Scanning electron microscopy of the gills of a freshwater catfish,Rita Rita

Variations in the gross morphology and surface architecture of the gill filaments and secondary lamellae of a freshwater catfish (Rita rita) have been investigated using scanning electron microscopy. Heterogeneity of the gill has been correlated with the distribution of lamellar water-flow at different regions of a gill filament. Higher lamellar water flow (cc/pore/cmH₂O/sec) was estimated for the middle region of the filaments. The filaments are covered with epithelial cells whose surface is provided with well-developed microridges. The lamellae are generally covered with microvillous epithelial cells. The variations in surface architecture of the gill filaments and secondary lamellae have been correlated with their probable functions.     

Ornithodoros moubata: spermateleosis and secretory activity of the sperm

Cytological aspects of spermateleosis in the tick Ornithodoros moubata were studied by electron microscopy. During spermateleosis, detachment of the operculum from the outer sheath of the prospermium results from the fusion of the plasma and the cisternal membranes. The fusion occurs between the shoulder of the acrosomal vesicle and the electron-dense layer of the operculum. A factor inducing vitellogenesis and egg-laying is secreted by the sperm cell after spermateleosis, and begins after the cell is almost completely devaginated. In vitro, fully devaginated spermiophores secrete most of this factor during the first 12 hr of incubation. The vitellogenesis-inducing activity of the secretion is sensitive to proteinase K (EC 3.4.21.14) digestion and correlates with the presence of two high-molecular-weight proteins in the sperm cell incubation medium.     

The mucous coat on gill lamellae of rainbow trout (Oncorhynchus mykiss)

The existence of a layer of mucus covering the gill lamellae of healthy rainbow trout (Oncorhynchus mykiss) was investigated. Using cryo-scanning electron microscopy, a smooth, undulating, thin layer was observed which completely covered gill filaments and lamellae, thereby obscuring epithelial microridges. After processing cryopreserved gill arches in glutaraldehyde for conventional scanning electron microscopy, the layer was no longer present and epithelial microridges were clearly visible. The identity of this layer was investigated using cryopreserved gills which were treated in one of two ways. First, gills were incubated with a rabbit antiserum to gill mucus, with normal rabbit serum, or with phosphate-buffered saline. Following fixation in glutaraldehyde and processing, only the gill tissue incubated with the mucus-specific antiserum was still covered with the smooth layer. The layer was also retained on the gills of fish anesthetized in a solution containing mucusspecific antiserum and then processes in glutaraldehyde for conventional scanning electron microscopy. The tenacious nature of the mucous layer was demonstrated by its stability following exposure to formalin and a cationic detergent. Second, the presence of this layer was confirmed on gill tissue which was cryopreserved, followed by freeze-substitution and vapor fixation, and then examined by transmission electron microscopy.     

Scanning electron microscopy of post-ejaculatory spermiogenesis in the tick Ornithodoros moubata

The final stages of spermiogenesis in ticks occur in the female genital tract. Scanning electron microscopy was used to follow the morphologic changes that occur in the sperm during this post-ejaculatory spermiogenesis in the African soft tick, Ornithodoros moubata, and to determine a time sequence for its occurrence in vivo. Characteristic features of the maturing and mature cell described include (1) differentiation and detachment of the operculum, (2) changes in cell shape corresponding to different developmental stages, (3) passive migration of the nucleus and acrosome from an anterior to a posterior position, and (4) eversion of that portion of the acrosomal canal containing the nucleus and acrosome. A possible fate for the remainder of the acrosomal canal is suggested by extrusion and detachment of spherical structures, the ‘posterior bubbles’, from the posterior end of the mature supermatozoon. A mechanism for cellular elongation resulting from contractions of the outer sheath is proposed.     

