The Molecular Resistance Mechanisms of European Earwigs from Apple Orchards Subjected to Different Management Strategies.

To date, apple orchards are among the most treated crops in Europe with up to 35 chemical treatments per year. Combining control methods that reduce the number of pesticide treatments is essential for agriculture and more respectful of the environment, and the use of predatory insects such as earwigs may be valuable to achieve this goal. European earwigs, Forficula auricularia (Dermaptera: Forficulidae) are considered beneficial insects in apple orchards where they can feed on many pests like aphids. The aim of this study was to investigate the potential impact of orchards' insecticide treatments on resistance-associated molecular processes in natural populations of earwigs. Because very few molecular data are presently available on earwigs, our first goal was to identify earwig resistance-associated genes and potential mutations. Using earwigs from organic, integrated pest management or conventional orchards, we identified mutations in acetylcholinesterase 2, α1 and ß2 nicotinic acetylcholine receptors. In addition, the expression level of these targets and of some essential detoxification genes were monitored using RT-qPCR. Unexpectedly, earwigs collected in organic orchards showed the highest expression for acetylcholinesterase 2. Four cytochromes P450, one esterase and one glutathione S-transferases were over-expressed in earwigs exposed to various management strategies in orchards. This first study on resistance-associated genes in Forficula auricularia paves the way for future experimental studies aimed at better understanding the potential competition between natural enemies in apple orchards in order to optimize the efficiency of biocontrol.

Impacts of neonicotinoids on biodiversity: a critical review.

Neonicotinoids are the most widely used class of insecticides in the world, but they have raised numerous concerns regarding their effects on biodiversity. Thus, the objective of this work was to do a critical review of the contamination of the environment (soil, water, air, biota) by neonicotinoids (acetamiprid, clothianidin, imidacloprid, thiacloprid, thiamethoxam) and of their impacts on terrestrial and aquatic biodiversity. Neonicotinoids are very frequently detected in soils and in freshwater, and they are also found in the air. They have only been recently monitored in coastal and marine environments, but some studies already reported the presence of imidacloprid and thiamethoxam in transitional or semi-enclosed ecosystems (lagoons, bays, and estuaries). The contamination of the environment leads to the exposure and to the contamination of non-target organisms and to negative effects on biodiversity. Direct impacts of neonicotinoids are mainly reported on terrestrial invertebrates (e.g., pollinators, natural enemies, earthworms) and vertebrates (e.g., birds) and on aquatic invertebrates (e.g., arthropods). Impacts on aquatic vertebrate populations and communities, as well as on microorganisms, are less documented. In addition to their toxicity to directly exposed organisms, neonicotinoid induce indirect effects via trophic cascades as demonstrated in several species (terrestrial and aquatic invertebrates). However, more data are needed to reach firmer conclusions and to get a clearer picture of such indirect effects. Finally, we identified specific knowledge gaps that need to be filled to better understand the effects of neonicotinoids on terrestrial, freshwater, and marine organisms, as well as on ecosystem services associated with these biotas.

Main conclusions and perspectives from the collective scientific assessment of the effects of plant protection products on biodiversity and ecosystem services along the land-sea continuum in France and French overseas territories.

Preservation of biodiversity and ecosystem services is critical for sustainable development and human well-being. However, an unprecedented erosion of biodiversity is observed and the use of plant protection products (PPP) has been identified as one of its main causes. In this context, at the request of the French Ministries responsible for the Environment, for Agriculture and for Research, a panel of 46 scientific experts ran a nearly 2-year-long (2020-2022) collective scientific assessment (CSA) of international scientific knowledge relating to the impacts of PPP on biodiversity and ecosystem services. The scope of this CSA covered the terrestrial, atmospheric, freshwater, and marine environments (with the exception of groundwater) in their continuity from the site of PPP application to the ocean, in France and French overseas territories, based on international knowledge produced on or transposable to this type of context (climate, PPP used, biodiversity present, etc.). Here, we provide a brief summary of the CSA's main conclusions, which were drawn from about 4500 international publications. Our analysis finds that PPP contaminate all environmental matrices, including biota, and cause direct and indirect ecotoxicological effects that unequivocally contribute to the decline of certain biological groups and alter certain ecosystem functions and services. Levers for action to limit PPP-driven pollution and effects on environmental compartments include local measures from plot to landscape scales and regulatory improvements. However, there are still significant gaps in knowledge regarding environmental contamination by PPPs and its effect on biodiversity and ecosystem functions and services. Perspectives and research needs are proposed to address these gaps.

