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Systemic insecticides are applied to plants using a wide variety of methods, ranging from foliar sprays to seed treatments and soil drenches. Neonicotinoids and fipronil are among the most widely used pesticides in the world. Their popularity is largely due to their high toxicity to invertebrates, the ease and flexibility with which they can be applied, their long persistence, and their systemic nature, which ensures that they spread to all parts of the target crop. However, these properties also increase the probability of environmental contamination and exposure of nontarget organisms. Environmental contamination occurs via a number of routes including dust generated during drilling of dressed seeds, contamination and accumulation in arable soils and soil water, runoff into waterways, and uptake of pesticides by nontarget plants via their roots or dust deposition on leaves. Persistence in soils, waterways, and nontarget plants is variable but can be prolonged; for example, the half-lives of neonicotinoids in soils can exceed 1,000 days, so they can accumulate when used repeatedly. Similarly, they can persist in woody plants for periods exceeding 1 year. Breakdown results in toxic metabolites, though concentrations of these in the environment are rarely measured. Overall, there is strong evidence that soils, waterways, and plants in agricultural environments and neighboring areas are contaminated with variable levels of neonicotinoids or fipronil mixtures and their metabolites (soil, parts per billion (ppb)-parts per million (ppm) range; water, parts per trillion (ppt)-ppb range; and plants, ppb-ppm range). This provides multiple routes for chronic (and acute in some cases) exposure of nontarget animals. For example, pollinators are exposed through direct contact with dust during drilling; consumption of pollen, nectar, or guttation drops from seed-treated crops, water, and consumption of contaminated pollen and nectar from wild flowers and trees growing near-treated crops. Studies of food stores in honeybee colonies from across the globe demonstrate that colonies are routinely and chronically exposed to neonicotinoids, fipronil, and their metabolites (generally in the 1–100 ppb range), mixed with other pesticides some of which are known to act synergistically with neonicotinoids. Other nontarget organisms, particularly those inhabiting soils, aquatic habitats, or herbivorous insects feeding on noncrop plants in farmland, will also inevitably receive exposure, although data are generally lacking for these groups. We summarize the current state of knowledge regarding the environmental fate of these compounds by outlining what is known about the chemical properties of these compounds, and placing these properties in the context of modern agricultural practices.
Neonicotinoids are subjected to vigilance because of environmental contaminations and deleterious effects on bees. Imidacloprid (IMI) is one of the most representative insecticides of this family. At chronic exposure, concentration-effect relationships are non linear. An insect model should allow a better description of this toxicity. We compared the lethal concentration 50% (LC50) of IMI for a Drosophila-field strain, after acute and chronic exposure. Relative to the acute LC50, the chronic LC50 was lowered by a factor of 29 for males (1.3 mM/45 muM), 52 for larvae (157 muM/3 muM) and more than 172 for females (>3.1 mM/18 muM). Chronic exposure also revealed significant lethal and sublethal effects, at concentrations 3-5 orders of magnitude lower than the chronic LC50. Mean mortalities reached 28% (at 3.91 nM) and 27% (at 39.1 nM) for females and males, respectively. Fecundity decreased of 16% at 1.96 nM. Mating increased of 30% at 0.391 nM. The LOEC (lowest observed effect concentration: 0.391 nM) was 46 000 times lower than the chronic LC50 for males; it was 115 000 times lower than the chronic LC50 for females. This study illuminates effects that neonicotinoids can induce at very low concentrations. This is of particular interest for nontarget insects and for insect dependent species.