The occurrence of biomineralization products in four lichen species growing on sandstone in western Norway

High performance thin-layer chromatography/thin-layer chromatography, X-ray diffraction, and scanning electron microscopy analysis of thallus and lichen-rock interface samples, were undertaken to characterize biomineralization products in Fuscidea cyathoides, Ochrolechia tartarea, Ophioparma ventosa, and Pertusaria corallina, growing on sandstone in western Norway. Whewellite (monohydrate form of Ca oxalate) was found in the thallus of all species, but not in any of the weathering rinds beneath the species. A significantly higher amount of whewellite was detected in the thalli of F. cyathoides and O. ventosa than in the other two species. There were only a few differences in whewellite occurrence between the thallus edge and centre samples in the four species. HPTLC/TLC and SEM analysis indicate that lichen compounds occur within the rock beneath some of the lichen specimens. Only divaricatic acid was observed within the weathering rind beneath O. ventosa. No lichen substances were found in the weathering rind beneath F. cyathoides and P. corallina, whereas gyrophoric and lecanoric acids were found in the weathering rind beneath O. tartarea.     

Transmission Dynamics of Borrelia turicatae from the Arthropod Vector

Background

With the global distribution, morbidity, and mortality associated with tick and louse-borne relapsing fever spirochetes, it is important to understand the dynamics of vector colonization by the bacteria and transmission to the host. Tick-borne relapsing fever spirochetes are blood-borne pathogens transmitted through the saliva of soft ticks, yet little is known about the transmission capability of these pathogens during the relatively short bloodmeal. This study was therefore initiated to understand the transmission dynamics of the relapsing fever spirochete Borrelia turicatae from the vector Ornithodoros turicata, and the subsequent dissemination of the bacteria upon entry into murine blood.

Methodology/Principal Findings

To determine the minimum number of ticks required to transmit spirochetes, one to three infected O. turicata were allowed to feed to repletion on individual mice. Murine infection and dissemination of the spirochetes was evaluated by dark field microscopy of blood, quantitative PCR, and immunoblotting against B. turicatae protein lysates and a recombinant antigen, the Borrelia immunogenic protein A. Transmission frequencies were also determined by interrupting the bloodmeal 15 seconds after tick attachment. Scanning electron microscopy (SEM) was performed on infected salivary glands to detect spirochetes within acini lumen and excretory ducts. Furthermore, spirochete colonization and dissemination from the bite site was investigated by feeding infected O. turicata on the ears of mice, removing the attachment site after engorment, and evaluating murine infection.

Conclusion/Significance

Our findings demonstrated that three ticks provided a sufficient infectious dose to infect nearly all animals, and B. turicatae was transmitted within seconds of tick attachment. Spirochetes were also detected in acini lumen of salivary glands by SEM. Upon host entry, B. turicatae did not require colonization of the bite site to establish murine infection. These results suggest that once B. turicatae colonizes the salivary glands the spirochetes are preadapted for rapid entry into the mammal.     

An actin-like component in spermatocytes of a crane fly (Nephrotoma suturalis Loew)

Actin-like filaments are seen at the cell periphery after crane fly spermatocytes are glycerinated and then treated with rabbit skeletal muscle heavy meromyosin. ATP and pyrophosphate inhibit the reaction with heavy meromyosin. From prometaphase through metaphase the filaments are all parallel to the cell surface, extending 0.5–1 μ. beneath the plasma membrane in a continuous layer of parallel filaments enveloping the cell; considering the poles of the spindle as north and south poles of the cell, the actin-like filaments at the cell periphery are all arranged as meridians. In late-anaphase, too, actin-like filaments are parallel to the cell surface, but here this includes bundles of filaments oriented as parallels in the furrow and adjacent regions of the cell periphery, as well as filaments oriented as meridians in the rest of the cell periphery. — Actin-like filaments are seen in the cellular projections associated with the spindle poles.     

Cutaneous and systemic cellular responses induced by the feeding of the argasid tick Ornithodoros parkeri