Cross effects of heat stress and three insecticides on the survival of the codling moth Cydia pomonella (L.): Investigating the molecular and biochemical mechanisms.

As temperature is expected to strongly increase in the future, understanding temperature-mediated toxicity of insecticides is determinant to assess pest management efficiency in a warming world. Investigating molecular and biochemical mechanisms associated with cross mechanisms of temperature and insecticides on pests' tolerance would also be useful in this context. This study aimed to investigate cross effects between temperature and insecticides on the survival of a major pest, the codling moth Cydia pomonella, and their underlying mechanisms. The effect of three insecticidal active ingredients, i.e. chlorantraniliprole, emamectin and spinosad, was assessed at different temperatures on: (i) C. pomonella larval survival; (ii) detoxification enzymes activities (cytochrome P450 multi-function oxygenases, carboxylesterases and glutathione S-transferases) and (iii) genes expression of some detoxification enzymes, heat shock proteins and receptors targeted by the insecticides. We observed a decreased efficiency of emamectin and spinosad at high temperature to control the codling moth while no influence of temperature on chlorantraniliprole efficacy was observed. Detoxification enzymes activities were improved by heat stress alone but not by double stress (temperature + insecticides). Moreover, two detoxification genes (Cyp9A61 and Gst1) were over-expressed by a single stress but not by two stresses while Hsp70 and Cyp6B2 genes may be involved in tolerance to two stresses in C. pomonella. These results confirmed the cross effects of temperature and insecticides on C. pomonella for emamectin and spinosad and provided clues to understand how temperature affects the susceptibility of C. pomonella to insecticides. They illustrate however the complexity of molecular and biochemical responses of individuals facing multiple stresses.

Field mixtures of currently used pesticides in agricultural soil pose a risk to soil invertebrates.

Massive use of pesticides in conventional agriculture leads to accumulation in soil of complex mixtures, triggering questions about their potential ecotoxicological risk. This study assessed cropland soils containing pesticide mixtures sampled from conventional and organic farming systems at La Cage and Mons, France. The conventional agricultural field soils contained more pesticide residues (11 and 17 versus 3 and 11, respectively) and at higher concentrations than soils from organic fields (mean 6.6 and 10.5 versus 0.2 and 0.6 µg kg-1, respectively), including systemic insecticides belonging to neonicotinoids, carbamate herbicides and broad-spectrum fungicides mostly from the azole family. A risk quotient (RQi) approach evaluated the toxicity of the pesticide mixtures in soil, assuming concentration addition. Based on measured concentrations, both conventional agricultural soils posed high risks to soil invertebrates, especially due to the presence of epoxiconazole and imidacloprid, whereas soils under organic farming showed negligible to medium risk. To confirm the outcome of the risk assessment, toxicity of the soils was determined in bioassays following standardized test guidelines with seven representative non-target invertebrates: earthworms (Eisenia andrei, Lumbricus rubellus, Aporrectodea caliginosa), enchytraeids (Enchytraeus crypticus), Collembola (Folsomia candida), oribatid mites (Oppia nitens), and snails (Cantareus aspersus). Collembola and enchytraeid survival and reproduction and land snail growth were significantly lower in soils from conventional compared to organic agriculture. The earthworms displayed different responses: L. rubellus showed higher mortality on soils from conventional agriculture and large body mass loss in all field soils, E. andrei showed considerable mass loss and strongly reduced reproduction, and A. caliginosa showed significantly reduced acetylcholinesterase activity in soils from conventional agriculture. The oribatid mites did not show consistent differences between organic and conventional farming soils. These results highlight that conventional agricultural practices pose a high risk for soil invertebrates and may threaten soil functionality, likely due to additive or synergistic "cocktail effects".

Elucidating pesticide sensitivity of two endogeic earthworm species through the interplay between esterases and glutathione S-transferases.

