Application of PCR and probe hybridization techniques in detection of airborne fungal spores in environmental samples

Specific PCR amplification and probe hybridization techniques were applied to examine the compositions of airborne fungi in samples from three different environments. The results from microscopic and CFU counting were compared to those of the molecular-based detections. The detection sensitivity for PCR amplifications was 9 to 73 spores and 1.3 to 19.3 CFUs per PCR reaction. The hybridization detection limit was 2 to 4 spores and 0.2 to 1.2 CFU. The hybridization method was more sensitive than PCR amplification and showed less variation among samples. Using specific PCR primers and probes we identified the presence of several fungal groups and species in the air samples. Specific detections through probe hybridization to PCR products amplified with universal or group-specific fungal primers have promising applications in the examination of air samples for environmental monitoring.     

Air sampling of fungal spores on filters. An investigation on passive sampling and viability

In this study, glycerol was tested as a collection substrate for passive bioaerosol sampling. Filters (mixed cellulose acetate and nitrate) were soaked in glycerol and exposed for an aerosol from three different fungal species: Penicillum commune, Aspergillus versicolor and Paecilomyces variotii. The passive sampling method was compared with a closed-face polycarbonate filter sampling method. Exposure was performed in an exposure chamber. The total number of spores was determined by microscopic techniques, and the cultivable number was determined by cultivation on Malt Extract Agar dishes. The glycerol soaked filter demonstrated a good correlation with the closed-face sampler with regard to the total count. Spores stored in a pumped filter cassette were not affected by storage for up to 7 days. On the other hand, the culturability of the spores was markedly decreased after 1 day when stored on glycerol soaked filters.     

Coordinated gene expression for pheromone biosynthesis in the pine engraver beetle, <Emphasis Type="Italic">Ips pini</Emphasis> (Coleoptera: Scolytidae)

In several pine bark beetle species, phloem feeding induces aggregation pheromone production to coordinate a mass attack on the host tree. Male pine engraver beetles, Ips pini (Say) (Coleoptera: Scolytidae), produce the monoterpenoid pheromone component ipsdienol de novo via the mevalonate pathway in the anterior midgut upon feeding. To understand how pheromone production is regulated in this tissue, we used quantitative real-time PCR to examine feeding-induced changes in gene expression of seven mevalonate pathway genes: acetoacetyl-coenzyme A thiolase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate 5-diphosphate decarboxylase, isopentenyl-diphosphate isomerase, geranyl-diphosphate synthase (GPPS), and farnesyl-diphosphate synthase (FPPS). In males, expression of all these genes significantly increased upon feeding. In females, the expression of the early mevalonate pathway genes (up to and including the isomerase) increased significantly, but the expression of the later genes (GPPS and FPPS) was unaffected or decreased upon feeding. Thus, feeding coordinately regulates expression of the mevalonate pathway genes necessary for pheromone biosynthesis in male, but not female, midguts. Furthermore, basal mRNA levels were 5- to 41-fold more abundant in male midguts compared to female midguts. This is the first report of coordinated regulation of mevalonate pathway genes in an invertebrate model consistent with their sex-specific role in de novo pheromone biosynthesis.Electronic Supplementary Material  Supplementary material is available in the online version of this article at .     

Transport and transformation of sulfur compounds over East Asia during the TRACE-P and ACE-Asia campaigns

On the basis of the recently estimated emission inventory for East Asia with a resolution of 1×1°, the transport and chemical transformation of sulfur compounds over East Asia during the period of 22 February through 4 May 2001 was investigated by using the Models-3 Community Multi-scale Air Quality (CMAQ) modeling system with meteorological fields calculated by the regional atmospheric modeling system (RAMS). For evaluating the model performance simulated concentrations of sulfur dioxide (SO₂) and aerosol sulfate (SO₄²⁻) were compared with the observations on the ground level at four remote sites in Japan and on board aircraft and vessel during the transport and chemical evolution over the Pacific and Asian Pacific regional aerosol characterization experiment field campaigns, and it was found that the model reproduces many of the important features in the observations, including horizontal and vertical gradients. The SO₂ and SO₄²⁻ concentrations show pronounced variations in time and space, with SO₂ and SO₄²⁻ behaving differently due to the interplay of chemical conversion, removal and transport processes. Analysis of model results shows that emission was the dominant term in regulating the SO₂ spatial distribution, while conversion of SO₂ to SO₄²⁻ in the gas phase and the aqueous phase and wet removal were the primary factors that controlled SO₄²⁻ amounts. The gas phase and the aqueous phase have the same importance in oxidizing SO₂, and about 42% sulfur compounds (25% in SO₂) emitted in the model domain was transported out, while about 57% (35% by wet removal processes) was deposited in the domain during the study period.     

Airborne chemical compounds on dairy farms

The chemical environment that dairy farmers are exposed to during milking was investigated. Volatile organic compounds (VOCs) were analysed and identified, and the levels of formaldehyde, ammonia and carbon dioxide were measured in eight farms in northern Sweden. Both stationary and personal samples were taken. A total of 70 VOCs were identified from the adsorbent samples, with p-cresol, 2-butanone, ethyl acetate, alpha-pinene and delta 3-carene occurring at the highest levels. All monitored levels were significantly lower for compounds having a stated highest occupational exposure level (OEL). Using multivariate techniques some differences in the composition of the workplace air between and within the farms were found. No difference was found between personal exposure and the surrounding environment in the cowshed.     

Toward Explaining Nitrogen and Phosphorus Trends in Chesapeake Bay Tributaries, 1992–2012

Understanding trends in stream chemistry is critical to watershed management, and often complicated by multiple contaminant sources and landscape conditions changing over varying time scales. We adapted spatially referenced regression (SPARROW) to infer causes of recent nutrient trends in Chesapeake Bay tributaries by relating observed fluxes during 1992, 2002, and 2012 to contemporary inputs and watershed conditions. The annual flow‐normalized nitrogen flux to the bay from its watershed declined by 14% to 127,000 Mg (metric tons) between 1992 and 2012, due primarily (more than 80% of the decline) to reduced point sources. The remainder of the decline was due to reduced atmospheric deposition (13%) and urban nonpoint sources. Agricultural inputs, which contribute most nitrogen to the bay, changed little, although trends in the average nitrogen yield (flux per unit area) from cropland and pasture to streams in some settings suggest possible effects of evolving nutrient applications or other land management practices. Point sources of phosphorus to local streams declined by half between 1992 and 2012, while nonpoint inputs were relatively unchanged. Annual phosphorus delivery to the bay increased by 9% to 9,570 Mg between 1992 and 2012, however, due mainly to reduced retention in the Susquehanna River at Conowingo Reservoir.