Insecticidal resistance and cross-resistance in populations of Cydia pomonella (Lepidoptera: Tortricidae) in central Europe

Insecticide bioassays were used to investigate resistance of Cydia pomonella (L.) (Lepidoptera: Tortricidae) to insecticides with various types of active ingredients. The efficacy baselines of selected insect growth regulators (fenoxycarb), insect growth inhibitors (diflubenzuron and teflubenzuron), organophoshorous insecticides (phosalone), and neonicotinoids (thiacloprid) against the eggs and first and fifth instars of sensitive laboratory strains of codling moth were determined. According to concentration-mortality baseline, 50% lethality concentration values and 90% lethality concentration values were determined for all the tested insecticides. The lethal concentration ratio quantified the relation between the efficacy of selected insecticides against fifth instars found by topical application and against first instars found by diet-treated bioassay. No difference was detected when the efficacy of technical grade diflubenzuron diluted in tetrahydrofuran and diflubenzuron in the formulated product Dimilin 48 SC diluted in water was compared. However, just before the application of insecticide, the integument of larvae must be treated with acetone. Two bioassays were used to monitor the resistance of codling moths collected in 2003-2005 in two apple (Malus spp.) orchards with different intensities of chemical control. Resistance ratios (RRs) to the tested insecticides were determined for both field populations of codling moth. For the population of codling moth from a commercial apple orchard in Velké Bílovice, cross-resistance to fenoxycarb, teflubenzuron, and phosalone was detected after the topical application of insecticides to fifth instars. The population of codling moth from Prague-Ruzyne was slightly resistant to phosalone and teflubenzuron. No resistance to diflubenzuron was detected in either tested population.     

Effects of seawater ozonation on biofilm development in aquaculture tanks

Microbial biofilms developing in aquaculture tanks represent a reservoir for opportunistic bacterial pathogens, and procedures to control formation and bacterial composition of biofilms are important for the development of commercially viable aquaculture industries. This study investigated the effects of seawater ozonation on biofilm development on microscope glass slides placed in small-scale aquaculture tanks containing the live feed organism Artemia. Fluorescence in situ hybridization (FISH) demonstrated that ozonation accelerated the biofilm formation cycle, while it delayed the establishment of filamentous bacteria. Gammaproteobacteria and Alphaproteobacteria were the most abundant bacterial groups in the biofilm for both water types, but ozonation influenced their dynamics. With ozonation, the bacterial community structure was relatively stable and dominated by Gammaproteobacteria throughout the experiment (21–66% of total bacteria). Without ozonation, the community showed larger fluctuations, and Alphaproteobacteria emerged as dominant after 18 days (up to 54% of total bacteria). Ozonation of seawater also affected the dynamics of less abundant populations in the biofilm such as Betaproteobacteria, Planctomycetales and the Cytophaga/Flavobacterium branch of phylum Bacteroidetes. The abundance of Thiothrix, a bacterial genus capable of filamentous growth and fouling of larvae, increased with time for both water types, while no temporal trend could be detected for the genus Vibrio. Denaturing gradient gel electrophoresis (DGGE) demonstrated temporal changes in the dominant bacterial populations for both water types. Sequencing of DGGE bands confirmed the FISH data, and sequences were related to bacterial groups commonly found in biofilms of aquaculture systems. Several populations were closely related to organisms involved in sulfur cycling. Improved Artemia survival rates in tanks receiving ozonated water suggested a positive effect of ozonation on animal health. Although the used ozonation protocol did not hinder biofilm formation, the results suggest ozonation as a promising approach for manipulation of bacterial populations in aquaculture systems, which can prove beneficial for cultured animals.     

The application of multi-parameter flow cytometry to the study of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> batch fermentation processes

Multi-parameter flow cytometric techniques coupled with dual colour fluorescent staining were used to study the physical and metabolic consequences of inclusion body formation in batch cultures of the recombinant Escherichia coli strain MSD3735. This strain contains a plasmid coding for the isopropylthiogalactopyranoside-inducible model eukaryotic protein AP50. It is known that the synthesis of foreign proteins at high concentrations can exert a severe metabolic stress on the host cell and that morphological changes can occur. In this work, using various points of induction, it was shown that inclusion body formation is followed immediately by measurable changes in the characteristic intrinsic light scatter patterns for the individual cell (forward scatter, 90° side scatter) and a concomitant progressive change in the individual cell physiological state with respect to both cytoplasmic membrane polarisation and permeability. This work establishes flow cytometry as a potentially valuable tool for monitoring recombinant fermentation processes, providing important information for scale-up. Further, we discuss the possibility of optimising inclusion body formation by manipulating the fermentation conditions based on these rapid real-time measurements.     

