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.     

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

Journal of the History of Biology -     

Placental permeability and energy metabolism enzymes in fetuses of lipemic rats

A model of maternal lipemia without hyperglycemia, in the rat, produced by high-fat feedings, was developed to study the effects of an abnormal maternal lipid homeostasis on placental transport of nutrients and possible alterations of key enzymes of energy metabolism in the liver and brain of the fetuses. Pregnant rats fed lower concentrations of fat served as controls. All studies were carried out in dams and fetuses one day prior to delivery. The dietary treatment of the dams and fetuses produced in the fetuses ketonemia as well as lipemia. Following a bolus of ¹⁴C-3-0-methyl-D-glucose to the dams, the levels of the tracer remained higher in the blood and brain of lipemic than in control fetuses. By contrast, there was a decrease in the fluxes of ¹⁴C--amino-isobutryic acid in the fetuses of lipemic dams as compared to controls. Among enzymes of energy metabolism, fetal liver glucose-6-phosphatase and succinic dehydrogenase were enhanced by lipemia. Fetal brain glucose-6-phosphatase was depressed. Thus, lipemia, as occuring in poorly controlled maternal diabetes, may be a factor in determining the access to the fetus of essential, neutral amino acids and alter the normal activity of energy metabolism enzymes in the fetus.