Several methods for analyzing pesticides in honey have been developed. However, they do not always reach the sufficiently low limits of quantification (LOQ) needed to quantify pesticides toxic to honey bees at low doses. To properly evaluate the toxicity of pesticides, LOQ have to reach at least 1 ng/g. In this context, we developed extraction and analytical methods for the simultaneous detection of 22 relevant insecticides belonging to three chemical families (neonicotinoids, pyrethroids, and pyrazoles) in honey. The insecticides were extracted with the QuEChERS method that consists in an extraction and a purification with mixtures of salts adapted to the matrix and the substances to be extracted. Analyses were performed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) for the pyrazoles and the pyrethroids and by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) for the neonicotinoids and ethiprole. Calibration curves were built from various honey types fortified at different concentrations. Linear responses were obtained between 0.2 and 5 ng/g. Limits of detection (LOD) ranged between 0.07 and 0.2 ng/g, and LOQ ranged between 0.2 and 0.5 ng/g. The mean extraction yields ranged between 63 % and 139 % with RSD <25 %. A complete validation of the methods also examined recovery rates and specificity. These methods were applied to 90 honey samples collected during a 2009-2010 field study in two apiaries placed in different anthropic contexts.
In less than 20 years, neonicotinoids have become the most widely used class of insecticides with a global market share of more than 25%. For pollinators, this has transformed the agrochemical landscape. These chemicals mimic the acetylcholine neurotransmitter and are highly neurotoxic to insects. Their systemic mode of action inside plants means phloemic and xylemic transport that results in translocation to pollen and nectar. Their wide application, persistence in soil and water and potential for uptake by succeeding crops and wild plants make neonicotinoids bioavailable to pollinators at sublethal concentrations for most of the year. This results in the frequent presence of neonicotinoids in honeybee hives. At field realistic doses, neonicotinoids cause a wide range of adverse sublethal effects in honeybee and bumblebee colonies, affecting colony performance through impairment of foraging success, brood and larval development, memory and learning, damage to the central nervous system, susceptibility to diseases, hive hygiene etc. Neonicotinoids exhibit a toxicity that can be amplified by various other agrochemicals and they synergistically reinforce infectious agents such as Nosema ceranae which together can produce colony collapse. The limited available data suggest that they are likely to exhibit similar toxicity to virtually all other wild insect pollinators. The worldwide production of neonicotinoids is still increasing. Therefore a transition to pollinator-friendly alternatives to neonicotinoids is urgently needed for the sake of the sustainability of pollinator ecosystem services.
Virus-like particles, 27 nm in diameter, were observed in extracts of individual Varroa destructor mites and in sections of mite tissue. Application of a purification procedure resulted in virus preparations that were used to prepare an antiserum to detect the virus in individual mites. Immunohistology studies showed that the gastric caecae were heavily infected, whereas no immunostaining could be detected in other mite tissues or organs, like the salivary glands, brain, rectum or reproductive organs. By electron microscopy large aggregates of virus-like particles in para-crystalline lattices were found in cells of the gastric caecae. The particles, reminiscent to picorna-like viruses, occurred mainly in the cytoplasm, whereas some virus particles were sparsely scattered in vacuoles. Occasionally, particles were observed in membrane-bound vesicles or in long tubular membrane structures in the cytoplasm. The accumulation of the picornalike virus particles in the cytoplasm and the presence of the virus in membrane structures give a strong indication that the virus replicates in the mite. (c) 2007 Elsevier Inc. All rights reserved.
The systemic imidacloprid is one of the most used insecticides in the world for field and horticultural crops. This neurotoxicant is often used as seed-dressing, especially for maize, sunflower, and rape. Using a LC/MS/MS technique (LOQ = 1 mu g/kg and LOD = 0.1 mu g/kg), the presence of imidacloprid has been measured in maize from field samples at the time of pollen shed, from less than 0.1 mu g/kg up to 33.6 mu g/kg. Numerous random samples were collected throughout France from 2000 to 2003. The average levels of imidacloprid measured are 4.1 mu g/kg in stems and leaves, 6.6 mu g/kg in male flowers (panicles), and 2.1 mu g/kg in pollen.
