Diversity and catalytic potential of PAH-specific ring-hydroxylating dioxygenases from a hydrocarbon-contaminated soil.

International audienceRing-hydroxylating dioxygenases (RHDs) catalyze the initial oxidation step of a range of aromatic hydrocarbons including polycyclic aromatic hydrocarbons (PAHs). As such, they play a key role in the bacterial degradation of these pollutants in soil. Several polymerase chain reaction (PCR)-based methods have been implemented to assess the diversity of RHDs in soil, allowing limited sequence-based predictions on RHD function. In the present study, we developed a method for the isolation of PAH-specific RHD gene sequences of Gram-negative bacteria, and for analysis of their catalytic function. The genomic DNA of soil PAH degraders was labeled in situ by stable isotope probing, then used to PCR amplify sequences specifying the catalytic domain of RHDs. Sequences obtained fell into five clusters phylogenetically linked to RHDs from either Sphingomonadales or Burkholderiales. However, two clusters comprised sequences distantly related to known RHDs. Some of these sequences were cloned in-frame in place of the corresponding region of the phnAIa gene from Sphingomonas CHY-1 to generate hybrid genes, which were expressed in Escherichia. coli as chimerical enzyme complexes. Some of the RHD chimeras were found to be competent in the oxidation of two- and three-ring PAHs, but other appeared unstable. Our data are interpreted in structural terms based on 3D modeling of the catalytic subunit of hybrid RHDs. The strategy described herein might be useful for exploring the catalytic potential of the soil metagenome and recruit RHDs with new activities from uncultured soil bacteria

Evaluation des impacts environnementaux liés aux pratiques phytosanitaires en vergers de pommiers : approches écotoxicologique et écologique utilisant un auxiliaire des cultures, le forficule