Carolyn M. Johnston and Stephen J. Brown 1985. Cutaneous and systemic cellular responses induced by the feeding of the argasid tick Ornithodoros parkeri. International Journal for Parasitology15: 621–628. Initial feeding by Ornithodoros parkeri ticks induced a significant blood basophilia in guinea pigs, with a minimal cutaneous basophil response. Hosts challenged 14 days later, however, exhibited significantly depressed blood basophil levels associated with a marked accumulation of these cells at tick feeding sites in the tissue. Blood eosinophil levels in primary and secondary hosts were comparable, but eosinophil levels at tick feeding sites in challenged animals were significantly greater than levels in primary hosts. Furthermore, challenge tick feeding resulted in the activation of primary tick feeding sites on the opposite flank that became erythematous 90 min after challenge and indurated within 24 h. Histologically, these activated primary feeding sites 90 min after challenge on the opposite flank were marked by a dominant eosinophil response (314 ± 128 cells, 59% of the infiltrate) with a marked basophil component (145 ± 67 cells, 28% of the infiltrate) that resembled the active challenge feeding sites 24 h after infestation (24 ± 52 cells, 76% of the infiltrate); 90 min after challenge active feeding sites had a weak basophil response (2 ± 1 cells) similar to uninfested controls. These results suggest the chronic nature of tick bites with an apparent continual recruitment of basophils that is probably a result of slow antigen release over time by appropriately sensitized antigen presenting cells. Primary tick feeding sites in guinea pigs previously exposed to Xenopsylla cheopis fleas, on the opposite flank, contained a marked eosinophilia (63 ± 25 cells) compared to primary tick feeding sites in naive guinea pigs (2 ± 0 cells); suggesting the possibility of cross reactivity between flea and tick antigens     

Fat body is the site of vitellogenin synthesis in the soft tick,Ornithodoros moubata

Summary Vitellogenin (Vg) synthesis in cultured tissues was analysed biochemically in a soft tick,Ornithodoros moubata. Nine tissue fractions dissected from reproductive females were incubated in vitro in a specially designed Ringer containing³⁵S-methionine. The synthesis of total protein and Vg was assayed by the radioactivity incorporated into precipitates with trichloroacetic acid and antivitellin (Vn)-serum, respectively. Fat body was the most active tissue in Vg synthesis, which comprised 46% of the Vg synthesis by all tissues and 42% of total protein synthesis by fat body. Protein synthesized by the fat body and precipitated with anti-Vn-serum was shown by electrophoresis and fluorography, to consist of six radioactive polypeptides corresponding to the components of Vg. Vg synthesized in cultured fat body was first accumulated in the tissue and secreted into the medium during incubation. Some tissues other than fat body showed low Vg synthesis (in each, less than 12% of total protein synthesis) which, however, may be due to contamination by fat body cells as seen with the scanning electron microscope (SEM). SEM also showed that fat body cells in the active stage of Vg synthesis expanded about 10-fold in length. Immunohistochemical analysis showed a very strong reaction with anti-Vn-IgG in the cytoplasm of fat body from reproductive females. Fat body from unfed females and other tissues including midgut, did not show any specific fluorescence. A positive reaction was obtained with developing oocytes. These results indicate that the fat body is the only site of Vg synthesis in this tick.Abbreviations Vg vitellogenin - Vn vitellin - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - SEM scanning electron microscopy - TCA trichloroacetic acid     

The mucous coat on gill lamellae of rainbow trout (Oncorhynchus mykiss)

The existence of a layer of mucus covering the gill lamellae of healthy rainbow trout (Oncorhynchus mykiss) was investigated. Using cryo-scanning electron microscopy, a smooth, undulating, thin layer was observed which completely covered gill filaments and lamellae, thereby obscuring epithelial microridges. After processing cryopreserved gill arches in glutaraldehyde for conventional scanning electron microscopy, the layer was no longer present and epithelial microridges were clearly visible. The identity of this layer was investigated using cryopreserved gills which were treated in one of two ways. First, gills were incubated with a rabbit antiserum to gill mucus, with normal rabbit serum, or with phosphate-buffered saline. Following fixation in glutaraldehyde and processing, only the gill tissue incubated with the mucus-specific antiserum was still covered with the smooth layer. The layer was also retained on the gills of fish anesthetized in a solution containing mucusspecific antiserum and then processes in glutaraldehyde for conventional scanning electron microscopy. The tenacious nature of the mucous layer was demonstrated by its stability following exposure to formalin and a cationic detergent. Second, the presence of this layer was confirmed on gill tissue which was cryopreserved, followed by freeze-substitution and vapor fixation, and then examined by transmission electron microscopy.     

