Fast and efficient discrimination of the Isotoma viridis group (Insecta: Collembola) by PCR-RFLP

Identification of collembolan species is generally based on specific morphological characters, such as chaetotaxy and pigmentation pattern. However, some specimens do not match to described characters because these refer to adult specimens, often of one specific sex, or the characters are highly variable in adults (e.g. pigmentation, setae or furcal teeth). Isozymes have frequently assisted species discrimination, and also these may vary with developmental stage or environmental conditions. For identification of single species of the Isotoma viridis group, we present both direct sequencing of the cytochrome oxidase subunit II (COII) gene and a simple DNA-based molecular method.

Five PCR primers amplifying the COII region (717 bp) of the mitochondrial DNA were used. The sequences clearly separated the species I. viridis, I. riparia and I. anglicana, irrespective of colour varieties within the first species. DNA amplification products of different species can also be distinguished by digestion with restriction endonucleases, followed by gel electrophoresis for separation of fragments. This restriction fragment length polymorphism (RFLP), obtained after digestion with the endonucleases TaqI, VspI, MvaI and Bsp143I, revealed specific fragments that separated the three species from each other. Since restriction enzymes are sensitive to single base mutations, we suggest to use a combination of enzymes with at least two species-specific restriction sites when using the RFLP technique. For the I. viridis complex, VspI and Bsp143I appear to be an appropriate combination.     

Effects of input management and crop diversity on non-renewable energy use efficiency of cropping systems in the Canadian Prairie

Although producers’ prime objective may be to increase net returns, many are also interested in conserving and enhancing the quality the soil, water and air resources through adopting more environmentally friendly production practices. This study compared non-renewable energy inputs, energy output, and energy use efficiency of nine dryland cropping systems comprised of a factorial combination of three methods of input management [high (HIGH), i.e., conventional tillage plus full recommended rates of fertilizer and pesticides; reduced (RED), i.e., conservation tillage plus reduced rates of fertilizer and pesticides; and organic (ORG), i.e., conventional tillage plus N-fixing legumes and non-chemical means of weed and pest control]; and three crop rotation systems with varying levels of cropping diversity [a fallow-based rotation with low crop diversity (LOW); a diversified rotation using annual cereal, oilseed and pulse grains (DAG); and a diversified rotation using annual grains and perennial forages (DAP)]. The study was conducted over the 1996–2007 period on a Dark Brown Chernozemic soil (Typic Boroll) in the Canadian Prairies. As expected, total direct plus indirect energy input was the highest for the HIGH and RED input treatments (3773 MJ ha^?1 year^?1), and 50% less for ORG management. Most of the energy savings came from the non-use of inorganic fertilizers and pesticides in the ORG management treatments. Further, total energy use was the highest for the DAG treatments (3572 MJ ha^?1 year^?1), and similar but about 18% lower for the DAP and LOW crop diversity treatments compared to DAG. Thus, overall, the HIGH/DAG and RED/DAG systems had the highest energy requirements (4409 MJ ha^?1 year^?1) and ORG/DAP had the lowest (1806 MJ ha^?1 year^?1). Energy output (calorimetric energy content) was typically the highest for the HIGH input treatments (26,541 MJ ha^?1 year^?1), was about 4% less with RED, and 37% less with ORG management. The latter reflected the lower crop yields obtained with organic management. Similarly, energy output was the highest for the DAP treatments (25,008 MJ ha^?1 year^?1), about 5% less for DAG, and 20% less for the LOW crop diversity treatments. The higher energy output with the DAP treatments largely reflected that the entire harvested biomass of the forage crops was included in energy output, while for grain crops only the seed was included. The straw and crop residues from annual crops were returned to the land to protect the soil from erosion and to maintain soil organic matter as this is the recommended practice in this semi-arid region. In contrast to energy output and to net energy produced, energy use efficiency (measured as yield of grain plus forage produced per unit of energy input or as energy output/energy input ratio) was the highest for the ORG input treatments (497 kg of harvested production GJ^?1 of energy input, and an energy output/energy input ratio of 8.8). We obtained lower, but generally similar energy use efficiency for the HIGH and RED input treatments (392 kg GJ^?1 and ratio of 7.1). Thus, overall, ORG/DAP was the most energy efficient cropping system, while RED/LOW and RED/DAG generally ranked the lowest in energy use efficiency. Our findings support the current movement of producers toward ORG management as a means of reducing the reliance on non-renewable energy inputs and improving overall energy use efficiency of their cropping systems. Our results also suggest that moving away from traditional monoculture cereal rotations that employ frequent summer fallowing, toward extended and diversified crop rotations that use reduced tillage methods, although resulting in an increase in energy output, will not significantly reduce the overall reliance on non-renewable energy inputs, nor enhance energy use efficiency, unless perennial legume forages and/or legume grain crops are included in the cropping mix.     

