Ultrastructural differentiation during embryonic Drosophila myogenesis in vitro

Summary Cultures of embryonicDrosophila melanogaster cells were examined by electron microscopy and events in myogenesis were recorded. Thick and thin myofilaments, T-tubules and sarcoplasmic reticulum all appeared at about the same time, 10.5 hr. This was about 5 hr after the final division of myoblasts and about the time that muscle cells were elongating, aligning and fusing. Sarcoplasm typical of insect muscle was detected by 18.5 hr, as were myotendonal and tendocuticular junctions. Two populations of myocytes were detected, the cytoplasm of one more electron-dense than the other. The only previous report of myofibrilogenesis in invertebrate embryos had described novel mechanisms. InDrosophila embryonic material, however, the sequence of myofibrilogenesis resembled that in post-embryonic insect or vertebrate material. Mrs. Pilar Toribio-Fiorio provided excellent technical assistance, and Patricia Minter, the secretarial expertise. This investigation was supported, in part, by NIH Grant NS9330 and the James Douglas Research Fund to Robert L. Seecof and NIH Grant No. 1 RO1 CA17223-01 to Raymond L. Teplitz.     

Eimeria crotalviridis sp. n. from Prairie Rattlesnakes, Crotalus viridis viridis, in New Mexico with Data on Excystation of Sporozoites and Ultrastructure of the Oocyst Wall

SYNOPSIS Oocysts of Eimeria crotalviridis sp. n. are described from prairie rattlesnakes, Crotalus viridis viridis in New Mexico on the basis of light and electron microscopy and in vitro excystation of sporozoites. Sporulated oocysts of E. crotalviridis are elliptical, 26.4 × 22.3 (23–29 × 20–24) μm with ovoid sporocysts 11.7 × 8.1 (11–13 × 7–9) μm. A micropyle, micropyle cap and polar bodies are absent, but oocyst and sporocyst residua and Stieda and substieda bodies are present. Excysted sporozoites are 12.4 × 2.8 (11–13 × 2–3) μm and have 1 large posterior refractile body and a nucleus with a prominent nucleolus. Ultrastructurally, the oocyst wall has 2 layers, a thick, electron-dense, highly sculptured outer layer composed of a fine granular matrix and a thin, granular, osmiophilic inner layer, separated from the outer layer by at least one unit membrane. These layers are 441 (353–510) and 21.6 (19–29) nm thick, respectively. Within 15 min after exposure to a trypsin-sodium taurocholate fluid, sporozoites of E. crotalviridis excysted from 5-month-old sporocysts.     

Scanning electron microscopy of pathogenic and non-pathogenic Naegleria cysts

Scanning electron microscopy of pathogenic and non-pathogenic Naegleria cysts. International journal for Parasitology4: 139–142. Cysts of 4 strains of non-pathogenic Naegleria gruberi and 5 strains of pathogenic Naegleria fowleri were examined in the scanning electron microscope. Excystment of the Naegleria gruberi amoebae occurred via preformed exit pores in the cyst wall. Similar structures were not found in the cysts of Naegleria fowleri, and excystment occurred by rupture of the cyst wall. The sequence of cyst wall rupture is illustrated for one of the pathogenic strains.     

Some Ultrastructural and Functional Aspects of the Golgi Apparatus of Thelohania sp. (Microsporida) in the Shrimp Pandalus jordani Rathbun

SYNOPSIS. Electron microscope observations on Thelohania sp. in the shrimp Pandalus jordani support the view that the Golgi complex in Microsporida is a classical one, composed of vesicular, vacuolar, and cisternal components. During development of the sporoblast, a portion of the Golgi complex is seen as an electron-dense reticulum enmeshing the core of the polar filament. Associated with the reticulum are electron-dense bodies. The reticulum and dense bodies, reported in several previous publications, have not been well understood and have been given a variety of names. The evidence favors the view that these structures have secretory activity in which the reticulum concentrates or synthesizes material, some of which takes the form of membrane-bounded granules. It is suggested that the most appropriate name for the reticulum is reticulum golgien, and that the correct name for the dense bodies is the standard cytologic term, secretion granules. The secretion granules apparently remain in the posterior part of the spore, and may be stored there for some as yet undetermined use.     