Fine structure and assemblyin vitro of nuclear lamina in plant cells

The fine structure of the nuclear lamina (NL) in sperm cells ofGinkgo biloba was visualised using high resolution low-voltage scanning electron microscopy (LVSEM). It was shown that the nuclear lamina was composed of 10 nm filaments which formed a fine network. Lamins were purified from cultured carrot suspension cells and assembledin vitro. Long 8–12 nm diameter filaments were seen and sometimes subfilaments could be distinguished. Western blot of filament preparations showed that these contained the 66 and 84 ku lamins. These data demonstrate that plant lamins are capable of assembling into filamentsin vitro.     

Eggshell fine structure of Bradysia aprica (Winnertz) (Diptera : Sciaridae)

The eggshell fine structure of the dark-winged fungus-gnat Bradysia aprica (Winnertz) (Diptera : Sciaridae) was investigated by scanning and transmission electron microscopy. At the anterior pole of the ovoid egg is a single micropyle, centrally located in a well-defined micropylar area. The latter is covered by many long drumstick-like chorionic processes that are longer and more numerous than those of the rest of the egg surface. Cross-sections of the eggshell show 3 concentric envelopes: the vitelline envelope, wax layer and chorion. The chorion consists of 3 components with different morphological features: the inner, intermediate and outer chorion. The latter 2 layers, involved in the organization of the drumstick-like processes, have homogeneous features, whereas the former is crystalline and resembles the innermost chorionic layer of other Diptera.     

The microscopy of P-protein filaments in freeze-etched sieve pores

Intact vascular bundles from Nymphoides peltata (S.G. Gmel.) O. Kuntze, shown to have translocated carbon-14, were freeze-fractured and etched for electron microscopy. The interpretation of freezefractured and etched sieve pores and P-protein filaments seen in them is discussed. The entire widths of most of the sieve pores seen contained filaments separated by less than 100 nm. Their arrangement indicates too high a resistance to flow for pressure flow alone to drive translocation at known rates; pumps would be necessary at places along sieve tubes. However, calculations are presented to show that during the time taken to fix pores, by fast freezing or chemically, the filaments in them could rearrange and move further by Brownian and other motion than the distances between filaments which we need to measure. These calculations show that it is not possible, by microscopy alone, to answer the outstanding question “How are filaments arranged in translocating sieve pores?” with enough certainty to tell us whether pressure flow is adequate to explain translocation where filaments are present. The calculations are relevant also to microscopy of other cell structures which may move.     

Fine structure and development of the sperm of the tick Ornithodoros (Pavlovskyella) erraticus (Ixodoidea, Argasidae)

Sperm development in Ornithodoros (Pavlovskyella) erraticus includes the formation of subsurface cisternae in the primary spermatocytes, which divide meiotically to secondary spermatocytes and ultimately to spermatids. During spermiogenesis the spermatid undergo morphological transformation including polarization of the nucleus and subsurface cisternae, formation of a cisternal tube, and modification of the subsurface cisternae to cellular processes surrounded by cisternal vesicles. Further transformation occurs after spermatids are introduced into the female. The spermatid cisternal tube now invaginates to form an inner cord surrounded by an outer sheath. The invaginated inner cord elongates anteriorly as the outer sheath continues to invaginate posteriorly during spermiogenesis. With further elongation, the spermatid membrane ruptures anteriorly, leaving the inner cord exposed as the outer surface of the maturing sperm. Posteriorly, the original plasma membrane invaginates to form an acrosomal canal which becomes surrounded by an acrosome. The hemispherical anterior end of the mature sperm is covered with rows of projections separated from the remainder of the sperm by a row of fringed processes. Except for the posterior end, the rest of the sperm is covered by longitudinally distributed electron-dense cellular processes and an outer mat of more electron-lucent tubular elements. Mitochondria and bundles of microfibrils are found beneath the cellular processes. Microfibrils are suggested to be the principal contractile organelles responsible for sperm motility. Cellular processes appear to be the main external motile structures, while movements of tubular elements and fringed processes may also contribute to sperm motility.     

