-4,5-bisphosphate together with four other molecules crucial for genome function, which proves its suitability for a wide range of biomedical applications.

Glycogen, poly(3-hydroxybutyrate) and pigment accumulation in three Synechocystis strains when exposed to a stepwise increasing salt stress.

The cyanobacterial genus Synechocystis is of particular interest to science and industry because of its efficient phototrophic metabolism, its accumulation of the polymer poly(3-hydroxybutyrate) (PHB) and its ability to withstand or adapt to adverse growing conditions. One such condition is the increased salinity that can be caused by recycled or brackish water used in cultivation. While overall reduced growth is expected in response to salt stress, other metabolic responses relevant to the efficiency of phototrophic production of biomass or PHB (or both) have been experimentally observed in three Synechocystis strains at stepwise increasing salt concentrations. In response to recent reports on metabolic strategies to increase stress tolerance of heterotrophic and phototrophic bacteria, we focused particularly on the stress-induced response of Synechocystis strains in terms of PHB, glycogen and photoactive pigment dynamics. Of the three strains studied, the strain Synechocystis cf. salina CCALA192 proved to be the most tolerant to salt stress. In addition, this strain showed the highest PHB accumulation. All the three strains accumulated more PHB with increasing salinity, to the point where their photosystems were strongly inhibited and they could no longer produce enough energy to synthesize more PHB.

Ultrastructure of spermatozoa of tench Tinca tinca observed by means of scanning and transmission electron microscopy.

Structure of tench (Tinca tinca L.) spermatozoa was investigated by means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Spermatozoa of 26.1+/-3.8 microm total length possessed typical primitive simple structure, called "aqua sperm", without acrosomal head structures. It was probably the smallest spermatozoon described among cyprinid fishes. Heads were mostly composed of dense and slightly granular material, which appeared to be fairly homogeneous except for the occasional appearance of vacuoles. The midpiece remained separated from the flagellum by the cytoplasmic channel; it was cylindric/cone-shaped, 0.86+/-0.27 microm in length and 1.17+/-0.24 microm in width at proximal part. The proximal centriole was located in the "implantation fossa". The distal centriole appeared almost tangential to the nucleus and it functioned as a basal body for the flagellum. It had an orientation of 140 degrees with respect to the distal centriole. The sperm flagellum with 25.45+/-2.47 microm of total length had no any fin. The diameter of the flagellum perpendicular to the plane of the doublet of central microtubules was 173.67+/-20.45 nm and horizontal plane of the central microtubules was 200.71+/-20.45 nm. Peripheral doublets and the central doublet of microtubules measured 23.39+/-3.18 and 35.88+/-4.44 nm in width, respectively. The diameter of a microtubule was only 9.14+/-2.97 nm. A vesicle was attached to the most basal region of the flagellum and located just under plasma membrane of the flagellum.

Lectin-binding characteristics of a Lyme borreliosis spirochete Borrelia burgdorferi sensu stricto.

Borrelial glycoconjugates were localized by labeled lectins on ultrathin cryosections and on surfaces of intact negatively stained bacteria. Protein-saccharide complexes in these glycoconjugates were partially characterized by means of enzyme deglycosylation and mild alkali pretreatment of cryosections. The results of labeling were examined by transmission electron microscopy. Statistically evaluated results (relative labeling index, chi2 test) of gold labeling indicated that surfaces of Borrelia burgdorferi strain B31 and external (outer) membrane vesicles (MVs) were covered with glycoconjugates containing O-glycosidically linked N-acetyl-D-galactosamine (GalNAc) and N-glycosidically linked N-acetyl-D-glucosamine (GlcNAc). The presence of N-linked GalNAc, sialic acid, mannose and fucose on the surfaces of outer membranes and MVs was probably due to an adherence of BSK-H medium components, especially rabbit serum, to Borrelia surfaces.

Imaging of tissue sections with very slow electrons.