Ultrastructural study of the epidermis in two free-living Platyhelminthes, Mesostoma viaregginum and M. productum (Rhabdocoela, Typhloplanida)

Abstract. The epidermis of the free-living typhloplanids Mesostoma viaregginum and M. productum (Mesostominae) is described. In both species, the epidermis has polarized cells with nuclei located at the basal part of the cell, whereas mitochondria are in the apical one. The epidermis is entirely covered by microvilli and locomotory cilia anchored in the cytoplasm by vertical and horizontal rootlets. Rootlets exhibit distinct length and periodic structure in the two species. Furthermore, in each species vertical and horizontal rootlets possess different periodic structure. The pattern of termination of microtubules in epidermal cilia is described for the first time in the Typhloplanida; central microtubules shift along one axonemal side, doublets 1 and 6–9 lose their microtubule B, and gradually peripheral doublets become singlets. Finally, an electron-dense material caps the tip of the cilia. This pattern of termination closely resembles that of Temnocephalida, Kalytorhynchia, and Dalyelliida examined so far, but differences exist.     

Ultrastructural changes in the oviduct of the laying hen during the laying cycle

The ultrastructural changes occurring in the fully functional oviduct of Isa Brown laying hens were studied during various stages of the laying cycle. Hens were killed at different positions of the egg in the oviduct. The oviduct was lined by ciliated and non-ciliated cells (also referred to as granular cells). The granular cells in the infundibulum contributed to secretion during egg formation, whereas ciliated cells showed little evidence of secretion. Ultrastructural changes were recorded in the granular and glandular cells of the distal infundibulum. In the magnum, the surface ultrastructure revealed glandular openings associated with the ciliated and granular cells. Cyclic changes were recorded in the glandular cells of the magnum. With respect to the three observed types of glands, the structure of gland type A and C cells varied at different egg positions in the oviduct, whereas type B cells represented a different type of gland cell containing amorphous secretory granules. The surface epithelium of the isthmus was also lined by mitochondrial cells. Two types of glandular cell (types 1 and 2) were recorded in the isthmus during the laying cycle. Intracisternal granules were found in type 2 cells of the isthmus. A predominance of glycogen particles occurred in the tubular shell gland. The granular cells in the shell gland contain many vacuoles. During egg formation, these vacuoles regressed following the formation of extensive rough endoplasmic reticulum; the reverse also occurred. The disintegrated material found in the vacuoles may have been derived from the disintegrating granules. The Physiology Teaching Unit, University of New England, provided financial support to K. Chousalkar for this study.     

Fine Structure of the Oocyst Walls of Isospora serini and Isospora canaria and Excystation of Isospora serini From the Canary, Serinus canarius L.

SYNOPSIS. Oocysts of Isospora serini and Isospora canaria , from the canary Serinus canarius , were broken, added to a cell suspension, fixed in Karnovsky's fluid, and studied in the electron microscope. The oocyst wall of each species had an electronlucent inner layer, a more osmiophilic middle layer and an outer layer of electron-lucent ( I. serini ) or electron-dense material interspersed with some electron-lucent material ( I. canaria ). A few, relatively large lipid-like bodies were present in the outer or middle layer of the oocyst wall of I. canaria. As many as 9 membranes were present in the oocyst wall of I. canaria and 3 in that of I. serini. When exposed to a trypsin-sodium taurocholate fluid, sporozoites of I. serini excysted from 5-month-old sporocysts in vitro , but not from sporocysts stored for more than 6 months. No excystation occurred in 15-month-old I. canaria sporocysts. Similarities and differences in excystation between I. serini and other Isospora, Eimeria , and Sarcocystis species are discussed.     

Ultrastructural alterations in ciliary cells exposed to ionizing radiation

Summary Early effects of ionizing radiation were investigated in an experimental in vitro system using the ciliary cells of the tracheal mucous membrane of the rabbit, irradiated at 30° C and at more than 90% humidity. The changes in physiological activities of the ciliary cells caused by irradiation were continuously registered during the irradiation. The specimens were examined immediately after irradiation electron microscopically. The morphological changes in irradiated material after 10–70 Gy are compared with normal material. After 40–70 Gy, scanning electron microscopy revealed the formation of vesicles on cilia, and club-like protrusions and adhesion of their tips. After 30–70 Gy, a swelling of mitochondrial membranes and cristae was apparent transmission electron microscopically. The membrane alterations caused by irradiation are assumed to disturb the permeability and flow of ATP from the mitochondria, which in turn leads to the recorded changes in the activity of the ciliated cells.This investigation was supported by grants from Konung Gustaf V:s Jubileumsfond, John and Augusta Perssons Stiftelse, B. Kamprads Fond, the Faculty of Medicine, University of Lund, Sweden and the Swedish Medical Research Council (No. B77-17X-03897-05)The authors are greatly indebted to Miss Inger Norling, Miss Marianne Palmegren and Miss Birgitta Sandström for their excellent technical assistance     

Ultrastructural and Cytochemical Observations on Sporogenesis of Myxobolus sp. (Myxosporida: Myxobolidae) from the Common Shiner Notropis cornutus*

