Effects of surrounding landscape on parasitism of alfalfa aphids in an IPM crop system in northern Catalonia

The parasitism (Hymenoptera, Braconidae, Aphidiinae) of alfalfa aphids (Hemiptera, Aphididae) was determined during three crop growing seasons. The highest rates of parasitism of Acyrthosiphon pisum Harris and of Therioaphis trifolii Monell occurred during the first-second and the fourth alfalfa intercuts, respectively. The landscape surrounding the fields affected only parasitism rates of A. pisum. During the second intercut the forest area had a negative influence on parasitism rates, whereas the bordering surfaces of alfalfa and winter cereals had a positive influence. Positive correlations were found between the rates of parasitism of A. pisum and the rate of aphid population increase in the first intercut. By contrast, negative correlations were found in the second intercut, when the aphid population peaked, suggesting that parasitoids contribute to the regulation of the aphid. This study stresses that regulation of alfalfa aphids cannot be explained solely on the basis of one functional group of natural enemies.     

Back from the Brink: The Holocene History of the Carpathian Barbel Barbus carpathicus

As a result of specific adaptations and habitat preferences strongly rheophilic fish species may show high levels of endemism. Many temperate rheophilic fish species were subjected to a series of range contractions during the Pleistocene, and then successfully expanded during the Holocene, colonising previously abandoned areas. The Carpathian barbel (Barbus carpathicus Kotlík, Tsigenopoulos, Ráb et Berrebi 2002) occurs in the montane streams in three basins of the main Central European rivers in the northern part of the Carpathian range. We used genetic variation within 3 mitochondrial and 9 microsatellite loci to determine a pattern of postglacial expansion in B. carpathicus. We found that overall genetic variation within the species is relatively low. Estimate of time to the most recent common ancestor (tMRCA) of mitochondrial sequences falls within the Holocene. The highest levels of genetic variation found in upper reaches of the Tisa river in the Danube basin suggest that glacial refugia were located in the south-eastern part of the species range. Our data suggest that the species crossed different watersheds at least six times as three genetically distinct groups (probably established in different expansion episodes) were found in northern part of the species range. Clines of genetic variation were observed in both the Danube and Vistula basins, which probably resulted from subsequent bottlenecks while colonizing successive habitats (south eastern populations) or due to the admixture of genetically diverse individuals to a previously uniform population (Vistula basin). Therefore, B. carpathicus underwent both demographic breakdowns and expansions during the Holocene, showing its distribution and demography are sensitive to environmental change. Our findings are important in the light of the current human-induced habitats alterations.     

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds.     

The effects of local and landscape-scale habitat characteristics and prey availability on corridor use by carnivores: A comparison of two contrasting farmlands

Corridors are thought to reduce the negative biological effects of habitat loss and fragmentation by providing connectivity and suitable habitat for many species, including carnivores. Although corridor structure maintenance is considered to be an essential tool for carnivore conservation in a human-dominated landscape, surprisingly little is known about the effects of different factors at various spatial scales. The main aim of this study was to determine how local and landscape-scale habitat characteristics and prey availability influence the corridor use by carnivores in a Central European agricultural landscape. Moreover we investigated carnivore corridor occurrence in two contrasting landscapes that differ in level of habitat loss and fragmentation to evaluate relative effect of species-specific response to regional context. Results show that the availability of principal prey (small mammals) was the most crucial factor affecting carnivore corridor use. Other important factors influencing corridor use were corridor width (positive), proportion of shrubs (positive) and presence of local, low-traffic, roads (positive). Single species models revealed interspecific and area-specific differences in carnivore preference at both spatial scales. Our findings confirm the general importance of multi-level approach to evaluating species-specific habitat requirements as a crucial tool for determining suitable methods for carnivore efficient conservation in human-dominated agricultural landscape.     

High Molecular Weight Forms of Mammalian Respiratory Chain Complex II

Mitochondrial respiratory chain is organised into supramolecular structures that can be preserved in mild detergent solubilisates and resolved by native electrophoretic systems. Supercomplexes of respiratory complexes I, III and IV as well as multimeric forms of ATP synthase are well established. However, the involvement of complex II, linking respiratory chain with tricarboxylic acid cycle, in mitochondrial supercomplexes is questionable. Here we show that digitonin-solubilised complex II quantitatively forms high molecular weight structures (CII_hmw) that can be resolved by clear native electrophoresis. CII_hmw structures are enzymatically active and differ in electrophoretic mobility between tissues (500 – over 1000 kDa) and cultured cells (400–670 kDa). While their formation is unaffected by isolated defects in other respiratory chain complexes, they are destabilised in mtDNA-depleted, rho0 cells. Molecular interactions responsible for the assembly of CII_hmw are rather weak with the complexes being more stable in tissues than in cultured cells. While electrophoretic studies and immunoprecipitation experiments of CII_hmw do not indicate specific interactions with the respiratory chain complexes I, III or IV or enzymes of the tricarboxylic acid cycle, they point out to a specific interaction between CII and ATP synthase.     

Preferential Binding of Hot Spot Mutant p53 Proteins to Supercoiled DNA In Vitro and in Cells

Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed.     

Adverse responses of Cabera pusaria caterpillars to high dietary manganese concentration

The adaptation of insects to environmental changes can constitute a crucial factor in their development and activity. The response of Cabera pusaria L. (Lepidoptera: Geometridae) caterpillars to high manganese (Mn) concentrations in the diet was studied. Birch leaves were treated by dipping in MnCl₂·4H₂O solutions, thereby achieving Mn contents of 370 (T0), 695 (T1), 3 198 (T2), and 6 302 mg kg⁻¹ (T3). The reactions were determined by observing caterpillar mortality, development time, food consumption, and pupal weight. Manganese concentrations in larval excrement, pupae, and food were determined. Manganese in the diet at unnaturally increased concentrations caused great stress for caterpillars. All individuals in the treatment with the highest Mn concentration (T3) died during rearing and successful pupation occurred in only four individuals in T2. Even in the case of caterpillars from T1 (twofold higher than T0) a negative reaction (increased food consumption and prolonged development) was recorded. We also determined significantly increased Mn concentration in pupae from T1 (T2 and T3 were not included in this evaluation due to mortality) and excrement (T1‒T3) compared with T0 having a natural Mn concentration. Caterpillars were seen to eliminate negatively acting dietary Mn by its translocation to excrement. However, the highest mortality rate in T2 and T3 and negative reactions of individuals in T1 very likely demonstrate energy insufficiency and the high energy requirements of Mn elimination mechanisms.