The examination of thin sections of tissues with electron microscopes is an indispensable tool. Being composed of light elements, samples of living matter illuminated with electrons at the usual high energies of tens or even hundreds of kiloelectronvolts provide very low image contrasts in transmission or scanning transmission electron microscopes. Therefore, heavy metal salts are added to the specimen during preparation procedures (post-fixation with osmium tetroxide or staining). However, these procedures can modify or obscure the ultrastructural details of cells. Here we show that the energy of electrons used for the scanned transmission imaging of tissue sections can be reduced to mere hundreds or even tens of electronvolts and can produce extremely high contrast even for samples free of any metal salts. We found that when biasing a sufficiently thin tissue section sample to a high negative potential in a scanning transmission electron microscope, thereby reducing the energy of the electrons landing on the sample, and collecting the transmitted electrons with a grounded detector, we obtain a high contrast revealing structure details not enhanced by heavy atoms. Moreover, bombardment with slow electrons sensitively depolymerises the resin in which the tissue is embedded, thereby enhancing the transmitted signal with no observable loss of structure details. The use of low-energy electrons requires ultrathin sections of a thickness of less than 10nm, but their preparation is now possible. Ultralow energy STEM provides a tool enabling the observation of very thin biological samples without any staining. This method should also be advantageous for examination of 2D crystals, thin films of polymers, polymer blends, etc.

Bacillary band ultrastructure of the fish parasite Capillaria pterophylli (Nematoda: Capillariidae).

In Capillaria pterophylli Heinze, 1933, two lateral bacillary bands extend along the whole body in female and male worms. A ventral bacillary band is present in females only. The bacillary bands consist of glandular and non-glandular cells, in the region between the nerve ring and the end of the stichosome, ciliated sense receptors in tight connection with gland are present.

Egg shell ultrastructure of the fish nematode Huffmanela huffmani (Trichosomoididae).

The egg shell of Huffmanela huffmani Moravec, 1987 forms three main layers: an outer vitelline layer, a middle chitinous layer, and an inner lipid layer. The vitelline layer, forming the superficial projections of the egg shell, comprises two parts: an outer electron-dense, and an inner electron-lucid part. The chitinous layer is differentiated into three parts: an outer homogenous electron-dense part, a lamellated part, and an inner electron-dense net-like part. The lipid layer comprises an outer net-like electron-lucid part, and an inner homogenous electron-lucid part. The polar plugs are formed by electron-lucid material with fine electron-dense fibrils.

Exposed and hidden lectin-binding epitopes at the surface of Borrelia burgdorferi.

The presence of surface- and subsurface-located lectin-binding epitopes of Borrelia burgdorferi was examined by electron microscopy using a variety of gold-labeled lectins. Concanavalin A reacted predominantly with extracellular material adjacent to the spirochetes. Wheat germ agglutinin bound weakly to the surface of borreliae; however, alterations of the outer membrane by preincubation in 100 ppm Triton X-100 or boiling uncovered numerous periplasmic sites recognized by the lectin. The periplasmic flagella liberated by some cells after detergent treatment were labeled with concanavalin A, wheat germ agglutinin and Ulex europaeus agglutinin UEA-I. No surface-exposed or periplasmic epitopes for the lectins from Glycine max, Dolichos biflorus or Helix pomatia were detected.

Preferential banding of secondary veins in strawberry is caused by mixed virus infection.

Leaves of Fragaria ananassa Duch. cv. Redgauntlet with mottle and mild dwarf symptoms were grafted onto F. vesca indicator clones. The youngest leaves developed specific vein banding pattern located preferentially on secondary veins near the edge of the leaves. Electron microscopy of ultrathin sections and negatively stained purified virus preparations from symptom-bearing strawberry leaves revealed presence of different-sized isometric virions. Particles of about 50 nm and 23 nm in diameter were identified as strawberry vein banding virus (SVBV) and tobacco necrosis virus (TNV) D strain. Based on results of electron microscopy, DNA hybridization, enzyme-linked immunosorbent assay (ELISA), and DNA sequencing we propose that the anomalous "leaf edge vein banding" symptoms are caused by a mixed virus infection with SVBV and other viruses such as TNV.

Distribution and ultrastructure of two types of scolex gland cells in adult Proteocephalus macrocephalus (Cestoda, Proteocephalidea).

In the scolex-neck region of the adult Proteocephalus macrocephalus two types of eccrine gland cells are present. The first type of gland cells, localized in the frontal part of the scolex only, contains large, more or less round electron lucid granules. The second type of unicellular glands produces large electron dense granules. These electron dense granular gland cells are localized primarily in the neck region, only few are present in the scolex apex. The secretion of both types of gland cells is concentrated in the ducts opening to the exterior. The ducts are fixed to the plasmalemma of the tegument by septate junctions. The function of both types of gland cells is discussed.

