Candidate natural enemies for control of Rhizoglyphus robini Claparède (Acari: Astigmata) in lily bulbs: exploration in the field and pre-selection in the laboratory

To find suitable candidates for biological control of the bulb mite, Rhizoglyphus robini Claparède (Acari: Astigmata) on lilies, exploration was undertaken in areas where the bulb mite is an established pest (The Netherlands, Taiwan and Japan). Among the predators, found in association with R. robini in the field and under storage conditions, mesostigmatic mites predominate. The most abundant species were Hypoaspis aculeifer (Canestrini), Lasioseius bispinosus Evans and Parasitus fimetorum (Berlese). These predators appeared to feed and reproduce on a diet of exclusively R. robini and they were able to control the bulb mite in small-scale population experiments initiated with a 1:20 predator-prey ratio. Under laboratory conditions corresponding to lily bulb propagation (lily scales mixed with vermiculite and stored at 23°C and >90% RH) the laelapid mite, H. aculeifer, was the most effective predator; the ascid predator, L. bispinosus, was much less effective, but being relatively small and being successful in attacking the juvenile stages of the bulb mite it may be better able to search for bulb mites hidden inside the lily bulb. The parasitid predator, P. fimetorum, failed to control the bulb mite when vermiculite was used as a medium, but turned out to suppress this prey when peat was used instead. Various strains of H. aculeifer or closely related species were compared with respect to their impact and performance on bulb mites as prey: two Dutch strains, one obtained from Breezand and the other from 'tZand, a Taiwanese strain, a German strain that in contrast to the previously mentioned strains was not collected from lily bulbs, but from agricultural areas near Bremen and, in addition, a Canadian strain of a related species (Hypoaspis miles Berlese), known to control sciarid fly larvae. These comparative experiments showed that H. miles died out without noticeable impact on the bulb mite population whereas all strains of H. aculeifer were able to suppress the bulb mites to very low numbers. However, the numerical responses of the H. aculeifer strains differed in that those collected in association with the pest (Breezand > Taiwan > 'tZand) were superior to the strain from Bremen. These results do not provide support to the Hokkanen and Pimentel hypothesis, which states that predators forming an evolutionary new association with the pest are often more effective in biological control.     

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.     

Genetic microheterogeneity and phenotypic variation of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> arginase in clinical isolates

Background  

Clinical isolates of the gastric pathogen Helicobacter pylori display a high level of genetic macro- and microheterogeneity, featuring a panmictic, rather than clonal structure. The ability of H. pylori to survive the stomach acid is due, in part, to the arginase-urease enzyme system. Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea, and urease hydrolyzes urea to carbon dioxide and ammonium, which can neutralize acid.     

Speckled-like Pattern in the Germinal Center (SLIP-GC), a Nuclear GTPase Expressed in Activation-induced Deaminase-expressing Lymphomas and Germinal Center B Cells