SYNOPSIS. The structure and cytochemistry of spores of Myxobolus sp. from plasmodia which occur in the gill filaments of the common shiner Notropis cornutus were studied by light microscopy and by scanning and transmission electron microscopy. The thin-walled valves of the pyriform spores are thickened in the lateral sutural and apical regions. Mucous material is associated predominantly with the posterior end of many spores. The plasmodium is surrounded by a syncytial wall bounded by 2 membranes. Pinocytotic channels are formed by the inner membrane and numerous dense vesicles are pinched off at the distal ends of the channels. Sporogenesis is initiated by the envelopment of one vegetative cell by another. The larger, enveloped cell divides to form a disporous pansporoblast, which contains 2 pairs of capsulogenic and valvogenic cells and 2 binucleate sporoplasm cells. Each capsular primordium and connecting external tubule gives rise to a polar capsule which houses a helically coiled polar tubule. The apical end of each polar capsule is plugged by a stopper. The valvogenic cells surround the capsulogenic and posteriorly situated sporoplasm cells to form the spore valves. Iodinophilic (glycogen) inclusions were not seen in spores stained with iodine or Best's carmine. A darkly stained band was observed around the posterior region of most spores stained with Best's carmine. In the electron microscope large aggregates of β glycogen particles were seen in the cytoplasm of sporoplasm cells in mature spores.     

The three-dimensional structure of the cyanobacterium Synechocystis sp. PCC 6803

To advance our knowledge of the model cyanobacterium Synechocystis sp. PCC 6803 we investigated the three-dimensional organization of the cytoplasm using standard transmission electron microscopy and electron tomography. Electron tomography allows a resolution of ~5 nm in all three dimensions, superior to the resolution of most traditional electron microscopy, which is often limited in part by the thickness of the section (70 nm). The thylakoid membrane pairs formed layered sheets that followed the periphery of the cell and converged at various sites near the cytoplasmic membrane. At some of these sites, the margins of thylakoid membranes associated closely along the external surface of rod-like structures termed thylakoid centers, which sometimes traversed nearly the entire periphery of the cell. The thylakoid membranes surrounded the central cytoplasm that contained inclusions such as ribosomes and carboxysomes. Lipid bodies were dispersed throughout the peripheral cytoplasm and often juxtaposed with cytoplasmic and thylakoid membranes suggesting involvement in thylakoid maintenance or biogenesis. Ribosomes were numerous and mainly located throughout the central cytoplasm with some associated with thylakoid and cytoplasmic membranes. Some ribosomes were attached along internal unit-membrane-like sheets located in the central cytoplasm and appeared to be continuous with existing thylakoid membranes. These results present a detailed analysis of the structure of Synechocystis sp. PCC 6803 using high-resolution bioimaging techniques and will allow future evaluation and comparison with gene-deletion mutants.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Ultrastructural in situ hybridization: A review of technical aspects

Detection of nucleic acid sequence at the ultrastructural level has allowed us to better understand the expression of genes in some fields of application in cell biology. In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultrathin frozen section, or on ultrathin section of tissue embedded in hydrophilic resin such as Lowicryl. Before starting the detection of nucleic acid sequences at the electron microscope level, the experimenter has to choose various parameters: the type of tissue fixation, the probe and its label, and the in situ hybridization method, depending on the sensitivity, the resolution and the ultrastructural preservation required. This review of technical aspects, by describing the different methods of ultrastructural in situ hybridization, will help the experimenter to optimize each step of the hybridization procedure.     

Isonema papillatum sp. n., a New Colorless Marine Flagellate: A Light- and Electronmicroscopic Study

SYNOPSIS. Isonema papillatum sp. n. is described from observations with the light and electron microscope. It has 2 short emergent subapical flagella without appendages, a specialized ingestion apparatus, and a nonrigid naked cell body capable of pronounced changes in shape similar to euglenoid movements. It lacks a stigma and flagellar swelling and does not form paramylon granules. The nucleolus persists throughout nuclear division.     

Ultrastructural topography of trematode eggshell

Scanning electron micrographs show that the eggshell of the digenetic trematode, Cryptocotyle lingua, is a concavo-convex ellipsoid with a longitudinal ridge-like demarcation extending along the convex, but not the concave surface. Numerous apical villosities contribute to an undulating appearance of the shell surface. Transmission electron microscopy reveals a fuzzy coat on the shell surface. Cytoplasmic flaps protruding from the parasite's uterus appear to intertwine with the shell surface villosities.     