Imidacloprid, a new systemic insecticide used as seed-dressing, has been widely used in France since 1994. Its application mode and its efficiency allow a significant reduction in comparison with the usual quantity of chemicals used during pulverising treatment. But the insecticide imidacloprid is suspected to have harmful effects on the pollinators as many bees have died since its introduction. Recent studies have shown that imidacloprid has chronic and sub-lethal toxicities at levels of micro g/kg or less. It was therefore necessary to detect imidacloprid at these levels in soils, plants, flowers, and pollens. With this aim, we characterised the bio-availability of imidacloprid in the environment using a new quantitative analytical method, as a basis for the evaluation of the risk for bees.
Aggregations of 27 nm virus-like particles were observed in electron microscopy images of sectioned Varroa destructor mite tissue. The scattered occurrence of individual particles and accumulation of the virions in lattices in the cytoplasm gave an apparent indication that the virus replicates in the mite. Sequence analysis of the RNA of the purified virus revealed a genome organization with high similarity to that of members of the genus Iflavirus. Phylogenetic analysis of the polymerase showed that the virus was related most closely to Deformed wing virus (DWV) and Kakugo virus (KV) of bees.
The assessment of agrophannaceuticals’ side effects requires more realistic simulations of field conditions than those deduced from the dose-lethality relation obtained under laboratory conditions. Because the presence of sublethal doses or concentrations may also alter the behavior of foraging insects, we attempted to devise a quantifiable and accurate protocol for evidencing various alterations in free-flying bees. Such a protocol was illustrated by testing new classes of systemic insecticides. The protocol focused on video recording to quantify the foraging activity of small colonies of honey bees confined in insect-proof tunnels. The basis of the protocol was not the colony itself but the change in each colony on a specific day and between days.
Razi or Rhazes for Latin people is one of the great scientists of ninth century. He left major works in medicine, pharmacy and in alchemy. We examine here, the use of honey by this scientist as a simple drug and as one of the essential substances included in composed medicines. That leads us to examine the notion of simple and of composed in this physician and alchemist.
Electuaries and theriacs include an important proportion of honey in their ingredients This one has a double purpose. It is an exicipient and a curative agent. On the other hand it is used as preservative, blinder and sweetener and on the other hand it is recommended because of its numerous healing virtues. Physicians take care of the selection and preparation’s conditions to have an optimal quality of the honey and they consider its real nature got during its elaboration. Honey will be a perfect food and drug if it was not subjected to the digestion’s action after its absorption.
Imidacloprid, the most used systemic insecticide, is suspected of having harmful effects on honeybees at nanogram per bee or at microgram per kilogram levels. However, there is a lack of methodology to detect imidacloprid and its metabolites at such low levels. We developed a method for the determination of low amounts of imidacloprid in soils, plants (leaves and flowers), and pollens by using HPLC coupled to tandem mass spectrometry (APCI-MS/MS). Extraction, separation, and detection were performed according to quality assurance criteria, to Good laboratory Practice, and to criteria from the directive 96/23/EC, which is designed for banned substances. The linear range of application is 0.5-20 mug/kg imidacloprid in soils, in plants, and in pollens, with a relative standard deviation of 2.9% at 1 mug/kg. The limits of detection and of quantification are LOD = 0.1 mug/kg and LOQ = 1 mug/kg, respectively. For the first time, this study permitted us to follow the fate of imidacloprid in the environment. When treated, flowers of sunflower and maize contain average values of similar to10 mug/kg imidacloprid. Ibis explains that pollens from these crops are contaminated at levels of a few micrograms per kilogram, suggesting probable deleterious effects on honeybees.
The enzyme spectrum of an ectoparasitic mite of the honeybee, Varroa destructor (Anderson and Trueman) was studied using a semi-quantitative method, especially designed for complex samples which have not been purified. Exopeptidases and phosphatases are shown present. A chitinase and enzymes able to transform beta carbohydrates are also present with a large range in the intensity of the reaction. The role of the chitinase can be related to the supply of nutritional needs or/and the piercing and sucking behaviour of the adult parasite. Chitinase activity could be one factor influencing the balance between the parasite and its host.