In the European context which aims at reducing risk and uses of pesticides, the development of tools to evaluate environmental impacts is a major focus. This thesis uses ecotoxicological and ecological approaches, from tissue level to population level. The model species is a generalist predator, the earwig, and the study site is apple orchards in Southeastern France. Pesticides expositions were tested both in laboratory or real conditions (in orchards). Lab studies allowed us to characterize and investigate the tissular distribution of B-esterases (AChE and CbE) of Forficula auricularia and to study its predatory behavior on the aphid Dysaphis plantaginea after exposure. CbE were more sensitive than AChE and males B-esterases were more sensitive than females ones to organophosphorus pesticides. The overall results allowed us to validate the use of B-esterases as biochemical biomarkers on F. auricularia. The predation behavior of F. auricularia was impacted by some insecticides with males more sensitive than females. The behavior results were supported by inhibition of biochemical biomarkers (B-esterases) validating predation behavior as a new biomarker. Taking into account the individual level, the reproduction and development of first larval stages were carried out thanks to a breeding set up with earwigs trapped in orchards conducted under different pest management (organic, integrated and conventional management). Eggs number by clutch was reduced and clutch date was delayed for earwigs trapped in orchards conducted under conventional agriculture. To assess the impact of pesticides on population level, the abundance and diversity of earwig (F. auricularia and F. pubescens) were carried out in different orchard management considering small-scale landscape parameters. Pesticide use had clearly much higher effects on earwig abundances and diversity than landscape characteristics. F. pubescens was more sensitive than F. auricularia, with the same sensitivity difference between sex on both species (males more sensitive) making earwigs a relevant bioindicator species of pesticides use in apple orchards. This study enhances the need of complementary biomarkers able to highlight physiological and behavioral modifications which influence natural enemy populations and the natural regulation of pests.Dans le contexte européen actuel visant la réduction des risques et de l‘utilisation des pesticides, le développement d‘outils permettant d‘évaluer les impacts environnementaux est un objectif majeur. Cette thèse recouvre approche écotoxicologique et approche écologique sous forme d‘études de l‘échelle tissulaire à l‘échelle des populations. L‘espèce modèle est un prédateur généraliste, le forficule, et le site d‘étude les vergers de pommiers du Sud-est de la France. Les effets des pesticides ont été testés aussi bien en condition de laboratoire qu‘en conditions réelles (en vergers). Les études en laboratoire nous ont permis de caractériser et d‘étudier la localisation tissulaire des B-estérases (AChE et CbE) et d‘évaluer la capacité prédatrice chez Forficula auricularia après exposition, sur le puceron Dysaphis plantaginea. Les CbE sont plus sensibles que l‘AChE chez F. auricularia et les B-estérases des mâles sont toujours plus sensibles aux insecticides organophosphorés que celles des femelles. Ces résultats nous permettent de valider l‘utilisation des B-estérases comme biomarqueurs biochimiques chez F. auricularia. La capacité prédatrice de F. auricularia est diminuée par certains insecticides avec les mâles plus impactés que les femelles. Les mesures d‘activité des B-estérases ont corroboré les résultats validant le comportement de prédation comme un nouveau biomarqueur. A l‘échelle de l‘organisme, la reproduction et le développement des premiers stades larvaires ont été suivis grâce à un élevage mis en place à partir d‘insectes prélevés dans des vergers soumis à différentes pratiques phytosanitaires (agriculture biologique, raisonnée et conventionnelle). Le nombre d‘oeufs par ponte est réduit et la date de ponte retardée pour les forficules provenant des vergers à agriculture « conventionnelle ». Pour évaluer l‘effet des pesticides à l‘échelle des populations, l‘abondance et la diversité du forficule (F. auricularia et F. pubescens) ont été suivies en vergers en prenant également en compte des caractéristiques paysagères. L‘impact des pratiques phytosanitaires est plus important que celui des caractéristiques paysagères. L‘espèce F. pubescens est plus sensible que F. auricularia, avec la même différence de sensibilité entre sexes chez les deux espèces (mâles plus sensibles). Le forficule devient alors une espèce bioindicatrice pertinente de l‘utilisation des pesticides en verger de pommiers. Cette étude soulève le besoin de biomarqueurs complémentaires capables de mettre en évidence les modifications physiologiques et comportementales qui influencent les populations d‘auxiliaires et la régulation naturelle des ravageurs

Evaluation of environmental impacts associated to plant protection practices in apple orchards : ecotoxicological and ecological approaches using a biocontrol agent, the earwig