Earthworms are common organisms in soil toxicity-testing framework, and endogeic species are currently recommended due to their ecological role in agroecosystem. However, little is known on their pesticide metabolic capacities. We firstly compared the baseline activity of B-esterases and glutathione-S-transferase in Allolobophora chlorotica and Aporrectodea caliginosa. Secondly, vulnerability of these species to pesticide exposure was assessed by in vitro trials using the organophosphate (OP) chlorpyrifos-ethyl-oxon (CPOx) and ethyl-paraoxon (POx), and by short-term (7 days) in vivo metabolic responses in soil contaminated with pesticides. Among B-esterases, acetylcholinesterase (AChE) activity was abundant in the microsomal fraction (80% and 70% of total activity for A. caliginosa and A. chlorotica, respectively). Carboxylesterase (CbE) activities were measured using three substrates to examine species differences in isoenzyme and sensitivity to both in vitro and in vivo exposure. CbEs were mainly found in the cytosolic fraction (80% and 60% for A. caliginosa and A. chlorotica respectively). GST was exclusively found in the soluble fraction for both species. Both OPs inhibited B-esterases in a concentration-dependent manner. In vitro trials revealed a pesticide-specific response, being A. chlorotica AChE more sensitive to CPOx compared to POx. CbE activity was inhibited at the same extent in both species. The 7-d exposure showed A. chlorotica less sensitive to both OPs, which contrasted with outcomes from in vitro experiments. This non-related functional between both approaches for assessing pesticide toxicity suggests that other mechanisms linked with in vivo OP bioactivation and excretion could have a significant role in the OP toxicity in endogeic earthworms.

How lasting are the effects of pesticides on earwigs? A study based on energy metabolism, body weight and morphometry in two generations of Forficula auricularia from apple orchards.

Widespread use of pesticides to control pests is the dominant system in conventional apple orchards. To avoid adverse side effects, there is a growing interest in promoting alternative methods including biological control based on the use of natural enemies. The European earwig Forficula auricularia L. (Dermaptera: Forficuidae) is an effective predator in apple orchards. Pesticide pressure has been shown to divert energy resources which could have a negative impact on life history traits. In this study we assessed (i) whether variations in pesticide exposure could differentially impact energy reserves, body weight and morphometric parameters of F. auricularia, and (ii) whether these effects persist into the next generation reared under optimal conditions. Individuals from the first generation were collected in late October from organic, IPM and conventional orchards. The next generation was obtained under a rearing program, in the absence of pesticide exposure. Earwigs collected from conventional orchards exhibited lower values for all morphometric parameters compared to those collected in organic orchards. However, a relaxed period without pesticide exposure (in autumn) appears to have allowed the females to recover their energy reserves to ensure reproduction and maternal care. Glycogen contents were the reserves that were more easily restored. However, probably due to the rearing conditions (food ad libitum), all the earwigs from the second generation exhibited higher body weights and energy reserves than their parents.

Sublethal exposure to deltamethrin impairs maternal egg care in the European earwig Forficula auricularia.

The application of pesticides typically leads to lethal and sublethal exposure of non-target insects. Whereas our current understanding of these sublethal effects typically focuses on reproductive and physiological parameters, recent works emphasize that sublethal effects on behaviors such as maternal care could be of major importance in non-target species. However, it remained unknown whether these sublethal effects occur in insects. Here, we tested if exposure to sublethal doses of deltamethrin - a pyrethroid insecticide commonly used in crops - alters the expression of maternal egg care in females of the European earwig Forficula auricularia, a predator insect and pest control. Our results first reveal that deltamethrin exposure impaired the expression of three forms of maternal egg care: It decreased the likelihood of mothers to gather their otherwise scattered clutch of eggs, increased the time during which the female abandoned the clutch after a predator attack and reduced egg grooming duration. These sublethal effects did not reflect a lower activity of deltamethrin-exposed females, as these females increased their expression of self-grooming, and deltamethrin exposure did not affect females' exploration and mobility. Finally, we found that the negative effects of deltamethrin on egg care did not modify egg development, hatching rate and juvenile weight, possibly due to the transient effects of deltamethrin on maternal behaviors. Overall, our results reveal that sublethal exposure to a pesticide may diminish maternal egg care in a natural pest control and call for the integration of this measurement in assays on pesticides application.

Metabolic mechanisms and acetylcholinesterase sensitivity involved in tolerance to chlorpyrifos-ethyl in the earwig Forficula auricularia.