Ordering in a fluid inert gas confined by flat surfaces

High-resolution transmission electron microscopy images of room-temperature fluid xenon in small faceted cavities in aluminum reveal the presence of three well-defined layers within the fluid at each facet. Such interfacial layering of simple liquids has been theoretically predicted, but observational evidence has been ambiguous. Molecular dynamics simulations indicate that the density variation induced by the layering will cause xenon, confined to an approximately cubic cavity of volume approximately 8 cubic nanometers, to condense into the body-centered cubic phase, differing from the face-centered cubic phase of both bulk solid xenon and solid xenon confined in somewhat larger (>/=20 cubic nanometer) tetradecahedral cavities in face-centered cubic metals. Layering at the liquid-solid interface plays an important role in determining physical properties as diverse as the rheological behavior of two-dimensionally confined liquids and the dynamics of crystal growth.     

Dynamic visualization of thrombopoiesis within bone marrow

Platelets are generated from megakaryocytes (MKs) in mammalian bone marrow (BM) by mechanisms that remain poorly understood. Here we describe the use of multiphoton intravital microscopy in intact BM to visualize platelet generation in mice. MKs were observed as sessile cells that extended dynamic proplatelet-like protrusions into microvessels. These intravascular extensions appeared to be sheared from their transendothelial stems by flowing blood, resulting in the appearance of proplatelets in peripheral blood. In vitro, proplatelet production from differentiating MKs was enhanced by fluid shear. These results confirm the concept of proplatelet formation in vivo and are consistent with the possibility that blood flow-induced hydrodynamic shear stress is a biophysical determinant of thrombopoiesis.     

Cytomegalovirus recruitment of cellular kinases to dissolve the nuclear lamina

The passage of large-sized herpesviral capsids through the nuclear lamina and the inner nuclear membrane to leave the nucleus requires a dissolution of the nuclear lamina. Here, we report on the functions of M50/p35, a beta-herpesviral protein of murine cytomegalovirus. M50/p35 inserts into the inner nuclear membrane and is aggregated by a second viral protein, M53/p38, to form the capsid docking site. M50/p35 recruits the cellular protein kinase C for phosphorylation and dissolution of the nuclear lamina, suggesting that herpesviruses target a critical element of nuclear architecture.     

Multielemental fingerprinting as a tool for authentication of organic wheat, barley, faba bean, and potato

The multielemental composition of organic and conventional winter wheat, spring barley, faba bean, and potato was analyzed with inductively coupled plasma-optical emission spectrometry (ICP-OES) and -mass spectrometry (ICP-MS). The crops were cultivated in two years at three geographically different field locations, each accommodating one conventional and two organic cropping systems. The conventional system produced the highest harvest yields for all crops except the nitrogen-fixing faba bean, whereas the dry matter content of each crop was similar across systems. No systematic differences between organic and conventional crops were found in the content of essential plant nutrients when statistically analyzed individually. However, chemometric analysis of multielemental fingerprints comprising up to 14 elements allowed discrimination. The discrimination power was further enhanced by analysis of up to 25 elements derived from semiquantitative ICP-MS. It is concluded that multielemental fingerprinting with semiquantitative ICP-MS and chemometrics has the potential to enable authentication of organic crops.     