Molecular ecology of nifH genes and transcripts in the eastern Mediterranean Sea

The eastern Mediterranean Sea is one of the most extreme oligotrophic oceanic regions on earth in terms of nutrient concentrations and primary productivity. Nitrogen fixation has been suggested to contribute to the high N : P molar ratios of approximately 28:1 found in this region. Surprisingly, no molecular biological work has been performed in situ to assess whether N(2) fixation genes actually occur in the eastern Mediterranean Sea, or to determine which organisms are responsible for this process. In this study, we examined the presence and expression of nitrogenase genes (nifH) in the upper water layer of the eastern Mediterranean. Clone libraries constructed from both DNA and reverse-transcribed PCR-amplified mRNA were examined and compared. We observed different nifH genes from diverse microbial groups, such as Cyanobacteria, Proteobacteria and methanogenic Archaea. Interestingly, numerous phylotypes were observed in coastal stations at the DNA level but none were active. However, in far offshore stations, the phylotypes observed at the DNA level were the ones that were actually active. Our preliminary study revealed diverse diazotrophs that possess and express nifH genes, which may support N(2) fixation in the eastern Mediterranean Sea.     

X-Ray Crystal Structure of a Highly Functionalized Thiophene as a New Backbone Amide Linker for Solid-phase Peptide Synthesis. Relationship between Crystal Structure and Reactivity

The development of new linkers (handles) for solid-phase synthesis provides new chemical opportunities for peptide synthesis. To understand the chemical properties of a recently developed backbone amide linker from a structural perspective, the crystal structure of S-((5-formyl-3,4-ethylenedioxy)thiophene-2-yl)-3-thiopropionic acid (T-BAL2) was studied. Specifically, we wished to address whether this highly substituted thiophene retained planarity in the aromatic ring as well as between the aromatic ring and the aldehyde carbonyl. Furthermore, we sought an explanation for the relatively low reactivity in reductive aminations of the thienylaldehyde with amines in solution and on solid phase. Based on the crystal structure of T-BAL2, the thienyl-C (aldehyde) and C–O (aldehyde) bond lengths were applied as measures for the electron-deficiency (electrophilicity) of the aldehyde and compared to similar bond lengths found in previously reported formylated homo- and hetero-aromatic systems, which show significantly higher reactivity towards imine formation. The bond lengths found in the present structure are in accordance with normal C–C single bond and C–O double bond lengths. The high similarity in aldehyde bond lengths in the present system and in the reported systems indicates similar electron distribution in these systems. The lower reactivity of the present system may therefore not be attributed to electronic factors.     

Predominant <Emphasis Type="Italic">Bacillus</Emphasis> spp. in Agricultural Soil under Different Management Regimes Detected via PCR-DGGE

A PCR system for studying the diversity of species of Bacillus and related taxa directly from soil was developed. For this purpose, a specific 24-bp forward primer located around position 110 of the 16S ribosomal RNA gene was designed and combined with a reverse bacterial primer located at the end of the gene. The specificity of this PCR system for bacilli and related taxons was confirmed on the basis of tests with diverse strains as well as with soil DNA. Analysis of a soil DNA derived clone library showed that the amplified fragments affiliated exclusively with sequences of gram-positive bacteria, with up to 95% of the sequences originating from putative Bacillus species. In particular, sequences affiliated to those of B. mycoides, B. pumilus, B. megaterium, B. thuringiensis, and B. firmus, as well as to related taxa such as Paenibacillus, were obtained. A minority, i.e., less than 6%, of the clones affiliated with other gram-positive bacteria, such as Arthrobacter spp., Frankia spp., and uncultured gram-positives. The amplified fragments were used as templates for a second PCR using bacterial 16S rDNA primers, yielding PCR products of about 410 bp, which were separated by denaturing gradient gel electrophoresis (DGGE). Amplicons indicating Bacillus spp. were found in the gel between 45% and roughly 60% denaturant, whereas those representing other, high-G+C% bacteria, were localized in gel regions with denaturant concentrations exceeding about 60%, thus allowing the distinction between these two groups of sequences. We applied this system to compare the group-specific diversity in bacterial communities in an agricultural soil under different regimes, i.e., permanent grassland, grassland recently turned to arable land, and arable land under agricultural rotation. Differences in the Bacillus-related community structures between the treatments were clearly detected. Higher diversities, as judged by Shannon–Weaver indices calculated on the basis of the molecular profiles, were consistently observed in the permanent grassland and the grassland turned into arable land, as compared to the arable land.     