The name surfactin refers to a bacterial cyclic lipopeptide, primarily renowned for its exceptional surfactant power since it lowers the surface tension of water from 72 mN m(-1) to 27 mN m(-1) at a concentration as low as 20 mu M. Although surfactin was discovered about 30 years ago, there has been a revival of interest in this compound over the past decade, triggered by an increasing demand for effective biosurfactants for difficult contemporary ecological problems. This simple molecule also looks very promising as an antitumoral, antiviral and anti-Mycoplasma agent.
A series of 9 lactonic lipopeptide biosurfactants was isolated from Bacillus licheniformis IM 1307 as representatives of the lichenysin group and we propose to name them lichenysins G. They were recovered from the culture medium as complex mixtures of molecules having different peptide sequences and different structures of P-hydroxy fatty acids. Their separation was achieved by a reversed-phase HPLC method leading to eight well-separated compounds. The complete structure of individual isoforms was proposed following the results of amino acid and fatty acid analysis, LSI-MS and 2D NMR spectroscopies. Compared to surfactin, lichenysins G are at least 10 fold more efficient biosurfactants.
Androctonin is a highly cationic antimicrobial peptide from scorpion exhibiting a broad spectrum of activities against bacteria and fungi. It contains 25 amino acids including four cysteine residues forming two disulfide bridges. We report here on the determination of its solution structure by conventional two-dimensional (2D) H-1-NMR spectroscopy and molecular modelling using distance geometry and molecular dynamics methods. The structure of androctonin involves a well-defined highly twisted anti-parallel beta-sheet with strands connected by a more variable positively charged turn. A comparison with the structure of tachyplesin I (horseshoe crab) reveals that the amphiphilic character of the protein surface of this homologous peptide is not observed in androctonin. We have undertaken a 200-ps molecular dynamics simulation study on a system including one androctonin molecule and a monolayer of DMPG (1,2-dimyristoylphosphatidylglycerol) lipids. On the basis of this simulation, the first steps of the membrane permeabilization process are discussed.
Thanatin is the first inducible insect peptide that has been found to have, at physiological concentrations, a broad ranee of activity against bacteria and fungi. Thanatin contains 21 amino acids including two cysteine residues that form a disulfide bridge. Two-dimensional (2D) H-1-NMR spectroscopy and molecular modelling have been used to determine its three-dimensional (3D) structure in water. Thanatin adopts a well-defined anti-parallel beta-sheet structure from residue 8 to the C-terminus, including the disulfide bridge. In spire of the presence of two proline residues, there is a large degree of structural variability in the N-terminal segment. The structure of thanatin is quite different from the known structures of other insect defence peptides, such as antibactarial defensin and antifungal drosomycin. It has more similarities with the structures of various peptides from different origins, such as brevinins, protegrins and tachyplesins. which have a two-stranded beta-sheet stabilized by one or two disulfide bridges. Combined with activity test experiments on several truncated isoforms of thanatin, carried out by Fehlbaum et al. [Fehlbaum, P., Bulet, P., Chernysh, S., Briand, J. P., Roussel, J. P., Letellier. L., Hetru, C. & Hoffmann. J. (1996) Pmc. Natl Acad Sci. USA 93, 1221-1225], our structural study evidences the importance of the P-sheet structure and also sn nests hat anti-Gram-negative activity involves a site formed by the Arg20 side-chain embedded in a hydrophobic cluster.
The solution structure of the anti-mammal and anti-insect LqqIII toxin from the scorpion Leiurus quinquestriatus quinquestriatus was refined and compared with other long-chain scorpion toxins, This structure, determined by H-1-NMR and molecular modeling, involves an a-helix (18-29) linked to a three-stranded ß-sheet (2-6, 33-39, and 43-51) by two disulfide bridges, The average RMSD between the 15 best structures and the mean structure is 0.71 Angstrom for C a atoms, Comparison between LqqIII, the potent anti-mammal AaHII, and the weakly active variant-3 toxins revealed that the LqqIII three-dimensional structure is closer to that of AaHII than to the variant-3 structure, Moreover, striking analogies were observed between the electrostatic and hydrophobic potentials of LqqIII and AaHII.
