Obtaining and characterization of DNA-containing micromummies of yeasts and gram-positive bacteria with enhanced cell wall permeability: Application in PCR

The procedure of obtaining DNA-containing cell envelopes (“micromummies”) of bacteria, yeasts, and fungi using chaotropic salts has been developed previously and the possibility of their direct application in PCR has been demonstrated. The fine structure of micromummies has been studied by electron microscopic methods. This work has demonstrated that additional treatment of micromummies of yeasts and gram-positive bacteria with proteinase K results in hydrolytic degradation of cell proteins and drastic enhancement of cell wall permeability for macromolecules (DNA). Thus, the efficiency of PCR ex situ using resultant micromummies after washing off the products of protein hydrolysis and proteinase K can be increased. The results of electron microscopic study of ultrathin sections of yeasts (Pichia pastoris, Saccharomyces cerevisiae) and gram-positive bacteria (Micrococcus luteus, Arthrobacter globiformis, Bacillus subtilis) support the biochemical data that treatment with chaotropic salts and proteinase K results in the loosening of microbial cell walls and in a decrease in the intracellular protein content. At the same time, cell walls generally maintain their integrity (continuity) and initial spherical or rodlike shape. The optimal modes of treatment of the cells of different microbial species with chaotropic salts and proteinase K have been selected to obtain permeabilized cell envelopes containing denatured or native DNA.     

Ultrastructural study of oogenesis and yolk formation in an opisthobranch mollusc, Runcina

Runcina is a hermaphroditic opisthobranch mollusc of small size. It produces large eggs, rich in yolk substances, and it is thus ideal for studying the mechanisms of vitellogenesis. The rough endoplasmic reticulum and the Golgi apparatus appear to be involved in early build up of yolk precursors. Endocytotic vesicles carrying yolk proteins, taken up from the haemolymph, dominate yolk formation at a later stage. The findings presented in this study enhance the proposition of a dual pathway of auto- and heterosynthesis in yolk formation. It is not found that the yolk bodies in Runcina acquire lysosomal enzymes in order to digest the perivitelline fluid, as has been described for some pulmonates. The importance of the role played by the follicle cells in oocyte development is discussed.     

Ultrastructure of the Circulatory System of the Phoronid Phoronopsis harmeri Pixell, 1912. 2. Main vessels

The ultrastructure of the wall of the main blood vessels of the phoronid Phoronopsis harmeri is described. The walls of the lophophoral and left lateral vessels consist of myoepithelial cells of the coelomic lining (peritoneal cells), a thin basal lamina, and an incomplete endothelial lining. In the head region of the body, the wall of the medial vessel consists of myoepithelial cells of the coelomic lining (peritoneal cells), a basal lamina, and true muscular endothelial cells. The anterior part of the medial vessel functions as the heart. In the anterior part of the body, the medial vessel wall consists of five layers: the external nonmuscular coelothelium, a layer of the extracellular matrix, the internal muscular coelothelium, an internal layer of the extracellular matrix, and an incomplete endothelial lining. The complicated structure of the medial vessel wall may be explained by the superimposition of the lateral mesentery on the ordinary vessel wall.     

Mycoparasitism of Rhizoctonia solani by Endophytic Chaetomium spirale ND35: Ultrastructure and Cytochemistry of the Interaction

The interaction between endophytic biocontrol agent Chaetomium spirale ND35 and the soil‐borne plant pathogen Rhizoctonia solani was studied by light microscopy and transmission electron microscopy (TEM), as well as further investigated by gold cytochemistry to assess the potential role of cell wall degrading enzymes (CWDEs) during the mycoparasitic process. Macroscopic observations of fungal growth in dual cultures revealed that pathogen growth inhibition occurred soon after contact with the antagonist, followed by the overgrowth of C. spirale on the colony of R. solani. The coiling of C. spirale around R. solani and intracellular growth of the antagonist in its host occurred frequently. Moreover, in advanced stage of interaction between the antagonist and the pathogen, The growth and development of C. spirale were associated with highly morphological changes of the host fungal cell, characterized by retraction of plasma membrane and cytoplasm disorganization. Further, TEM investigations through localization by gold immunocytochemistry showed that contact between the two fungi was mediated by an amorphous β‐1,3‐glucan‐enriched matrix originating from cell wall of the antagonist C. spirale and sticking to its host surface. At the same time, the hemispherical wall appositions which were intensely labeled by the antibodies of β‐1, 3‐glucan in cell wall of R. solani were induced to form at sites of potential antagonist entry. However, the antagonist was capable of penetrating this barrier, indicating that β‐1,3‐glucanases were produced during the mycoparasitic process. Localization of N‐acetylglucosamine residues (chitin) with the gold‐labelled wheat germ agglutinin (WGA) implicated that chitinases might be involved in the CWD of R. solani in this antagonistic process as well. This report is the first evidence about mechanisms of the interactions between C. spirale and R. solani in ultrastructural and cytochemical aspects.     