Analysis of the effect of temperature changes combined with different alkaline pH on the β-lactoglobulin trypsin hydrolysis pattern using MALDI-TOF-MS/MS

Temperature and pH influence the conformation of the whey protein β-lactoglobulin (β-Lg) monomer, dimer, and octamer formation, its denaturation, and solubility. Most hydrolyses have been reported at trypsin (EC 3.4.21.4) optimum conditions (pH 7.8 and 37 °C), while the hydrolysate mass spectrometry was largely limited to peptides with <4 kDa. There are few reports on trypsin peptide release patterns away from optimum. This work investigated the influence of alkaline (8.65 and 9.5) and optimum (7.8) pH at different temperatures (25, 37.5, and 50 °C) on β-Lg (7.5%, w/v) hydrolysis. Sample aliquots were drawn out before the addition of trypsin (blank sample) and at various time intervals (15 s to 10 min) thereafter. Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) was used to monitor peptide evolution over time with the use of two matrixes: α-cyano-4-hydroxycinnamic acid (HCCA) and 2.5-dihydroxyacetophenone (DHAP). Mass analysis showed that the N- and C-terminals (Lys(8)-Gly(9), Lys(100)-Lys(101), Arg(124)-Thr(125), Lys(141)-Ala(142), and Arg(148)-Leu(149)) of β-Lg were cleaved early (15 s) implying the ease of trypsinolysis at the exposed terminals. Hydrolyses at 25 °C and pH 7.8 as well as at 50 °C and pH 9.5 were slowed down and ordered. Nonspecific chymotrypsin-like behavior occurred more at higher temperatures (50 °C) than at lower ones (25 and 37.5 °C). In addition to our earlier work in the acid pH region, it can be concluded that there is potential for controlled hydrolysis outside the trypsin optimum, where different target peptides with predictable biofunctionalities could be produced.     

EMS and UV irradiation induce unstable resistance against CAA fungicides in <Emphasis Type="Italic">Bremia lactucae</Emphasis>

Wild type (WT) field isolates of Bremia lactucae failed to germinate in vitro or infect lettuce leaves in the presence of CAA (carboxylic acid amide) fungicides. Minimal inhibitory concentrations (MIC) for mandipropamid, dimethomorph, benthiavalicarb and iprovalicarb were 0.005, 0.5, 0.5 and 5 μg ml⁻¹, respectively. Mutagenesis experiments showed that spores exposed to EMS (ethyl methane sulphonate) or UV irradiation (254 nm) could infect lettuce leaves in the presence of up to 100 μg ml⁻¹ CAA. The proportion of infected leaves relative to the number of spores inoculated (infection frequency) was inversely related to the concentration of CAA used, ranging between 0 and 160 per 1 × 10⁶ spores. Resistant mutants (RM) lost their resistance within 1–14 reproduction cycles on CAA-treated plants. Crosses were made between RMxWT isolates and RMxRM isolates with an attempt to obtain stable homozygous resistant off-springs. Such crosses yielded few resistant but unstable progeny isolates. Mutagenic treatments given to hybrid isolates also failed to produce stable resistance. Previous gene sequencing data showed that stable resistance to CAAs is based on a single SNP in the cellulose synthase 3 (CesA3) gene of Plasmopara viticola. Therefore, we sequenced a 582 bp DNA fragment of Ces3A of WT, RM and hybrid isolates of B.lactucae. No mutation in this gene fragment was found. We conclude that mutagenic agents like EMS or UV may induce resistance to CAA in Bremia lactucae but this resistance is not stable and not linked to mutations in CesA3 gene.