Oviposition preferences of Maculinea alcon as influenced by aphid (Aphis gentianae) and fungal (Puccinia gentianae) infestation of larval host plants

Abstract 1. The influence of infestation of the larval host plant Gentiana cruciata on the egg‐laying preferences of the xerophilous ecotype of Alcon Blue butterfly (Maculinea alcon) was studied in a semi‐dry grassland area (Aggtelek Karst Region, Northern Hungary). 2. We examined whether oviposition patterns of females differed when G. cruciata stems were uninfested compared with when they were infested by an aphid (Aphis gentianae) or a rust (Puccinia gentianae) species. 3. Females laid more than 90% of their eggs on fertile, uninfested G. cruciata stems, although these stems comprised only ～ 50% of the total stems available. Stems infested by aphids were similar to uninfested ones in properties that had a strong correlation with egg numbers, and yet there were significantly fewer eggs on infested stems than on intact ones. 4. Females never laid eggs on parts of Gentiana stems infested by aphids, and the presence of Lasius paralienus ants, which have a mutualistic interaction with Aphis gentianae, did not increase the repulsive effect of aphids. Infection of Gentiana by Puccinia did not influence the egg‐laying behaviour of females, even though the flowers and buds of infested stems exhibited a delayed development. 5. Aphid infestation can influence butterfly oviposition patterns through both direct and indirect effects. The presence of aphids directly excluded oviposition, but our data also indicated the possibility of an indirect effect of aphid infestation. Stems that had no aphids at the last egg counting, but were infested prior to it, had significantly fewer eggs than those that were never infested.     

Recolonization history and large-scale dispersal in the open sea: the case study of the North Atlantic cod, Gadus morhua L.

Most studies of the genetic structure of Atlantic cod have focused on small geographical scales. In the present study, the genetic structure of cod sampled on spawning grounds in the North Atlantic was examined using eight microsatellite loci and the Pan I locus. A total of 954 cod was collected from nine different regions: the Baltic Sea, the North Sea, the Celtic Sea, the Irish Sea and Icelandic waters during spring 2002 and spring 2003, from Norwegian waters and the Faroe Islands (North and West spawning grounds) in spring 2003, and from Canadian waters in 1998. Temporal stability among spawning grounds was observed in Icelandic waters and the Celtic Sea, and no significant difference was observed between the samples from the Baltic Sea and between the samples from Faroese waters. F -statistics showed significant differences between most populations and a pattern of isolation-by-distance was described with microsatellite loci. The Pan I locus revealed the presence of two genetically distinguishable basins, the North-west Atlantic composed of the Icelandic and Canadian samples and the North-east Atlantic composed of all other samples. Permutation of allele sizes at each microsatellite locus among allelic states supported a mutational component to the genetic differentiation, indicating a historical origin of the observed variation. Estimation of the time of divergence was approximately 3000 generations, which places the origin of current genetic pattern of cod in the North Atlantic in the late Weichselian (Wisconsinian period), at last glacial maximum.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 315–329.     

Separation of cellular nonpolar neutral lipids by normal-phase chromatography and analysis by electrospray ionization mass spectrometry

Neutral lipids are an important class of hydrophobic compounds found in all cells that play critical roles from energy storage to signal transduction. Several distinct structural families make up this class, and within each family there are numbers of individual molecular species. A solvent extraction protocol has been developed to efficiently isolate neutral lipids without complete extraction of more polar phospholipids. Normal-phase HPLC was used for the separation of cholesteryl esters (CEs), monoalkylether diacylglycerols, triacylglycerols, and diacylglycerols in a single HPLC run from this extract. Furthermore, minor lipids such as ubiquinone-9 could be detected in RAW 264.7 cells. Molecular species that make up each neutral lipid class can be analyzed both qualitatively and quantitatively by on-line LC-MS and LC-MS/MS strategies. The quantitation of >20 CE molecular species revealed that challenging RAW 264.7 cells with a Toll-like receptor 4 agonist caused a >20-fold increase in the content of CEs within cells, particularly those CE molecular species that contained saturated (14:0, 16:0, and 18:1) fatty acyl groups. Longer chain CE molecular species did not change in response to the activation of these cells.     