Structural and functional properties of chimeric EspA-FliCi filaments of EPEC

Enteropathogenic Escherichia coli utilise a filamentous type III secretion system to translocate effector proteins into host gut epithelial cells. The primary constituent of the extracellular component of the filamentous type III secretion system is EspA. This forms a long flexible helical conduit between the bacterium and host and has a structure almost identical to that of the flagella filament. We have inserted the D3 domain of FliCi (from Salmonella typhimurium) into the outer domain of EspA and have studied the structure and function of modified filaments when expressed in an enteropathogenic E. coli espA mutant. We found that the chimeric protein EspA-FliCi filaments were biologically active as they supported protein secretion and translocation [assessed by their ability to trigger actin polymerisation beneath adherent bacteria (fluorescent actin staining test)]. The expressed filaments were recognised by both EspA and FliCi antisera. Visualisation and analysis of the chimeric filaments by electron microscopy after negative staining showed that, remarkably, EspA filaments are able to tolerate a large protein insertion without a significant effect on their helical architecture.     

Membrane skeleton in cultured chick cardiac myocytes revealed by high resolution immunocytochemistry

Distribution of cytoskeletal proteins with emphasis on the membrane-cytoskeleton interface was examined in cultured cardiac myocytes. Using specific antibodies recognizing α-sarcomeric actin, desmin, β-tubulin, spectrin/α-fodrin and ankyrin, respectively, the cellular localization of these cytoskeletal proteins was detected by laser scanning confocal microscopy. In addition, the fine filamentous structure of these proteins was identified by combining silver-enhanced immunogold labelling with electron microscopy. The latter technique employed the sequence of quick-freezing, deep-etching and rotary shadowing of the specimens. Conventional transmission electron microscopy of the spherical cardiac myocytes revealed a filamentous submembranous layer, approximately 100 nm thick. Specific immunolabelling of α-sarcomeric actin and spectrin/α-fodrin as well as ankyrin was seen beneath the plasmalemma. A three-dimensional meshwork of spectrin/α-fodrin was shown. Numerous desmin filaments that exhibited a tortuous course throughout the cells were also observed running in parallel with the surface in the submembranous area, whereas β-tubulin was infrequently detected in these areas. In conclusion, the present study shows that spherical cardiac myocytes contain a distinct and complex three-dimensional membrane skeleton. Major constituents of this distinct submembranous layer were spectrin/α-fodrin fibres as well as actin and desmin filaments. Accepted: 28 July 1999     

Microtubules and Filaments Beneath the Fission Furrow of Stentor coeruleus1

ABSTRACT. The ultrastructure of the cortex beneath the fission furrow of dividing Stentor coeruleus was examined using scanning and transmission electron microscopy. During division, basal bodies, axonemes, and km fibers beneath the furrow were absorbed near the moving primordial oral apparatus, and a circumferential band of microtubules and filaments was formed at the base of the furrow. The location and orientation of this fibrous band suggest that it may be an important component of the cytokinetic machinery. Treatment with vinblastine sulfate (4 × 10^-5 M) disrupted the circumferential microtubules and blocked division, which is consistent with this hypothesis.     

Surface features of Entamoeba histolytica and rabbit kidney (RK13) cell surface changes after trophozoite contact--observations by scanning electron microscopy.

Trophozoites of Entamoeba histolytica grown on glass, fixed in situ, and examined by scanning electron microscopy (SEM) were seen to possess microfilopodia on their lower sides and under surfaces, a hitherto undescribed feature. No surface lysosomes were seen. After trophozoites had been brought into contact with a host monolayer of cultured RK13 cells, immediate surface changes were observed. First the RK13 cell surface microvilli elongated, then decreased in number and finally disappeared. These changes were accompanied by erosion of the cell surface and cellular swelling.     

Ornithodoros sawaii (Ixodida: Argasidae) Larvae Collected from Hydrobates monorhis on Sogugul and Gaerin Islands,Jeollanam-do (Province), Republic of Korea

The 65th Medical Brigade and Public Health Command District-Korea, in collaboration with the Migratory Bird Research Center, National Park Research Institute, conducted migratory bird tick surveillance at Sogugul and Gaerin Islands (small rocky bird nesting sites), Jeollanam-do (Province), Republic of Korea (ROK), on 30 July and 1 August 2009. Breeding seabirds captured by hands in their nesting burrows were banded, identified to species, and carefully examined for ticks during the nesting season. A total of 9 Ornithodoros sawaii larvae were removed from 4 adult Hydrobates monorhis (Swinhoe’s storm petrel). The identification of the larvae of O. sawaii collected from migratory seabirds were molecularly confirmed using mitochondrial 16S rDNA primer sets.