Stichosome ultrastructure of the fish nematode Capillaria pterophylli Heinze, 1933.

The stichosome (posterior glandular esophagus) of Capillaria pterophylli Heinze, 1933 consists of large gland cells (stichocytes) and lumenal epithelium with cuticular lining. Both structures are enclosed in a reticulum of muscle cells. The stichocyte cytoplasm contains small cisternae of rough endoplasmic reticulum, Golgi complexes, one kind of electron dense secretory granules, mitochondria and a branching system of intracellular collecting ducts without filament bundles around them.

Field emission scanning electron microscopy (FE-SEM) as an approach for nanoparticle detection inside cells.

When developing new nanoparticles for bio-applications, it is important to fully characterize the nanoparticle's behavior in biological systems. The most common techniques employed for mapping nanoparticles inside cells include transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). These techniques entail passing an electron beam through a thin specimen. STEM or TEM imaging is often used for the detection of nanoparticles inside cellular organelles. However, lengthy sample preparation is required (i.e., fixation, dehydration, drying, resin embedding, and cutting). In the present work, a new matrix (FTO glass) for biological samples was used and characterized by field emission scanning electron microscopy (FE-SEM) to generate images comparable to those obtained by TEM. Using FE-SEM, nanoparticle images were acquired inside endo/lysosomes without disruption of the cellular shape. Furthermore, the initial steps of nanoparticle incorporation into the cells were captured. In addition, the conductive FTO glass endowed the sample with high stability under the required accelerating voltage. Owing to these features of the sample, further analyses could be performed (material contrast and energy-dispersive X-ray spectroscopy (EDS)), which confirmed the presence of nanoparticles inside the cells. The results showed that FE-SEM can enable detailed characterization of nanoparticles in endosomes without the need for contrast staining or metal coating of the sample. Images showing the intracellular distribution of nanoparticles together with cellular morphology can give important information on the biocompatibility and demonstrate the potential of nanoparticle utilization in medicine.

Vitellogenesis in Archigetes sieboldi Leuckart, 1878 (Cestoda, Caryophyllidea, Caryophyllaeidae), an intestinal parasite of carp (Cyprinus carpio L.).

Vitellogenesis in the caryophyllidean tapeworm Archigetes sieboldi Leuckart, 1878, from carp Cyprinus carpio L. in Slovakia, has been examined using transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) for glycogen. Vitelline follicles extend in two lateral bands in the medullary parenchyma along both sides of the monozoic body. They are surrounded by an external basal lamina and contain vitellocytes and an interstitial tissue. The general pattern of vitellogenesis is essentially like that of other caryophyllideans. It involves four stages: immature, early maturing, advanced maturing cells and mature vitellocytes. During vitellogenesis, a continuous increase in cell volume is accompanied by an extensive development of cell components engaged in shell globule formation, e.g. granular endoplasmic reticulum and Golgi. Shell globule clusters are membrane-bound. Nuclear and nucleolar transformation are associated with formation and storage of large amounts of intranuclear glycogen, a very specific feature of the Caryophyllidea. For the first time, (a) additional vitelline material in Archigetes is represented by lamellar bodies and (b) lipid droplets are described in the mature vitellocytes from vitelline follicles and vitelloduct of the Caryophyllidea. Our results indicate that there may be a double origin of lamellar bodies: either from the endoplasmic reticulum or through transformation of shell globule/shell globule clusters. Lamellar body clusters and some single lamellar bodies appear to have a membrane. Other ultrastructural features of vitellogenesis and/or vitellocyte in A. sieboldi from its vertebrate (fish) and invertebrate (oligochaete) hosts are briefly compared and contrasted with those in other caryophyllideans and/or Neodermata.

Transmission electron microscopy of the scolex and neck microtriches of Silurotaenia siluri (Batsch, 1786) (Cestoda: Proteocephalidea).

Microtriches on different parts of the scolex and neck of the gangesiine cestode Silurotaenia siluri (Batsch 1786) were studied. The neodermis of the central frontal scolex part (around the rostellar furrow) bears filamentous microtriches only. The lateral frontal part and the parts between and posterior to the suckers cover filamentous and spine-like microtriches. Within the suckers there are short spine-like microtriches with bases enforced by electron-dense ribs. The proximal neck part bears filamentous and spine-like microtriches, the middle part solitary distributed blade-like and spine-like microtriches among filamentous ones, and the distal part blade-like microtriches. The functions of different types of microtriches are discussed.