Apple orchards are highly treated crops, in which organophosphorus (OP) are among the most heavily sprayed insecticides. These pesticides are toxic to non-target arthropods and their repeated use increases the risk of resistance. We studied mechanisms involved in tolerance and resistance to OP insecticides in the earwig Forficula auricularia, an effective generalist predator in pomefruit orchards. Adult earwigs were sampled in three apple orchards managed under contrasting strategies: conventional, Integrated Pest Management, and organic. The threshold activities of enzyme families involved in pesticides tolerance: Glutathione-S-transferases (GSTs) and Carboxylesterases (CbEs) were measured in earwig extracts. Acetylcholinesterase (AChE) was monitored as a toxicological endpoint. Variations in these activities were assessed prior to and after exposure to chlorpyrifos-ethyl at the normal application rate. We observed that the mortality of earwigs exposed to chlorpyrifos-ethyl depended on the management strategy of orchards. Significantly lower mortality was seen in individuals sampled from conventional orchard. The basal activities of CbEs and GSTs of collected organisms were higher in conventional orchard. After in vivo exposure, AChE activity appeared to be inhibited in surviving males with no difference between orchards. However an in vitro inhibition trial with chlorpyrifos-oxon showed that AChE from earwigs collected in organic and IPM orchards were more sensitive than from conventional ones. These observations support the hypothesis of a molecular target modification in AChE and highlight the possible role of CbEs in effective protection of AChE. Our findings suggest that the earwigs with a high historic level of insecticide exposure could acquire resistance to chlorpyrifos-ethyl.

Nerve conduction velocity as a non-destructive biomarker in the earthworm Aporrectodea caliginosa exposed to insecticides.

Earthworms are important and useful soil organisms, but in agricultural soils, they are potentially exposed to a wide variety of pesticides. Insecticides represent the highest threat to earthworms and many are neurotoxic. There is a need for a reliable, relevant, simple biomarker to assess the sub-lethal effects of neurotoxic insecticides on earthworms under laboratory or field conditions. The Aporrectodea caliginosa earthworms were exposed to 0 (control), 0.5×, 1× (normal field application rate), and 5× concentrations of a carbamate (Pirimor®) and an organophosphate (Lorsban®) insecticides. The nerve conduction velocity (NCV) of the medial giant fibers of A. caliginosa earthworm was recorded on days 0, 1, 2, 3, 4, and 7 to quantify sub-lethal neurotoxic effects. Acetylcholinesterase (AChE) enzyme activity of A. caliginosa homogenates was measured at the conclusion of the experiment. Pirimor® but not Lorsban® induced a significant decrease in NCV on days 3, 4, and 7 at 1× and 5× doses. A significant dose-dependent decrease was observed on AChE activity to Pirimor® at the doses used but not Lorsban®. A clear relationship is observed between AChE activity and NCV in the case of Pirimor®. This study showed that NCV is a sensitive biomarker that correlates well with classical biomarker measurements such as AChE enzyme activity. This technique could be used to study the impact of insecticides on earthworms and also their recovery.

An exploratory study of energy reserves and biometry as potential tools for assessing the effects of pest management strategies on the earwig, Forficula auricularia L.

Apple orchards are heavily treated crops and some sprayed insecticides are recognized to have toxic effects on non-target arthropods. Earwigs are important natural enemies in pip-fruit orchards and contribute to the biological control of aphids. In addition, due to their ease of capture and identification, they are an interesting potential bioindicator of the possible detrimental effects of different orchard management strategies. In this study, we measured the energy reserves and some morphological traits of Forficula auricularia L. sampled in apple orchards under management strategies (organic versus integrated pest management (IPM)). We observed a significant decrease in mass (22 to 27%), inter-eye width (3%), and prothorax width (2 to 5%) in earwigs from IPM compared to organic orchards. Energy body reserves also confirmed these results with a significant decrease of 48% in glycogen and 25 to 42% in lipid content in earwigs from IPM compared to organic orchards. However, the protein content was approximately 70% higher in earwigs from IPM than in organic orchards. Earwigs sampled in IPM orchards may adapt to minimize the adverse toxic effects of pesticide treatments using a large number of strategies, which are reflected in changes to their energy reserves. These strategies could, in turn, influence the population dynamics of natural enemies and impair their role in the biological control of pests in apple orchards.

Interspecific differences in biochemical and behavioral biomarkers in endogeic earthworms exposed to ethyl-parathion.