The biosynthesis of bacterial isoleucyl-rich surfactins was controlled by supplementation of L-isoleucine to the culture medium. Two new variants, the [Ile4,7]- and [Ile2,4,7]surfactins, were thus produced by Bacillus subtilis and their separation was achieved by reverse-phase HPLC. Amino acids of the heptapeptide moiety were analysed by chemical methods, and the lipid moiety was identified to B-hydroxy anteiso pentadecanoic acid by combined GC/MS. Sequences were established on the basis of two-dimensional NMR data. Because conformational parameters issuing from NMR spectra suggested that the cyclic backbone fold was globally conserved in the new variants, structure-activity relationships were discussed in details on the basis of the three-dimensional model of surfactin in solution.
Defensin A is an inducible antibacterial protein isolated from the larvae of Phormia terranovae. The conformation of defensin A has been previously determined by two-dimensional(1)H-NMR for concentrations in the range of 4-8 mM in water (Bonmatin JM et al (1992)J Biomol NMR 2, 235-256). CD spectroscopic data of defensin A at lower concentrations (10(-5) to 10(-3) M) are reported herein. The ellipticity in the 200-240 nm wavelength range for various solvents varies as follows: acetonitrile
Background: Insect defensin A is a basic 4 kDa protein secreted by Phormia terranovae larvae in response to bacterial challenges or injuries. Previous biological tests suggest that the bacterial cytoplasmic membrane is the target of defensin A. The structural study of this protein is the first step towards establishing a structure-activity relationship and forms the basis for understanding its antibiotic activity at the molecular level. Results: We describe a refined model of the three-dimensional structure of defensin A derived from an extensive analysis of 786 inter-proton nuclear Overhauser effects. The backbone fold involves an N-terminal loop and an alpha-helical fragment followed by an antiparallel beta-structure.
Bacillus subtilis coproduces several surfactin variants that are powerful biosurfactants and have potential applications in biology and industry. A single amino acid substitution in the heptapeptide moiety of surfactins strongly modifies their properties. To better establish structure-activity relationships and to search new variants with enhanced properties, Bacillus subtilis was grown into two modified culture media. Two new variants were isolated by chromatographic methods and studied by NMR spectroscopy. As planned, modifications consisted in the substitution of the L-valine residue at the fourth position by a more hydrophobic residue, i.e., leucine or isoleucine. These [Leu(4)]- and [Ile(4)]surfactins have a higher affinity for hydrophobic solvents and a twice improved surfactant power. Structure-property correlations were confirmed by analysis of the hydrophobic residue distribution in the three-dimensional model of the structure of surfactin in solution.
Light scattering and P-31-NMR have been used to monitor the effect of the bee-toxin, melittin, on phosphatidylcholine (PC) bilayers of variable acyl chain length (from C-16:0 to C-20:0). Melittin interacts with all lipids provided the interaction is initiated in the lipid fluid phase. For low-to-moderate amounts of toxin (lipid-peptide molar ratios, R(i) greater than or equal to 15), the system takes the form of large spheroidal vesicles, in the fluid phase, whose radius increases from 750 Angstrom A with dipalmitoyl-PC (DPPC) to 1500 Angstrom A with diarachinoyl-PC (DAPC). These vesicles fragment into small discoids of 100-150 Angstrom A radius when the system is cooled down below T-c (the gel-to-fluid phase transition temperature).