Spermiogenesis in the Australian cockatiel Nymphicus hollandicus

Information on the ultrastructure of parrot spermatids and spermatozoa is limited to only four species with no comprehensive study of spermiogenesis conducted within the order Psittaciformes. The present study was undertaken to describe the development of the cockatiel spermatid using electron microscopy. Four phases of spermatid maturation were documented on the basis of nuclear morphology, development of the acrosome, perforatorium, and axial filament. These phases included 1) round nuclei, 2) irregular nuclei, 3) elongated nuclei with granular chromatin, and 4) elongated nuclei with homogenous chromatin. While development of the cockatiel spermatid was comparable to that of other domestic avian species, we have noted the hollow nature of some chromatin granules, an abnormal formation of the axoneme, the absence of the fibrous sheath around the axoneme of the principal piece, and the absence of an annulus. J. Morphol. , 2012. © 2012 Wiley Periodicals, Inc.     

Azadirachtin disrupts formation of organised microtubule arrays during microgametogenesis of Plasmodium berghei

Transmission of malaria parasites from vertebrate blood to the mosquito vector depends critically on the differentiation of the gametocytes into gametes. This occurs in response to environmental stimuli encountered by the parasite in the mosquito bloodmeal. Male gametogenesis involves three rounds of DNA replication and endomitosis, and the assembly de novo of 8 motile axonemes. Azadirachtin, a plant limnoid and insecticide with an unkown mode of action, specifically inhibits the release of motile gametes from activated microgametocytes but does not inhibit growth and replication of a sexual blood stages. We have combined confocal laser scanning microscopy and transmission electron microscopy to examine the effect of azadirachtin on the complex reorganisation of the microtubule cytoskeleton during gametogenesis in Plasmodium berghei. Neither the replication of the genome nor the ability of tubulin monomers to assemble into microtubules upon gametocyte activation were prevented by azadirachtin. However, the drug interfered with the formation of mitotic spindles and with the assembly of microtubules into typical axonemes. Our observations suggest that azadarachtin specifically disrupts the patterning of microtubules into more complex structures, such as mitotic spindles and axonemes.     

THE MORPHOLOGY AND DEVELOPMENT OF SOME PROMINENTLY STALKED SOUTHERN AUSTRALIAN HALYMENIACEAE (CRYPTONEMIALES,RHODOPHYTA). II. THE SPONGE-ASSOCIATED GENERA THAMNOCLONIUM KUETZING AND CODIOPHYLLUM GRAY1

Three members of the red algal family Halymeniaceae (Thamnoclonium dichotomum (J. Ag.) J. Ag., Codiophyllum flabelliforme (Sond.) Schmitz, and C. decipiens (J. Ag.) Schmitz) are investigated. All are endemic to southern and southwestern Australia, possess basal stalks of substantial size and firmness, and are consistently associated with specific sponge taxa. In each case, the sponges are bonded by collagen-like fibrils to the host cuticle without modifying the algal tissue at the ultrastructural level. Secondary cortication and prominent growth rings occur in the stalks of all three species, and in each the pit plugs between cells become wider, more convoluted and less electron dense with increasing distance from the surface. Such pit plugs are apparently a unique attribute of the stalked Halymeniaceae. The three species share pit plug, sponge association and stalk morphological features but are not otherwise closely related, as they actually represent three distinct genera.     

Variability in tolerance to UV-B radiation among Beauveria spp. isolates

Solar radiation, particularly the UV-B component, negatively affects survival of entomopathogenic fungi in the field. In an effort to identify Beauveria spp. isolates with promise for use in biological control settings with high insolation, we examined 53 Beauveria bassiana isolates, 7 isolates of 4 other Beauveria spp. and Engyodontium albus (=Beauveria alba). The origins of these fungi varied widely as to host/substrate and country, but approximately 30% of these isolates were B. bassiana from ticks in Brazil. A preliminary trial with three B. bassiana isolates (Bb 19, CG 310 and CG 481) at several UV-B dosages indicated that 2h of weighted UV-B irradiance at 978mWm(-2) (providing a total dose of 7.04kJm(-2)) allowed separation of isolates into low, medium or high UV-B tolerance. This dose, therefore, was selected as a single dose to compare UV-B tolerances of all 60 Beauveria spp. isolates. There was high variability in tolerance to UV-B radiation among the B. bassiana isolates, ranging from virtually zero tolerance (e.g., Bb 03) to almost 80% tolerance (e.g., CG 228). In addition, surviving B. bassiana conidia demonstrated delayed germination; and this is likely to reduce virulence. Conidia of the other species were markedly more sensitive to UV-B, with E. albus (UFPE 3138) being the least UV-B tolerant. Among B. bassiana isolates originating from 0 degrees to 22 degrees latitudes, those from lower latitudes demonstrated statistically significant greater UV-B tolerances than those isolates from higher latitudes. Isolates from above 22 degrees , however, were unaffected by latitude of origin. A similar analysis based on host type did not indicate a correlation between original host and UV-B tolerance. The identification in this study of several B. bassiana isolates with relatively high UV-B tolerance will guide the selection of isolates for future arthropod microbial control experiments.