Earthworms are common organisms in the soil toxicity-testing framework, and the epigeic Eisenia andrei and E. fetida are the recommended species. However, Eisenia species are rarely found in agricultural soils and recent studies have pointed out endogeic species are more sensitive to pesticides than Eisenia. Allolobophora chlorotica and Aporrectodea caliginosa are two endogeic soil-dwelling species that are abundant in the agroecosystem. However, knowledge on pesticide impact on this ecological group of earthworms is still incipient. Herein, we compared the biochemical (acetylcholinesterase [AChE] and carboxylesterase [CbE] activities) and behavioral (burrowing, casting and feeding) biomarker responses of these two endogeic earthworm species exposed for 7 days to soils contaminated with 0.1, 1 and 10 mg kg-1 ethyl-parathion. The results showed marked species-specific differences in both groups of biomarkers, suggesting A. caliginosa the most sensitive species to this organophosphorus pesticide under the exposure conditions in this study. Moreover, an in vitro inhibition trial with ethyl-paraoxon evidenced a higher sensitivity of A. caliginosa AChE activity compared with that of A. chlorotica. This finding suggested that this molecular target endpoint could contribute to the interspecific differences of behavioral responses rather than CbE activity; this latter considered a potent mechanism of OP removal. Our results suggest the inclusion of more than one endogeic earthworm species to assess toxicity from organophosphorus insecticides. However, the use of A. caliginosa in the environmental risk assessment framework of organophosphorus contamination is highly recommended because of its higher sensibility to this class of pesticides, in addition to its abundance in the agroecosystem.

Soil enzyme dynamics in chlorpyrifos-treated soils under the influence of earthworms.

Earthworms contribute, directly and indirectly, to contaminant biodegradation. However, most of bioremediation studies using these annelids focus on pollutant dissipation, thus disregarding the health status of the organism implied in bioremediation as well as the recovery of indicators of soil quality. A microcosm study was performed using Lumbricus terrestris to determine whether earthworm density (2 or 4individuals/kg wet soil) and the time of exposure (1, 2, 6, 12, and 18wk) could affect chlorpyrifos persistence in soil initially treated with 20mg active ingredientkg-1 wet soil. Additionally, selected earthworm biomarkers and soil enzyme activities were measured as indicators of earthworm health and soil quality, respectively. After an 18-wk incubation period, no earthworm was killed by the pesticide, but clear signs of severe intoxication were detected, i.e., 90% inhibition in muscle acetylcholinesterase and carboxylesterase (CbE) activities. Unexpectedly, the earthworm density had no significant impact on chlorpyrifos dissipation rate, for which the measured half-life ranged between 30.3d (control soils) and 44.5d (low earthworm density) or 36.7d (high earthworm density). The dynamic response of several soil enzymes to chlorpyrifos exposure was examined calculating the geometric mean and the treated-soil quality index, which are common enzyme-based indexes of microbial functional diversity. Both indexes showed a significant and linear increase of the global enzyme response after 6wk of chlorpyrifos treatment in the presence of earthworms. Examination of individual enzymes revealed that soil CbE activity could decrease chlorpyrifos-oxon impact upon the rest of enzyme activities. Although L. terrestris was found not to accelerate chlorpyrifos dissipation, a significant increase in the activity of soil enzyme activities was achieved compared with earthworm-free, chlorpyrifos-treated soils. Therefore, the inoculation of organophosphorus-contaminated soils with L. terrestris arises as a complementary bioremediation strategy in terms of recovery of soil biochemical performance and quality.

Does wastewater treatment plant upgrading with activated carbon result in an improvement of fish health?

In the present study, the efficiency of a wastewater treatment plant (WWTP) upgraded with a powdered activated carbon unit for the reduction of micropollutants and the related advantages for fish health have been analyzed by means of different biomarkers, i.e. histopathological investigations, analyses of glycogen content and stress proteins, as well as by chemical analyses in different matrices. Comparative analyses were conducted prior and subsequent to the installation of the additional purification unit. Chemical analyses revealed a significant reduction of several pharmaceuticals, including diclofenac, carbamazepine and metoprolol, in samples of effluent and surface water downstream of the WWTP after its upgrade. In addition, diminished concentrations of diclofenac and PFOS were detected in tissues of analyzed fish. Histopathological investigations of fish liver, gills, and kidney revealed improved tissue integrity in fish after improved wastewater treatment. In parallel, biochemical measurements of glycogen revealed increased energy resources in fish liver and, furthermore, hsp70 levels in livers of exposed rainbow trout and in kidneys of exposed brown trout were lower after than before the WWTP upgrade. In summary, additional treatment with powdered activated carbon led to a reduction of potentially hazardous chemicals in the effluent and the adjacent river and, consequently, to an improvement of fish health in the receiving water course.