Application of TILLING and EcoTILLING as Reverse Genetic Approaches to Elucidate the Function of Genes in Plants and Animals

With the fairly recent advent of inexpensive, rapid sequencing technologies that continue to improve sequencing efficiency and accuracy, many species of animals, plants, and microbes have annotated genomic information publicly available. The focus on genomics has thus been shifting from the collection of whole sequenced genomes to the study of functional genomics. Reverse genetic approaches have been used for many years to advance from sequence data to the resulting phenotype in an effort to deduce the function of a gene in the species of interest. Many of the currently used approaches (RNAi, gene knockout, site-directed mutagenesis, transposon tagging) rely on the creation of transgenic material, the development of which is not always feasible for many plant or animal species. TILLING is a non-transgenic reverse genetics approach that is applicable to all animal and plant species which can be mutagenized, regardless of its mating / pollinating system, ploidy level, or genome size. This approach requires prior DNA sequence information and takes advantage of a mismatch endonuclease to locate and detect induced mutations. Ultimately, it can provide an allelic series of silent, missense, nonsense, and splice site mutations to examine the effect of various mutations in a gene. TILLING has proven to be a practical, efficient, and an effective approach for functional genomic studies in numerous plant and animal species. EcoTILLING, which is a variant of TILLING, examines natural genetic variation in populations and has been successfully utilized in animals and plants to discover SNPs including rare ones. In this review, TILLING and EcoTILLING techniques, beneficial applications and limitations from plant and animal studies are discussed.Key Words: Reverse genetics, functional genomics, TILLING (target induced local lesions in genomes), EcoTILLING (Ecotype TILLING), sequencing, SNP (single nucleotide polymorphism), genetic stocks.     

Mass spectrometry advances in lipidomica: collision-induced decomposition of Kdo2-lipid A

Mass spectrometry has made significant advances in the analysis of lipid substances, both simple and complex present in extracts of eukaryotic and prokaryotic cells. The development of the ionization techniques of electrospray ionization and matrix-assisted laser desorption ionization (MALDI) have both been applied to the analysis of lipids. The example of the types of structural information that can be obtained from MALDI-TOF tandem mass spectrometry is exemplified by the analysis of Kdo2-lipid A, a complex lipopolysaccharide known to activate toll-like 4 receptors on mammalian cells. Analysis of Kdo2-lipid A obtained from an Escherichia coli WBB06 was found to generate an abundant [M-H]- ion at m/z 2236.4 and a more abundant carbon-13 isotope at m/z 2237.4. Furthermore, collisional activation of the lipid A portion of the molecule at m/z 1796.3 resulted in a series of ions corresponding to the loss of all four fatty acyl groups as neutral carboxylic acids. An altogether different challenge of mass spectrometry applied to the area of lipid analysis is that of quantitative analysis. Two rather different requirements have emerged. One with high precision and accuracy for the measurement of relatively few lipid species that are produced at very low concentrations and typically interact with specific receptor proteins. A rather different challenge is that for the analysis of abundant lipid classes, which are composed of multiple molecular species that can approach several hundred under certain circumstances.     

Catalytic mechanism of Escherichia coli isopentenyl diphosphate isomerase involves Cys-67, Glu-116, and Tyr-104 as suggested by crystal structures of complexes with transition state analogues and irreversible inhibitors

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase is a key enzyme in the biosynthesis of isoprenoids. The reaction involves protonation and deprotonation of the isoprenoid unit and proceeds through a carbocationic transition state. Analysis of the crystal structures (2 A) of complexes of Escherichia coli IPP.DMAPPs isomerase with a transition state analogue (N,N-dimethyl-2-amino-1-ethyl diphosphate) and a covalently attached irreversible inhibitor (3,4-epoxy-3-methyl-1-butyl diphosphate) indicates that Glu-116, Tyr-104, and Cys-67 are involved in the antarafacial addition/elimination of protons during isomerization. This work provides a new perspective about the mechanism of the reaction.     