Ultrastructural studies on diurnal variations in mitochondria in various regions of the hepatic lobule

Stereologic studies on diurnal variations in ultrastructural manifestations of hepatic mitochondrial function in various regions of the hepatic lobules were performed. The experiments were performed on Wistar rats in May when the liver shows the maximal content of glycogen. The materials for the studies were taken at 8 am. and 10 p.m. The stereologic examinations were done according to the method of Weibel et al. The results obtained may suggest a conversion of the orthodox hepatic mitochondria to the transitional state, that is during the glycogen minimum in the rats in May. In the morning, when the glycogen deposits increased, the pericentral mitochondria are in the orthodox state. In the evening glycogen consumption due to glycogenolysis was accompanied by conversion of the mitochondria from the orthodox state to the transitional ones, which may suggest their increased metabolic activity.     

Biological activity of hydroxyanthraquinones and their glucosides toward microorganisms

Five mono- and dihydroxyanthraquinones as well as 12 of their glucosides (both free and acetylated) were tested with six different microbial species using the plate-diffusion method. None of the tested substances was active againstEscherichia coli, 15 of the 17 substances displayed an activity towardBacillus subtilis, Bacillus cereus, Candida albicans, Saccharomyces cerevisiae andStreptomyces aurecfaciens. Relationships between the substance type and biological activity are discussed.     

Genetic and environmental influences of surfactant protein D serum levels

The collectin surfactant protein D (SP-D) is an important component of the pulmonary innate immune system, but SP-D is also present on extrapulmonary epithelial surfaces and in serum, where it has been used as a biomarker for pulmonary disease states. In this study, we investigate the mechanisms defining the constitutional serum level of SP-D and determine the magnitude of the genetic contribution to serum SP-D in the adult population. Recent studies have demonstrated that serum SP-D concentrations in children are genetically determined and that a single nucleotide polymorphism (SNP) located in the NH(2)-terminal region (Met11Thr) of the mature protein is significantly associated with the serum SP-D levels. A classic twin study was performed on a twin population including 1,476 self-reported healthy adults. The serum SP-D levels increased with male sex, age, and smoking status. The intraclass correlation was significantly higher for monozygotic (MZ) twin pairs than for dizygotic (DZ) twin pairs. Serum SP-D variance was influenced by nonshared environmental effects and additive genetic effects. Multivariate analysis of MZ and DZ covariance matrixes showed significant genetic correlation among serum SP-D and metabolic variables. The Met11Thr variant explained a significant part of the heritability indicating that serum SP-D variance could be decomposed into non-shared environmental effects (e(2) = 0.19), additive genetic effects (h(2) = 0.42), and the effect of the Met11Thr variations (q(2) = 0.39).     

Effects of the Commercial Flame Retardant Mixture DE-71 on Cytokine Production by Human Immune Cells

IntroductionAlthough production of polybrominated diphenyl ethers (PBDEs) is now banned, release from existing products will continue for many years. The PBDEs are assumed to be neurotoxic and toxic to endocrine organs at low concentrations. Their effect on the immune system has not been investigated thoroughly. We aimed to investigate the influence of DE-71 on cytokine production by peripheral blood mononuclear cells (PBMCs) stimulated with Escherichia Coli lipopolysaccharide (LPS) or phytohaemagglutinin-L (PHA-L).ResultsAt non-cytotoxic concentrations (0.01–10 μg/mL), DE-71 significantly enhanced secretion of IL-1β, IL-6, CXCL8, IL-10, and TNF-α (p<0.001–0.019; n = 6) from LPS-stimulated PBMCs. IFN-γ, TNF-α, IL-17A, and IL-17F (p = <0.001–0.043; n = 6) secretion were enhanced from PHA-L-stimulated PBMCs as well. Secretion of IL-1β, IL-2, IL-10, IL-8 and IL-6 was not significantly affected by DE-71.ConclusionsWe demonstrate an enhancing effect of DE-71 on cytokine production by normal human PBMCs stimulated with LPS or PHA-L ex vivo.