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

The 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.

Management strategies in apple orchards influence earwig community.

Our 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.

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

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 acetylthiocholine 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), 1mM 4-nitrophenyl valerate (4-NPV), and 2 mM α-naphtyl acetate (α-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 α-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.

Acetylcholinesterase activity in the terrestrial snail Xeropicta derbentina transplanted in apple orchards with different pesticide management strategies.

Apple orchards are highly manipulated crops in which large amounts of pesticides are used. Some of these pesticides lack target specificity and can cause adverse effects in non-target organisms. In order to evaluate the environmental risk of these products, the use of transplanted sentinel organisms avoids side-effects from past events and facilitate comparison of multiple sites in a short time. We released specimens of the terrestrial snail Xeropicta derbentina in each 5 of two kinds of apple orchards with either conventional or organic management strategies plus in a single abandoned orchard. After one month, individuals were retrieved in order to measure acetylcholinesterase (AChE) activity. Mean values of AChE activity were significantly reduced in all conventional apple orchards compared to the others. Results show that the measurement of biomarkers such as AChE inhibition in transplated X. derbentina could be useful in the environmental risk assessment of post-authorized pesticides.

Earthworm cast production as a new behavioural biomarker for toxicity testing.

There is currently a lack of ecotoxicity tests adapted to earthworm species of higher ecological relevance and whose endpoints could be directly related to their ecological role in the soil. We propose a new and relatively simple ecotoxicity test based on the estimation of cast production (CP) by Lumbricus terrestris under laboratory conditions. CP was found to be linearly correlated to earthworm biomass and to be greatly influenced by soil water content. Azinphos-methyl had no effect on CP at all the concentrations tested. Significant decreases were observed at the normal application rate for other pesticides with (imidacloprid, carbaryl, methomyl) or without (ethyl-parathion and chlorpyrifos-ethyl) a clear concentration-effect response. For the highest concentration tested, reduction in CP varied between 35 and 67%. CP is straightforward and rapidly measured and ecologically meaningful. We thus believe it to be of great use as an endpoint in ecotoxicity testing.

Carboxylesterase activity in earthworm gut contents: Potential (eco)toxicological implications.

Carboxylesterases (CbEs) are key enzymes in pesticide detoxification. These esterases are involved in the biochemical mechanism for pesticide resistance in some pest species, and further they are considered an efficient protective mechanism against acute toxicity by organophosphate (OP) pesticides in mammals. To gain knowledge on the role of CbEs in pesticide toxicity and natural tolerance in earthworms, we performed an enzyme kinetic analysis to investigate whether these annelids are able to secrete them into their gut lumen. We determined levels of CbE activity and isozyme abundance in the gut wall and ingested soil collected from different portions of the gastrointestinal tract of Lumbricus terrestris. Moreover, modulation of enzyme activity by selected substrates (alpha-naphthyl acetate [alpha-NA], 4-nitrophenyl valerate [4-NPV] and 4-nitrophenyl acetate [4-NPA]) and OP pesticides was examined to compare the response between tissue and soil CbEs. We found a high CbE activity in the ingested soil extracts from the crop/gizzard (alpha-NA-CbE=8.43+/-2.76U mg(-1) protein and 4-NPA-CbE=5.98+/-2.11U mg(-1) protein) compared to the gut wall. Three lines of evidences suggest that the gut epithelium is the main source of this luminal CbE activity. First, the effect of substrate concentrations on CbE activity from both the ingested soil extracts and gut tissues resulted in similar apparent K(m) and V(max) values. Second, native PAGE gels revealed that some of the CbE isozymes in the gut tissue were also present in the soil extracts. Third, tissue and soil CbEs showed the same sensitivity to inhibition by OPs. The concentrations of insecticide causing 50% of esterase inhibition (IC(50)) was comparable between tissue (IC(50)s range=4.01-9.67nM dichlorvos and 8480-6880nM paraoxon) and soil (IC(50)s range=6.01-11.5nM dichlorvos and 8400-7260nM paraoxon). Our results suggest a set of (eco)toxicological implications and environmental applications derived from the ability of earthworms to secrete these pesticide-detoxifying enzymes.