The Relative Abundance of Mountain Pine Beetle Fungal Associates Through the Beetle Life Cycle in Pine Trees

The mountain pine beetle (MPB) is a native bark beetle of western North America that attacks pine tree species, particularly lodgepole pine. It is closely associated with the ophiostomatoid ascomycetes Grosmannia clavigera, Leptographium longiclavatum, Ophiostoma montium, and Ceratocystiopsis sp.1, with which it is symbiotically associated. To develop a better understanding of interactions between beetles, fungi, and host trees, we used target-specific DNA primers with qPCR to assess the changes in fungal associate abundance over the stages of the MPB life cycle that occur in galleries under the bark of pine trees. Multivariate analysis of covariance identified statistically significant changes in the relative abundance of the fungi over the life cycle of the MPB. Univariate analysis of covariance identified a statistically significant increase in the abundance of Ceratocystiopsis sp.1 through the beetle life cycle, and pair-wise analysis showed that this increase occurs after the larval stage. In contrast, the abundance of O. montium and Leptographium species (G. clavigera, L. longiclavatum) did not change significantly through the MPB life cycle. From these results, the only fungus showing a significant increase in relative abundance has not been formally described and has been largely ignored by other MPB studies. Although our results were from only one site, in previous studies we have shown that the fungi described were all present in at least ten sites in British Columbia. We suggest that the role of Ceratocystiopsis sp.1 in the MPB system should be explored, particularly its potential as a source of nutrients for teneral adults.     

A rapid fluorometric DNA assay for the measurement of cell density and proliferation in vitro

Summary Many research efforts require the accurate determination of cell density in vitro. However, physical cell counting is inaccurate, time-intensive and requires removal of the cells from their growth environment, thereby introducing a host of potential artifacts. The current studies document a very simple method of determining cell density in microtiter wells via DNA-enhanced fluorescence. Fixed cells are stained with the A-T intercalating DNA stains DAPI or Hoechst 33342 and then fluorescence is quantified in a plate fluorometer. Fluorescence is shown to be linearly related to cell density as determined by two physical counting methods. The validity of the method is established in determining serum-stimulated growth of smooth muscle cells and in mitogen-induced growth of endothelial cells. The fixed cells can be stored for prolonged periods, thus allowing time-course proliferation assays without interassay variations. The fixed cells are also suitable for determinations of antigens of interest by ELISA. This method is potentially valuable in many in vitro systems where the quantification of cell density and proliferation is necessary. This work supported in part by NIH Cardiovascular Training Grant HL07423 and a grant from the American Federation for Aging Research to T. M. and HL35724 to B. W. EDITOR’S STATEMENT The technique described in this paper represents an approach to quantifying cell density in adherent monolayers of cultured cells in microtiter wells that is rapid and simple and does not require radioisotopes or removal of cells.     

Regulation of Phytochrome Message Abundance in Root Caps of Maize : Spatial, Environmental, and Genetic Specificity

In many cultivars of maize (Zea mays L.) red light affects root development via the photomorphogenetic pigment phytochrome. The site of perception for the light is the root cap. In the maize cultivar Merit, we investigated phytochrome-mediated events in the cap. We established that the message encoded by the phyA1 gene was most abundant in dark-grown tissue and was asymmetrically distributed in the root cap, with greatest expression in the cells which make up the central columella core of the cap. Phytochrome message was negatively autoregulated in a specific region within the root cap. This autoregulation was sensitive to very-low-fluence red light, and thus was characterized as a phytochrome-mediated, very-low-fluence event. The kinetics of message reaccumulation in the dark were also examined and compared to the kinetics of the light requirement for root gravitropism in this cultivar. Similarly, the degree of autoregulation present in two other maize cultivars with different light requirements for gravitropic sensitivity was investigated. It appears that the Merit cultivar expresses a condition of hypersensitivity to phytochromemediated light regulation in root tissues. We conclude that phytochrome regulates many activities within the cap, but the degree to which these activities share common phytochrome-mediated steps in not known.     

Causes of nondiffusing lipid in the plasma membrane of mammalian spermatozoa

In the plasma membranes of most mammalian somatic cells, lipid is nearly completely free to diffuse laterally in the plane of the membrane. In mammalian spermatozoa and certain other highly polarized mammalian cells, a significant fraction of the plasma membrane lipid is not free to diffuse laterally. Using the technique of fluorescence recovery after photobleaching, we have demonstrated that a variety of fluorescent lipid analogues exhibit a nondiffusing fraction in the plasma membrane of the anterior region of the ram sperm head. The possible causes of this nondiffusing fraction were investigated. The nondiffusing lipid fraction is not the result of lipid oxidation during handling, and it is not released by extensive enzymatic digestion of the membrane surface proteins or the "bleeding" of the membrane by hypoosmotic shock. When lipid bilayers were prepared from protein-free lipid extracts of the plasma membranes of spermatozoa, most of the nondiffusing fraction was retained. These results suggest that the nondiffusing lipid fraction results from lipid factors such as lateral phase separations, which can cause such a nondiffusing fraction in model systems.     

Conceptual models and analytical tools: The biology of physicist Max Delbrück

Journal of the History of Biology -     

Electron microscopy of stomatogastric ganglion in the lobster Homarus americanus

The stomatogastric ganglion and two of the associated afferent and efferent nerve trunks (stomatogastric and dorsal ventricular nerves) from Homarus americanus have been examined with light and electron microscopy after glutaraldehyde-osmium tetroxide fixation. The dorsally located neuron somata, rich in ribosomes and glycogen, are encased in multi-layered glial and fibrous sheaths. The synaptic neuropil regions occur scattered throughout the central and ventral part of the ganglion, interspersed amonglarger nerve fibres of extrinsic and intrinsic origin from which the neuropil is derived. Neural processes containing masses of small clear vesicles plus larger dense-core vesicles make apparent synaptic contacts at points of increased membrane density with smaller, non-vesicle-containing or sometimes other vesicle-containing nerve fibres.     

Sugar and Alcohol Stabilization of Yeast in Sweet Wine

Secondary fermentation of sweet wine was prevented by the Delle stabilization procedure. For this procedure, advantage is taken of the inhibitory effects of high concentrations of sugar as well as of alcohol. Thus, relatively small amounts of wine spirits were added to fermenting musts to obtain stability, as compared to the conventional procedure in which larger amounts of alcohol are added and the inhibitory effect of alcohol only is considered. The Delle value is a function of the concentrations in the wine, after spirits addition, of alcohol and sugar. Delle values which gave stable wine were dependent on time of alcohol addition, on strain of wine yeast, and on composition of wine spirits. Fractional addition of spirits, concentration of SO(2), and clarity of must had little effect on the Delle value. Sensory comparison of wines especially prepared for tasting by the Delle procedure and by the conventional procedure showed the wines made by the Delle procedure to be superior in quality. Under proper storage conditions, the Delle wines were shown to be microbiologically stable and resistant to wine spoilage organisms.