The solution three-dimensional structure of the protonated [Leu7]-surfactin, an heptapeptide extracted from Bacillus subtilis, has been determined from two-dimensional H-1-nmr performed in H-2(6)-dimethylsulfoxide and combined with molecular modeling. Experimental data included 9 coupling constants, 61 nuclear Overhauser effect derived distances, NH temperature coefficients, and C-13 relaxation times. Two distance geometry (DISMAN) protocols converged toward models of the structure and the best of them were refined by restrained and unrestrained molecular dynamics (GROMOS). Two structures in accord with the set of experimental constraints are presented. Both are characterized by a ’’horse saddle’’ topology for ring atoms on which are attached the two polar Glu and Asp side chains showing an orientation clearly opposite to that of the C-11-13 aliphatic chain. Amphipathic and surface properties of surfactin are certainly related to the existence of such minor polar and a major hydrophobic domains. The particular ’’claw’’ configuration of acidic residues observed in surfactin gives important clues for the understanding of its cation binding and transporting ability. (C) 1994 John Wiley and Sons, Inc.
When Bacillus subtilis S 499 was grown on a culture medium containing L-alanine as nitrogen source, a mixture of surfactins was obtained. Suitable chromatographic conditions allowed the separation of isoforms. Among these compounds, a new variant of surfactin was isolated and its structure was established by chemical and spectrometric methods, especially by NMR spectrometry. It contains a peptide sequence which differs from that of standard surfactin by the replacement of the L-valine residue by L-alanine residue in position 4. The folding mode of [Ala4]surfactin as deduced from NMR results was compared with that of standard surfactin and the structure/properties relationship issuing from the study of this new isoform is discussed.
Peptides which have the plasma membrane as a target may induce colloid osmotic lysis of cells. This is reflected by changes in phospholipid structure and dynamics as documented by spectroscopic methods and by modifications of their supramolecular properties such as phase stability, vesicularization, fusion and micellization. From similar changes induced both on model systems and on natural membranes, mechanisms are proposed for biological membrane destabilization.
2-D and 3-D NMR techniques were used to investigate the conformations in solution of several peptides and proteins for which crystalline structures are not available yet. Insect defensin A is a small (40 aa) antibiotic protein exhibiting a characteristic ’loop-helix-beta-sheet’ structure. A striking analogy was found with charybdotoxin, a scorpion toxin in which a CSH (cysteine stabilized alpha-helix) motif is also present. Wheat phospholipid transfer protein (PLTP) (90 aa) has a 3-D structure resulting from the packing of four helices and of a C-terminal less well-defined fragment. Preliminary results show that PLTP forms a complex with lyso-PC and that such an interaction results in a conformational change affecting principally the C-terminal half of the protein. A last example is given with surfactin, a lipopeptide biosurfactant from bacterial origin. Its protonated form shows a very compact structure in which the two acidic residues located on the top of a ’horse saddle’ topology face each other, whereas the ionized form could adopt a more extended conformation. A common property of these compounds is their capacity to interact with lipids. The present structural data open the way for a further establishment of structure-activity relationships.
A 500 MHz 2D H-1 NMR study of recombinant insect defensin A is reported. This defense protein of 40 residues contains 3 disulfide bridges, is positively charged and exhibits antibacterial properties. 2D NMR maps of recombinant defensin A were fully assigned and secondary structure elements were localized. The set of NOE connectivities, 3J(NH-alpha-H) coupling constants as well as H-1/H-2 exchange rates and DELTA-delta/DELTA-T temperature coefficients of NH protons strongly support the existence of an alpha-helix (residues 14-24) and of an antiparallel beta-sheet (residues 27-40). Models of the backbone folding were generated by using the DISMAN program and energy refined by using the AMBER program.
Two cyclic peptides have been prepared and characterized by MS and NMR. The linear precursors have been obtained by solid phase synthesis and the cyclization step was achieved using the BOP reagent. They increase markedly the rate of hydrolysis of oligoribonucleotides.
Reinvestigation of surfactin, a previously studied peptidolipid surfactant from Bacillus subtilis, by fast-atom-bombardment mass spectrometry and H-1-NMR spectroscopy, as well as by chemical methods, revealed the presence of a closely related second constituent. This new compound, [Val7]surfactin, differs from the known surfactin by the C-terminal amino acid residue which is valine instead of leucine.
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