Dans le contexte européen actuel visant la réduction des risques et de l‘utilisation des pesticides, le développement d‘outils permettant d‘évaluer les impacts environnementaux est un objectif majeur. Cette thèse recouvre approche écotoxicologique et approche écologique sous forme d‘études de l‘échelle tissulaire à l‘échelle des populations. L‘espèce modèle est un prédateur généraliste, le forficule, et le site d‘étude les vergers de pommiers du Sud-est de la France. Les effets des pesticides ont été testés aussi bien en condition de laboratoire qu‘en conditions réelles (en vergers). Les études en laboratoire nous ont permis de caractériser et d‘étudier la localisation tissulaire des B-estérases (AChE et CbE) et d‘évaluer la capacité prédatrice chez Forficula auricularia après exposition, sur le puceron Dysaphis plantaginea. Les CbE sont plus sensibles que l‘AChE chez F. auricularia et les B-estérases des mâles sont toujours plus sensibles aux insecticides organophosphorés que celles des femelles. Ces résultats nous permettent de valider l‘utilisation des B-estérases comme biomarqueurs biochimiques chez F. auricularia. La capacité prédatrice de F. auricularia est diminuée par certains insecticides avec les mâles plus impactés que les femelles. Les mesures d‘activité des B-estérases ont corroboré les résultats validant le comportement de prédation comme un nouveau biomarqueur. A l‘échelle de l‘organisme, la reproduction et le développement des premiers stades larvaires ont été suivis grâce à un élevage mis en place à partir d‘insectes prélevés dans des vergers soumis à différentes pratiques phytosanitaires (agriculture biologique, raisonnée et conventionnelle). Le nombre d‘oeufs par ponte est réduit et la date de ponte retardée pour les forficules provenant des vergers à agriculture « conventionnelle ». Pour évaluer l‘effet des pesticides à l‘échelle des populations, l‘abondance et la diversité du forficule (F. auricularia et F. pubescens) ont été suivies en vergers en prenant également en compte des caractéristiques paysagères. L‘impact des pratiques phytosanitaires est plus important que celui des caractéristiques paysagères. L‘espèce F. pubescens est plus sensible que F. auricularia, avec la même différence de sensibilité entre sexes chez les deux espèces (mâles plus sensibles). Le forficule devient alors une espèce bioindicatrice pertinente de l‘utilisation des pesticides en verger de pommiers. Cette étude soulève le besoin de biomarqueurs complémentaires capables de mettre en évidence les modifications physiologiques et comportementales qui influencent les populations d‘auxiliaires et la régulation naturelle des ravageurs.In the European context which aims at reducing risk and uses of pesticides, the development of tools to evaluate environmental impacts is a major focus. This thesis uses ecotoxicological and ecological approaches, from tissue level to population level. The model species is a generalist predator, the earwig, and the study site is apple orchards in Southeastern France. Pesticides expositions were tested both in laboratory or real conditions (in orchards). Lab studies allowed us to characterize and investigate the tissular distribution of B-esterases (AChE and CbE) of Forficula auricularia and to study its predatory behavior on the aphid Dysaphis plantaginea after exposure. CbE were more sensitive than AChE and males B-esterases were more sensitive than females ones to organophosphorus pesticides. The overall results allowed us to validate the use of B-esterases as biochemical biomarkers on F. auricularia. The predation behavior of F. auricularia was impacted by some insecticides with males more sensitive than females. The behavior results were supported by inhibition of biochemical biomarkers (B-esterases) validating predation behavior as a new biomarker. Taking into account the individual level, the reproduction and development of first larval stages were carried out thanks to a breeding set up with earwigs trapped in orchards conducted under different pest management (organic, integrated and conventional management). Eggs number by clutch was reduced and clutch date was delayed for earwigs trapped in orchards conducted under conventional agriculture. To assess the impact of pesticides on population level, the abundance and diversity of earwig (F. auricularia and F. pubescens) were carried out in different orchard management considering small-scale landscape parameters. Pesticide use had clearly much higher effects on earwig abundances and diversity than landscape characteristics. F. pubescens was more sensitive than F. auricularia, with the same sensitivity difference between sex on both species (males more sensitive) making earwigs a relevant bioindicator species of pesticides use in apple orchards. This study enhances the need of complementary biomarkers able to highlight physiological and behavioral modifications which influence natural enemy populations and the natural regulation of pests

Impact of insecticide exposure on the predation activity of the European earwig Forficula auricularia

International audienceThe European earwig Forficula auricularia is an effective predator in apple orchards. It is therefore crucial to study whether insecticides affect this natural pest control agent. Predation activity, i.e., the number of aphids eaten in 24 h, was determined under laboratory conditions after exposure of fourth-instar nymphs and adult earwigs to widely used insecticides (acetamiprid, chlorpyrifos-ethyl, deltamethrin, and spinosad), which were applied at the normal application rates. Inhibition of acetylcholinesterase and carboxylesterase activities were also measured as indicators of pesticide exposure. Predation activity decreased significantly in nymphs exposed to spinosad (62 %) and chlorpyrifos-ethyl (98 %) compared with controls. A similar response was found for both esterase activities. Spinosad had a stronger effect on AChE (-33 %) whereas chlorpyrifos-ethyl affected CbE activity preferentially (-59 %). Spinosad (20 % of controls), acetamiprid (28 %), and chlorpyrifos-ethyl (66 %) also significantly decreased the predation behavior of adult male but not female (5 to 40 %) earwigs. Adult AChE and CbE activities were also significantly reduced (28 to 67 % of controls) in pesticide-exposed earwigs. Our results suggest that earwigs should be included in the environmental risk assessment framework for authorization of newly marketed plant protection products. Their predation behavior appears to be a sensitive and complementary biomarker