We identified a novel GTPase, SLIP-GC, with expression limited to a few tissues, in particular germinal center B cells. It lacks homology to any known proteins, indicating that it may belong to a novel family of GTPases. SLIP-GC is expressed in germinal center B cells and in lymphomas derived from germinal center B cells such as large diffuse B cell lymphomas. In cell lines, SLIP-GC is expressed in lymphomas that express activation-induced deaminase (AID) and that likely undergo somatic hypermutation. SLIP-GC is a nuclear protein, and it localizes to replication factories. Reduction of SLIP-GC levels in the Burkitt lymphoma cell line Raji and in non-Hodgkin lymphoma cell lines resulted in an increase in DNA breaks and apoptosis that was AID-dependent, as simultaneous reduction of AID abrogated the deleterious effects of SLIP-GC reduction. These results strongly suggest that SLIP-GC is a replication-related protein in germinal center B cells whose reduction is toxic to cells through an AID-dependent mechanism.The germinal center (GC)³ is a transient structure formed during T-dependent B cell responses wherein B cell affinity maturation to a specific antigen occurs, leading to the formation of high affinity memory B cells (1–3). Many features of this reaction are unique in biology such as the somatic hypermutation (SHM) of immunoglobulin (Ig) genes, the genetic rearrangement of the constant domains in class switch recombination to generate B cells bearing receptors of downstream isotypes such as IgG, IgE, and IgA, and the cellular selection process that recruits high affinity variants generated via SHM. In SHM the variable (V) regions of the heavy and light chain loci of Ig genes undergo a directed process of hypermutation where base substitutions accumulate, particularly in regions encoding the antigen binding pockets of the B cell receptor. The molecular basis for SHM is not fully understood, but it is known to be triggered by a cytosine deaminase, AID (4, 5). However, it is clear that novel factors are yet to be discovered in SHM. For example, AID alone is not sufficient for proper targeting to the Ig locus, and it is likely that a novel factor targets AID to the Ig locus (6). In addition, AID-mediated deamination of cytosines explains only mutations at G:C base pairs, yet mutations at A:T base pairs occur at approximately the same rate as G:C mutations. Although A:T mutations have been linked to the activities of the mismatch repair (MMR) proteins MSH/MSH6 and the error-prone DNA polymerase η, hypermutating Burkitt lymphoma cell lines have intact MMR and polymerase η, yet mutations at A:T base pairs are markedly reduced (7). The class switch recombination reaction is also only partly understood. Targeting of AID, the DNA substrate subjected to AID deamination, and the subsequent DNA breaks and their repair also remain only partially defined for class switch recombination. Finally, it remains unclear how these reactions are coordinated in the GC environment with both cellular selection for increased affinity to foreign antigen and tolerance mechanisms to prevent or minimize autoreactivity acquired during hypermutation that can lead to high affinity pathogenic IgG antibodies (8, 9). Clearly, efforts to understand these mechanisms and to identify novel proteins that contribute to this unique environment are needed.To identify proteins that may contribute to SHM or other aspects of the GC reaction, we mined expression libraries generated by the I.M.A.G.E. Consortium (10) through informatics tools in the Cancer Genome Anatomy Group website (11). Given that BCL6 is a critical protein for the GC reaction (12, 13), we pooled libraries derived from GC B cells with BCL6 expression and compared them to all other libraries (see Fig. 1A for the scheme). This strategy led us to the discovery of a novel protein, SLIP-GC (speckled-like pattern in the germinal center), expressed in GC B cells, and its expression profile was similar to that of AID. Subsequent experiments showed that this protein is expressed in GC B cells and localizes to replication factories in the nucleus and when reduced in AID⁺ lymphoma cell lines results in an increase in DNA breaks and in cell death. These studies reveal SLIP-GC to be a novel factor that likely contributes to the unique reactions in GCs. The data also suggest that SLIP-GC reduction is toxic to B cells through an AID-mediated mechanism.Open in a separate windowFIGURE 1.Identification of a novel GTPase expressed in germinal center B cells. A, Shown is a schematic representation of the method used to identify novel proteins primarily expressed in germinal center B cells by mining EST libraries. B, shown is the amino acid sequence of SLIP-GC. Italic boldface motifs are potential nuclear localization signals. The underlined boldfaced motif is a GTPase motif (P-loop), whereas the underlined italic sequence near the C terminus is the coiled-coil region. C, shown is a representative graph of GTPase assay of immunoprecipitated SLIP-GC from lipopolysaccharide-activated and unstimulated B cells. CRL-2289 is a cell line with endogenous SLIP-GC. As a positive control, RhoA, a ubiquitous GTPase, was immunoprecipitated with specific antibodies and tested the same way as SLIP-GC. We also performed GTPase assays on CRL-2631, which does not express SLIP-GC. Accordingly, a Coomassie Blue gel of immunoprecipitated SLIP-GC only shows a SLIP-GC band in the CRL-2289 extracts (data not shown), and SLIP-GC GTPase activity in lipopolysaccharide (LPS)-activated CRL-2631 was negligible, whereas RhoA GTPase activity was high (SLIP-GC, 0.36 nmol GDP/min; RhoA, 28.48 nmol GDP/min). The assay was done at least two times.     

Genetic differentiation in the urban habitat: the great tits (Parus major) of the parks of Barcelona city

The increase of urban areas has led to a fragmentation of habitats for many forest‐living species. Man‐made parks might be a solution, but they can also act as sinks that are unable to maintain themselves without immigration from natural areas. Alternatively, parks might act as true metapopulations with extinctions and colonizations. In both cases, we can expect genetic variation to be reduced in the parks compared to the natural habitat. A third alternative is that the parks have sufficient reproduction to maintain themselves. To test these hypotheses, we analysed the pattern of genetic variation in the great tit (Parus major) in 12 parks in central Barcelona, and in an adjacent forest population using microsatellites. Genetic variation was not lower in the parks compared to the forest population, but larger, and gene flow was higher from the town to the forest compared to vice versa. We found a significant genetic differentiation among the parks, with a structure that only partly reflected the geographic position of the parks. Relatedness among individuals within parks was higher than expected by chance, although we found no evidence of kin groups. Assignment tests suggest that some parks are acting as net donors of individuals to other parks. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 9–19.     

Variation in seedling growth of 11 perennial legumes in response to phosphorus supply

Phosphorus (P) deficiency is a major problem for Australian agriculture. Development of new perennial pasture legumes that acquire or use P more efficiently than the current major perennial pasture legume, lucerne (Medicago sativa L.), is urgent. A glasshouse experiment compared the response of ten perennial herbaceous legume species to a series of P supplies ranging from 0 to 384 µg g^?1 soil, with lucerne as the control. Under low-P conditions, several legumes produced more biomass than lucerne. Four species (Lotononis bainesii Baker, Kennedia prorepens F.Muell, K. prostrata R.Br, Bituminaria bituminosa (L.) C.H.Stirt) achieved maximum growth at 12 µg P g^?1 soil, while other species required 24 µg P g^?1. In most tested legumes, biomass production was reduced when P supply was ≥192 µg g^?1, due to P toxicity, while L. bainesii and K. prorepens showed reduced biomass when P was ≥24 µg g^?1 and K. prostrata at ≥48 µg P g^?1 soil. B. bituminosa and Glycine canescens F.J.Herm required less soil P to achieve 0.5 g dry mass than the other species did. Lucerne performed poorly with low P supply and our results suggest that some novel perennial legumes may perform better on low-P soils.     

