Third contribution to the knowledge of the Raviniini (Diptera, Sarcophagidae), based on observations of the larvae, using scanning electron microscope

L1, L2 and L3 of Oxysarcodexia paulistanensis (Mattos), L3 of O. confusa Lopes, L2 of Ravinia belforti (Prado & Fonseca) and L2 of Oxyvinia excisa (Lopes) were described and figured using scanning electron microscope.

Indirect hydrogen analysis by gas chromatography coupled to mass spectrometry (GC-MS).

Gas chromatography (GC) is an analytical tool very useful to investigate the composition of gaseous mixtures. The different gases are separated by specific columns but, if hydrogen (H2 ) is present in the sample, its detection can be performed by a thermal conductivity detector or a helium ionization detector. Indeed, coupled to GC, no other detector can perform this detection except the expensive atomic emission detector. Based on the detection and analysis of H2 isotopes by low-pressure chemical ionization mass spectrometry (MS), a new method for H2 detection by GC coupled to MS with an electron ionization ion source and a quadrupole analyser is presented. The presence of H2 in a gaseous mixture could easily be put in evidence by the monitoring of the molecular ion of the protonated carrier gas. Copyright © 2013 John Wiley & Sons, Ltd.

Distal dendrites of frog motor neurons: a computer-aided electron microscopic study of cobalt-filled cells.

With the aid of the cobalt labelling technique, frog spinal cord motor neuron dendrites of the subpial dendritic plexus have been identified in serial electron micrographs. Computer reconstructions of various lengths (2.5-9.8 micron) of dendritic segments showed the contours of these dendrites to be highly irregular, and to present many thorn-like projections 0.4-1.8 micron long. Number, size and distribution of synaptic contacts were also determined. Almost half of the synapses occurred at the origins of the thorns and these synapses had the largest contact areas. Only 8 out of 54 synapses analysed were found on thorns and these were the smallest. For the total length of reconstructed dendrites there was, on average, one synapse per 1.2 micron, while 4.4% of the total dendritic surface was covered with synaptic contacts. The functional significance of these distal dendrites and their capacity to influence the soma membrane potential is discussed.

Rapid detection by transmission electron microscopy of mycoplasma contamination in sera and cell cultures

Transmission electon microscopy has been employed for the rapid detection of mycoplasma in sera and cell cultures. High speed centrifugation of sera or low speed centrifugation of cell debris, followed by negative staining of the resuspended pellet, detected mycoplasma contamination more frequently than a culture method followed by direct fluorescence (DAPI), which was used as a control procedure. The appearance of the mycoplasma cell border and content gives some information about particle viability.

Scanning electron microscopy of the egg and the first instar larva of Dermatobia hominis (Diptera, cuterebridae)

The egg and the first instar larva of Dermatobia hominis were described based on observation with a scanning electron microscope.

Morphological evidence by scanning electron microscopy of excretion of metacyclic forms of Trypanosoma cruzi in vector's urine

Comparision by scanning electron microscopy (SEM) of Trypanosoma cruzi flagellates attached to the cuticle of the rectal gland of infected Dipetalogaster maxima nymphs, showed marked differences before amd after feeding. Before feeding numerous metacyclic trypomastigotes were observed among the abundant epimastigotes that formed the carpet of flagellates. On the other hand, in insects that were allowed to urinate for 24 hours after a meal, the metacyclics were scarce,indicating that they had been detached by the urine flow. An asymetric type of cell division, probably originating both an epi-and a trypomastigote, was occasionally observed. The occurrence of swellings at different levels of the flagella of epimastigotes suggests that secondary sites of attachment may be common.

Studies on male genitalia of sarcophagidae (Diptera) based on scanning electron microscope observations

Male genitalia of Oxyvinia exicisa (Lopes), Oxysarcodexia thomax (Walker), O. fluminensis Lopes, Sarcodexia lambens (Wiedemann), Peckia chrysostoma (Wiedemann) and Liopygia ruficornis (Fabricius) were studied based on scanning electron microscope photography. Some important details were evidentiated with this method.

Visualisation of human rad52 protein and its complexes with hRad51 and DNA.