Calcium-Induced Ultrastructural Interactions in Adrenocorticocytes

Steroidogenesis in adrenocorticocytes is closely related to intracellular [Ca²⁺]. To detect ultrastructural changes induced by growth in cytosolic [Ca²⁺], we used a rat adrenocortical cell culture, which was examined with electron microscopy and morphometric analysis. We established that either KCl-induced membrane depolarization evoking Ca²⁺ influx into the cell via voltage-operated Ca channels and Ca²⁺ ionophore, A23187, induced remarkable ultrastructural interactions between several cytosolic organelles. Lipid droplets known as key elements for Ca²⁺-induced steroidogenesis directly contacted with organelles containing the enzymes providing steroidogenic reactions (mitochondria, smooth and rough endoplasmic reticulum, nucleus, peroxisomes, and lysosomes). In most cases, the lipid droplets formed a specialized morphological structure at the sites of contact with the partner organelles. These structures are interpreted as a specialized transporting system, which provides contacts between organelles and exchange of intermediate products of the steroidogenesis process between the droplet and organelles.     

Apical Feeding in the Karyorelictids (Protozoa, Ciliophora) Sultanophrys arabica and Tracheloraphis sp.

Sultanophrys arabica and Tracheloraphis sp., two interstitial karyorelictid ciliates, were cultivated in sealed 100–200 ml glass bottles half-filled with filtered interstitial water to which some millilitres of the natural organism community and a couple of wheat grains were added. Removing sand grains and sealing the bottles were crucial to achieve a low oxygen tension milieu, which was maintained by the algae contained in the community. This cultivation method provided, for the first time, rich cultures with many feeding, dividing, and conjugating cells. Both species were omnivorous and fed through the apical end, where a well-developed oral apparatus is present. Apical feeding was documented by micrographs of living specimens and by scanning electron microscopy of preserved cells.     

Ultrastructural studies of neutral lipid localisation inStreptomyces

Triacylglycerol is accumulated by Streptomyces spp. when grown in submerged culture. Ultrastructural studies using transmission electron microscopy (TEM), staining and freeze-fracture/freeze-etch procedures, and light microscopy confirmed the accumulation of neutral lipid by S. lividans and S. coelicolor during the stationary phase and its storage within membrane-bound globular structures within the cytoplasm. These structures were of various sizes and occupied up to approximately 80% of the total cell volume at that time. There was no evidence of such material within cells examined during the early exponential phase of growth. The globules visualised by TEM were electron-transparent since they comprised lipids containing saturated fatty acids that did not react with osmium tetroxide. The globules appeared to be bounded by a single membrane. Received: 6 June 1995 / Accepted: 4 September 1995     

Ultrastructural studies of the hearts in Arenicola marina L. (Annelida: Polychaeta)

Summary The two hearts in Arenicola are capable of great dilation and contraction. The heart wall consists of myoepithelial cells resting on a basal lamina. On the luminal side of the basal lamina is a layer of collagen fibrils. No true endothelium was observed but occasional haemocytes were observed, subjacent to the collagenous layer. A few chloragogen cells are also found peripherally.The myofibrils are of a non-striated type consisting of thick and thin filaments and scattered Z-bodies. The sarcoplasmic reticulum forms a three-dimensional network. Only peripheral couplings were observed. The myofibrils are in contact with the sarcolemma on the luminal side of the cells, constituting a kind of hemidesmosome. The myoepithelial heart muscle is compared with other muscle types described in invertebrates. Supercontraction is discussed.     

Ultrastructural study of experimental muscle degeneration and regeneration in the adult rat

Summary Methyl-bupivacaine is a local anaesthetic with a selective myotoxic action. A single subcutaneous injection of the drug into the hind leg of adult rats produces a uniform, complete and irreversible destruction of superficial layers of fibres in the underlying extensor digitorum longus muscle. The degeneration of muscle fibres is followed by phagocytosis and a rapid and complete regeneration.The first stage in the regeneration process is the appearance of presumptive myoblasts within the original basement membrane of the sarcolemmal tube. On the second day after injury aggregates of myoblasts are present and fusion is observed between the cells. The myotubes thus formed increase in size by fusing with additional myoblasts. Myotubes are also observed to fuse with one another. On the fifth day after injury the regeneration process has proceeded to the stage of early muscle fibres with fully differentiated myofibrils with typical sarcomere structures. By ten days only mature muscle fibres of about normal size are present and regeneration appears complete.In previously denervated and methyl-bupivacaine treated muscles the stages of regeneration are similar to those observed in innervated muscles, the only apparent difference being a slowing of cell differentiation and incomplete maturation.An electrophysiological study shows that the motor nerve at the third day after injury forms synaptic contacts with regenerating muscle cells. At that stage of myogenesis the myotubes are highly sensitive to applied acetylcholine.1 (1-n-butyl-DL-piperidine-2-carboxylic acid-2,6-dimethyl-anilide-hydrochloride); Marcaine®, manufactured by AB Bofors, Nobel-Pharma, Mölndal, Sweden.The study was carried out under the auspicies of The Czechoslovak Academy of Sciences and the Royal Academy of Sciences in Sweden.