Tissue distribution, characterization and in vitro inhibition of B-esterases in the earwig Forficula auricularia

International audienceTrue acetylcholinesterase located in the head as a membrane-bound enzyme in earwig. High carboxylesterase isoenzymes could play important role in detoxification. In vitro strongest inhibitory effect of chlorpyrifos-oxon on carboxylesterase. Different pesticides effects on male and female for carboxylesterase but not acetylcholinesterase. Carboxylesterase could be complementary biomarkers of pesticides exposure in earwig. a b s t r a c t Earwigs are important natural enemies of numerous pests in pome fruit orchards worldwide. Studying the effects of agricultural practices on these biological control agents is important for understanding its vulnerability in the field. The aim of this study was to characterize the B-esterase activities in the European earwig Forficula auricularia and to evaluate in vitro its sensitivity to organophosphate and carbamate pesticides. Acetylcholinesterase (AChE) activity was mainly measured with 1.5 mM acetylthi-ocholine as the substrate in the microsomal fraction of earwig heads (70% of total AChE activity). Carboxylesterase (CbE) activities were measured with three substrates [5 mM 4-nitrophenyl acetate (4-NPA), 1 mM 4-nitrophenyl valerate (4-NPV), and 2 mM a-naphtyl acetate (a-NA)] to examine different isoenzymes, which were present mainly in the cytosolic fraction (about 70–88% of total activities) of all earwig tissues. CbE activity was higher than AChE activity, especially with a-NA, then 4-NPA and lastly 4-NPV. Chlorpyrifos-oxon an organophosphate, and carbaryl a carbamate pesticide, inhibited AChE and CbE activities in a concentration-dependent manner. Earwig CbE activities showed a stronger sensitivity to organophosphate than AChE, with the strongest effect for chlorpyrifos-oxon on male carboxylesterase activities. CbE and AChE showed about the same sensitivity to carbamate pesticides regardless of sex. These results suggest that B-type esterases in the European earwig F. auricularia are suitable biomarkers of pesticide exposure

Management strategies in apple orchards influence earwig community

International audienceOur aim was to assess whether different apple orchard management strategies (low-input, organic, Integrated Pest Management (IPM)) would have an effect on earwigs, which are important natural enemies of apple pests. These commercial orchards were as well compared to abandoned orchards. The density of Forficula auricularia and Forficula pubescens was studied for three years in 74 orchards around Avignon. The pesticide usage, some orchard characteristics and two small-scale landscape parameters were characterized. Pesticide use was significantly different between low-input, organic and IPM orchards with particularly significant differences in the number of insecticide applications (2.2, 4.9 and 9.2 respectively). Pesticide use had a much stronger impact on earwig community than other characteristics. F. auricularia density was significantly lower in IPM orchards (0.47 individuals per tree) compared to organic, low-input and abandoned orchards (3.1, 4.5 and 1.6 individuals per tree, respectively). F. pubescens was almost absent from IPM orchards and its abundance was higher in abandoned or low-input orchards compared to organic orchards (1.5 and 2.8 vs 0.8 individuals per tree). The percentage of F. pubescens in the earwig community decreased from abandoned (52%) to low-input (40%), organic (15%) and IPM orchards (0.5%). These results were confirmed by LD50 assays showing that for the two pesticides causing mortality close to normal application rates (chlorpyrifos-ethyl and acetamiprid), F. pubescens was significantly more sensitive than F. auricularia. Since earwigs are also easy to capture and identify, they may be useful to estimate the effects of management strategies and their modification in pome fruit orchards. (C) 2014 Elsevier Ltd. All rights reserved

Les PCB dans les sols : comment les éliminer?

National audienc