Identification of an Archaeal type II isopentenyl diphosphate isomerase in methanothermobacter thermautotrophicus

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in Streptomyces sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon Methanothermobacter thermautotrophicus encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in Salmonella enterica serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (idi) and 1-deoxy-D-xylulose 5-phosphate (dxs). The dxs gene was interrupted with a synthetic operon containing the Saccharomyces cerevisiae genes erg8, erg12, and erg19 allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His6-tagged M. thermautotrophicus type II IPP isomerase was produced in Escherichia coli and purified by Ni2+ chromatography. The purified protein was characterized by matrix-assisted laser desorption ionization mass spectrometry. The enzyme has optimal activity at 70 degrees C and pH 6.5. NADPH, flavin mononucleotide, and Mg2+ are required cofactors. The steady-state kinetic constants for the archaeal type II IPP isomerase from M. thermautotrophicus are as follows: K(m), 64 microM; specific activity, 0.476 micromol mg(-1) min(-1); and k(cat), 1.6 s(-1).     

Baseline Levels of Potentially Toxic Elements in Pampas Soils

The soils of the Pampas are thought to be generally non-contaminated but there is growing evidence of trace element accumulation at some specific sites. The goal of this study was to measure the current levels of the main Potentially Toxic Elements (PTE) in the top horizon and in specific soil profiles so that we would establish the baseline concentrations of these elements. Eighty-eight top soils and three soil profiles were sampled. The samples were acid digested. Arsenic, boron, barium, cadmium, cobalt, chromium, copper, lead, manganese, mercury, molybdenum, nickel, silver, selenium and zinc were determined with inductively coupled argon plasma emission spectrometry (ICPES).

All of the values found are within the normal range for uncontaminated soils as reported from several continents. Elements with high environmental risk potential are lower than the admissible range of the European Union and some of them are orders of magnitude lower than those of the United States Environmental Protection Agency (US-EPA) 501 levels. Potentially Toxic Elements contents increased with depth or showed a maximum concentration at the B2 horizon. This is related to the parent material and the pedogenetic processes but not to recent contamination. Soil profiles showed higher concentrations of PTE in clayey horizons. However, these relationships did not appear in top soil samples in any soil Great Group studied. The shown data establishes a baseline for PTE concentrations for Pampas soils.     

Solution conformation of EcoRI restriction endonuclease changes upon binding of cognate DNA and Mg2+ cofactor

EcoRI endonuclease has two tryptophans at positions 104 and 246 on the protein surface. A single tryptophan mutant containing Trp246 and a single cysteine labeling site at the N-terminus was used to determine the position of the N-terminus in the protein structure. The N-termini of EcoRI endonuclease are essential for tight binding and catalysis yet are not resolved in any of the crystal structures. Resonance energy transfer was used to measure the distance from Trp246 donor to IAEDANS or MIANS acceptors at Cys3. The distance is 36 A in apoenzyme, decreasing to 26 A in the DNA complex. Molecular modeling suggests that the N-termini are located at the dimer interface formed by the loops comprising residues 221-232. Protein conformational changes upon binding of cognate DNA and cofactor Mg(2+) were monitored by tryptophan fluorescence of the single tryptophan mutant and wild-type endonuclease. The fluorescence decay of Trp246 is a triple exponential with lifetimes of 7, 3.5, and 0.7 ns. The decay-associated spectra of the 7- and 3.5-ns components have emission maxima at approximately 345 and approximately 338 nm in apoenzyme, which shift to approximately 340 and approximately 348 nm in the DNA complex. The fluorescence quantum yield of the single tryptophan mutant drops 30% in the DNA complex, as compared to 10% for wild-type endonuclease. Fluorescence changes of Trp104 upon binding of DNA were inferred by comparison of the decay-associated spectra of wild type and single tryptophan mutant. Fluorescence changes are related to changes in proximity and orientation of quenching functional groups in the tryptophan microenvironments, as seen in the crystal structures.