Ultrastructure of the spermatozoon of the proteocephalidean cestode Proteocephalus torulosus (Batsch, 1786).

The fine structure of the mature spermatozoon of the proteocephalidean tapeworm Proteocephalus torulosus, a parasite of cyprinid fishes, was studied by transmission electron microscopy for the first time. The mature spermatozoon of P. torulosus is filiform and tapered at both extremities. It contains two axonemes of the 9+"1" type and of unequal length. The anterior extremity of the spermatozoon bears a helicoidal crested body approximately 100 nm thick. Cortical microtubules of two types lie parallel to the spermatozoon axis. The nucleus is a fine cord of condensed chromatin. The slightly electron-dense cytoplasm contains electron-dense granules in regions II and III of the spermatozoon. The anterior and posterior extremities of the spermatozoon contain a single axoneme. The mature spermatozoon of P. torulosus differs from that of other Proteocephalusspecies in the presence of a single crested body and in the morphology of its posterior end.

Spermiogenesis in the proteocephalidean cestode Proteocephalus torulosus (Batsch, 1786).

Spermiogenesis of the proteocephalidean cestode Proteocephalus torulosus (Batsch, 1786) was examined for the first time using transmission electron microscopy. Spermiogenesis begins with the formation of a distal cytoplasmic protrusion, a differentiation zone, at the periphery of the early spermatid. This differentiation zone is lined with cortical microtubules and contains two centrioles aligned along the same axis. Subsequently, each centriole is associated with the striated root and the intercentriolar body appears between them. A flagellar bud arises from each centriole, growing later as a free flagellum. Simultaneously, a median cytoplasmic process (MCP) develops distally to the flagella. The two flagella, which are of unequal length, become longer and rotate towards the MCP. At this stage, two arching membranes appear at the base of the differentiation zone. The nucleus elongates and when both flagella are fused with the MCP, the nucleus subsequently migrates into the MCP. Finally, the advanced spermatids detach from a condensing residual cytoplasm at the level of the arching membranes.

Ultrastructure of the early rostellum of Silurotaenia siluri (Batsch, 1786) (Cestoda: Proteocephalidae).

The neodermis of the whole early rostellum of Silurotaenia siluri (Batsch, 1786) bears filamentous microtriches. At the base, there are five to six irregular rows of hooks and spine-like microtriches. The rostellar hooks of S. siluri originate through the enlargement of microtriches. Electron-dense hook substance is deposited along the edges of the microthrix to form the hook blade and basal plate. The blade does not become hollow as in the cyclophyllidean cestodes. The basal plate of the hook, corresponding to the handle and the guard of Cyclophyllidea, is formed by the deposition of hook substance around the base of the microthrix. Within the centre of the base, only a narrow cleft-like core is occupied by distal cytoplasm. The hook bases are more deeply inserted into the distal cytoplasm, as the bases of the spine-like microtriches are localized on the rostellum below the rows of hooks. No hemidesmosomes, fixing the hook base to the basal lamina, are present. Eccrine gland cells and uniciliate and nonciliate sensory receptors of the primitive rostellum are described.

Ultrastructure of the spermatozoon of the pseudophyllidean cestode Eubothrium crassum (Bloch, 1779).

The fine structure of the mature spermatozoon of the pseudophyllidean tapeworm Eubothrium crassum, a parasite of salmonid fishes, has been studied by transmission electron microscopy for the first time. The mature spermatozoon of E. crassum is filiform and tapered at both extremities. It contains two axonemes of unequal length showing the 9 + "1" pattern of Trepaxonemata. The anterior extremity exhibits a crested body 50-100 nm thick. It spirals around the outside of the anterior region of the spermatozoon. The nucleus is electron-dense, exhibiting a fibrous appearance in its middle (the broadest) region. The cortical microtubules are of two types and are situated parallel to the spermatozoon axis. The cytoplasm is slightly electron-dense and contains numerous electron-dense granules in region II of the spermatozoon. A ring of electron-dense, centred microtubules surrounds the axoneme, together with the underlying ring of moderately electron-dense, subjacent submicrotubular material in region V. The anterior and posterior extremities of the spermatozoon lack cortical microtubules and contain a single axoneme. Our results reveal several peculiarities, in which the spermatozoon of E. crassum differs from those of other pseudophyllidean cestodes.