The human Rad52 protein stimulates joint molecule formation by hRad51, a homologue of Escherichia coli RecA protein. Electron microscopic analysis of hRad52 shows that it self-associates to form ring structures with a diameter of approximately 10 nm. Each ring contains a hole at its centre. hRad52 binds to single and double-stranded DNA. In the ssDNA-hRad52 complexes, hRad52 was distributed along the length of the DNA, which exhibited a characteristic "beads on a string" appearance. At higher concentrations of hRad52, "super-rings" (approximately 30 nm) were observed and the ssDNA was collapsed upon itself. In contrast, in dsDNA-hRad52 complexes, some regions of the DNA remained protein-free while others, containing hRad52, interacted to form large protein-DNA networks. Saturating concentrations of hRad51 displaced hRad52 from ssDNA, whereas dsDNA-Rad52 complexes (networks) were more resistant to hRad51 invasion and nucleoprotein filament formation. When Rad52-Rad51-DNA complexes were probed with gold-conjugated hRad52 antibodies, the presence of globular hRad52 structures within the Rad51 nucleoprotein filament was observed. These data provide the first direct visualisation of protein-DNA complexes formed by the human Rad51 and Rad52 recombination/repair proteins.

Scanning electron microscopy of the antennal sensilla in female Culicoides paraensis (Diptera: Ceratopogonidae)

We studied by sanning electron microscopy the number, types, structure and distribution of the antennal sensilla of the medical important ceratopogonid Culicoides paraensis (Goeldi). There are about 174 sense organs on the antenmal flagellum which are classified as sensilla chaetica; sharp-tipped and blunt-tipped (type I and II) sensilla trichodea; sensilla basiconica; sensilla coeloconica; sensilla ampullacea and styloconic-type sensilla. The role of antennal sensory organs are discussed regarding the host preference of the biting midges.

Subcellular investigation of photosynthesis-driven carbon assimilation in the symbiotic reef coral Pocillopora damicornis.

Reef-building corals form essential, mutualistic endosymbiotic associations with photosynthetic Symbiodinium dinoflagellates, providing their animal host partner with photosynthetically derived nutrients that allow the coral to thrive in oligotrophic waters. However, little is known about the dynamics of these nutritional interactions at the (sub)cellular level. Here, we visualize with submicrometer spatial resolution the carbon and nitrogen fluxes in the intact coral-dinoflagellate association from the reef coral Pocillopora damicornis by combining nanoscale secondary ion mass spectrometry (NanoSIMS) and transmission electron microscopy with pulse-chase isotopic labeling using [(13)C]bicarbonate and [(15)N]nitrate. This allows us to observe that (i) through light-driven photosynthesis, dinoflagellates rapidly assimilate inorganic bicarbonate and nitrate, temporarily storing carbon within lipid droplets and starch granules for remobilization in nighttime, along with carbon and nitrogen incorporation into other subcellular compartments for dinoflagellate growth and maintenance, (ii) carbon-containing photosynthates are translocated to all four coral tissue layers, where they accumulate after only 15 min in coral lipid droplets from the oral gastroderm and within 6 h in glycogen granules from the oral epiderm, and (iii) the translocation of nitrogen-containing photosynthates is delayed by 3 h. IMPORTANCE: Our results provide detailed in situ subcellular visualization of the fate of photosynthesis-derived carbon and nitrogen in the coral-dinoflagellate endosymbiosis. We directly demonstrate that lipid droplets and glycogen granules in the coral tissue are sinks for translocated carbon photosynthates by dinoflagellates and confirm their key role in the trophic interactions within the coral-dinoflagellate association.

Scanning electron microscopy of the male genitalia of Sarcophagidae (Diptera)

The male genitalia of nine species of Sarcophagidae (Diptera) - Goniophyto honsuensis Rohdendorf, 1962, Tricharaea brevicornis (Wiedemann, 1830), Chaetoravinia derelicta (Walker, 1852), Austrohartigia spinigena (Rondani, 1864), Chrysagria duodecimpunctata Townsend, 1935, Boettcheria bisetosa Parker, 1914, Lipoptilocnema lanei Townsend, 1934, L. crispina (Lopes, 1938) and Euboettcheria alvarengai Lopes & Tibana, 1982 - were examined by scanning electron microscope (SEM) and the main morphological features are descirbed.