Helicobacter pylori thioredoxin is an arginase chaperone and guardian against oxidative and nitrosative stresses

The gastric human pathogen Helicobacter pylori faces formidable challenges in the stomach including reactive oxygen and nitrogen intermediates. Here we demonstrate that arginase activity, which inhibits host nitric oxide production, is post-translationally stimulated by H. pylori thioredoxin (Trx) 1 but not the homologous Trx2. Trx1 has chaperone activity that renatures urea- or heat-denatured arginase back to the catalytically active state. Most reactive oxygen and nitrogen intermediates inhibit arginase activity; this damage is reversed by Trx1, but not Trx2. Trx1 and arginase equip H. pylori with a "renox guardian" to overcome abundant nitrosative and oxidative stresses encountered during the persistence of the bacterium in the hostile gastric environment.     

Distribution of Carboxylates and Acid Phosphatase and Depletion of Different Phosphorus Fractions in the Rhizosphere of a Cereal and Three Grain Legumes

This study investigates the distribution of carboxylates and acid phosphatases as well as the depletion of different phosphorus (P) fractions in the rhizosphere of three legume crop species and a cereal, grown in a soil with two different levels of residual P. White lupin (Lupinus albus L.), field pea (Pisum sativum L.), faba bean (Vicia faba L.) and spring wheat (Triticum aestivum L.) were grown in small sand-filled PVC tubes to create a dense root mat against a 38-μm mesh nylon cloth at the bottom, where it was in contact with the soil of interest contained in another tube. The soil had either not been fertilised (P0) or fertilised with 15 (P15) kg P ha⁻¹ in previous years. The mesh size did not allow roots to grow into the soil, but penetration of root hairs and diffusion of nutrients and root exudates was possible, and a rhizosphere was established. At harvest, thin (1 mm) slices of this rhizosphere soil were cut, down to a 10-mm distance from the mesh surface. The rhizosphere of white lupin, particularly in the P0 treatment, contained citrate, mostly in the first 3 mm, with concentrations decreasing with distance from the root. Acid phosphatase activity was enhanced in the rhizosphere of all species, as compared with bulk soil, up to a distance of 4 mm. Phosphatase activity was highest in the rhizosphere of white lupin, followed by faba bean, field pea and wheat. Both citrate concentrations and phosphatase activities were higher in P0 compared with P15. The depletion of both inorganic (P_i) and organic (P_o) phosphorus fractions was greatest at the root surface, and decreased gradually with distance from the root. The soil P fractions that were most depleted as a result of root activity were the bicarbonate-extractable (0.5 M) and sodium hydroxide-extractable (0.1 M) pools, irrespective of plant species. This study suggests that differences among the studied species in use of different P pools and in the width of the rhizosphere are relatively small.     

Competitive environments induce shifts in host fidelity

Recent models support the idea of sympatric speciation as a result of the joint effects of disruptive selection and assortative mating. We present experimental data, testing models of speciation through frequency‐dependent selection. We show that under high competition on a mixture of resources/hosts, strains of the Seed beetle, Callosobruchus maculatus, change their host fidelity and evolve a more generalistic behaviour in resource utilization among females. The change in host fidelity did not result in disruptive selection and was not followed by assortative mating. This means that only one of three fundamental prerequisites for sympatric speciation evolved as a result of the frequency‐dependent selection. We conclude that for this process to work, a shift to a novel food resource as a result of selection must also lead to a loss of preference for the original resource such that individuals are only able to use either one of the two.     

Local selection modifies phenotypic divergence among Rana temporaria populations in the presence of gene flow

In ectotherms, variation in life history traits among populations is common and suggests local adaptation. However, geographic variation itself is not a proof for local adaptation, as genetic drift and gene flow may also shape patterns of quantitative variation. We studied local and regional variation in means and phenotypic plasticity of larval life history traits in the common frog Rana temporaria using six populations from central Sweden, breeding in either open‐canopy or partially closed‐canopy ponds. To separate local adaptation from genetic drift, we compared differentiation in quantitative genetic traits (Q_ST) obtained from a common garden experiment with differentiation in presumably neutral microsatellite markers (F_ST). We found that R. temporaria populations differ in means and plasticities of life history traits in different temperatures at local, and in F_ST at regional scale. Comparisons of differentiation in quantitative traits and in molecular markers suggested that natural selection was responsible for the divergence in growth and development rates as well as in temperature‐induced plasticity, indicating local adaptation. However, at low temperature, the role of genetic drift could not be separated from selection. Phenotypes were correlated with forest canopy closure, but not with geographical or genetic distance. These results indicate that local adaptation can evolve in the presence of ongoing gene flow among the populations, and that natural selection is strong in this system.