Scanning electron microscopy of the dorsal vessel of Panstrongylus megistus (Burmeister, 1835) (Hemiptera: Reduviidae)

In this study we analyzed the microanatomy of the dorsal vessel of the triatomine Panstrongylus megistus. The organ is a tuble anatomically divided into an anterior aorta anad a posterior heart, connected to the body wall through 8 pairs of alary muscles. The heart is divided in 3 chambers by means of 2 pairs of cardiac valves. a pair of ostia can be observed in the lateral wall of each chamber. A bundle of nerve fibers was found outside the organ, running dorsally along its major axis. A group of longitudinal muscular fibers was found in the ventral portion of the vessel. The vessel was found to be lined both internally and externally by pericardial cells covered by a thin laminar membrane. Inseide the vessel the pericardial cells were disposed in layers and on the outside they formed clusters or rows.

Structure of a multisubunit complex that promotes DNA branch migration.

The RuvA and RuvB proteins of Escherichia coli, which are induced in response to DNA damage, are important in the formation of heteroduplex DNA during genetic recombination and related recombinational repair processes. In vitro studies show that RuvA binds Holiday junctions and acts as a specificity factor that targets the RuvB ATPase, a hexameric ring protein, to the junction. Together, RuvA and RuvB promote branch migration, an ATP-dependent reaction that increases the length of the heteroduplex DNA. Electron microscopic visualization of RuvAB now provides a new insight into the mechanism of this process. We observe the formation of a tripartite protein complex in which RuvA binds the crossover and is sandwiched between two hexameric rings of RuvB. The Holliday junction within this complex adopts a square-planar structure. We propose a molecular model for branch migration, a unique feature of which is the role played by the two oppositely oriented RuvB ring motors.

Skeletal Muscle Mitochondrial and Lipid Droplet Volume Density: Validity of Electron Microscopy Point-counting Measurements

Mitochondrial (M) and lipid droplet (L) volume density (vd) are often used in exercise research. Vd is the volume of muscle occupied by M and L. The means of calculating these percents are accomplished by applying a grid to a 2D image taken with transmission electron microscopy; however, it is not known which grid best predicts these values. PURPOSE: To determine the grid with the least variability of Mvd and Lvd in human skeletal muscle. METHODS: Muscle biopsies were taken from vastus lateralis of 10 healthy adults, trained (N=6) and untrained (N=4). Samples of 5-10mg were fixed in 2.5% glutaraldehyde and embedded in EPON. Longitudinal sections of 60 nm were cut and 20 images were taken at random at 33,000x magnification. Vd was calculated as the number of times M or L touched two intersecting grid lines (called a point) divided by the total number of points using 3 different sizes of grids with squares of 1000x1000nm sides (corresponding to 1µm2), 500x500nm (0.25µm2) and 250x250nm (0.0625µm2). Statistics included coefficient of variation (CV), 1 way-BS ANOVA and spearman correlations. RESULTS: Mean age was 67 ± 4 yo, mean VO2peak 2.29 ± 0.70 L/min and mean BMI 25.1 ± 3.7 kg/m2. Mean Mvd was 6.39% ± 0.71 for the 1000nm squares, 6.01% ± 0.70 for the 500nm and 6.37% ± 0.80 for the 250nm. Lvd was 1.28% ± 0.03 for the 1000nm, 1.41% ± 0.02 for the 500nm and 1.38% ± 0.02 for the 250nm. The mean CV of the three grids was 6.65% ±1.15 for Mvd with no significant differences between grids (P>0.05). Mean CV for Lvd was 13.83% ± 3.51, with a significant difference between the 1000nm squares and the two other grids (P<0.05). The 500nm squares grid showed the least variability between subjects. Mvd showed a positive correlation with VO2peak (r = 0.89, p < 0.05) but not with weight, height, or age. No correlations were found with Lvd. CONCLUSION: Different size grids have different variability in assessing skeletal muscle Mvd and Lvd. The grid size of 500x500nm (240 points) was more reliable than 1000x1000nm (56 points). 250x250nm (1023 points) did not show better reliability compared with the 500x500nm, but was more time consuming. Thus, choosing a grid with square size of 500x500nm seems the best option. This is particularly relevant as most grids used in the literature are either 100 points or 400 points without clear information on their square size.