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Abstract: In order for cholinesterase (ChE) activity to be used as an effective biomarker in earthworms, the time course of enzyme activity inhibition and recovery must be fully characterized. A laboratory experiment was carried out using parathion as a model organophosphorus pesticide at the recommended dose (1mgkg−1) and a 10 fold higher dose (10mgkg−1), on two earthworm species (Allolobophora chlorotica and Aporrectodea caliginosa). ChE activity and weight were measured every week for a 14day period of exposure to parathion and then for 8weeks in uncontaminated soil. After 3days of exposure, the weight of both earthworm species had decreased by 10–15% compared to the control, regardless of the dose used. During the remainder of the exposure period, no differences were observed between the two doses for A. chlorotica; but A. caliginosa showed rapid weight recuperation for the lowest dose applied. After 28days and over, the control and both exposed species of worms lost similar amounts of weight. ChE inhibition was measured during and after the exposure period. ChE inhibition followed a different time course for the two species investigated. A. chlorotica appeared less sensitive to parathion than A. caliginosa. In this latter species, ChE inhibition was rapid at close to 70% of the control after 3days, for either dose, and reached 80–90% after 7days exposure. While A. chlorotica exhibited the same pattern of inhibition for 10mgkg−1 of parathion, the inhibition process was slower for the recommended dose with 50% inhibition after 7days of exposure and 70% after 14days. ChE activity recovery, after transfer to uncontaminated soil, also followed a different pattern for the two species. After exposure to 1mgkg−1 parathion, ChE activity from A. chlorotica underwent a slow but constant recovery process to regain the control value after 8weeks in unpolluted conditions. On the other hand, the ChE activity from A. caliginosa remained strongly inhibited. The differential susceptibility to parathion found in this study could be related to differences in the specificity of the total ChE activities between those two species. [Copyright &y& Elsevier]

The present study used cholinesterase (ChE) activity in earthworms as a biomarker of pesticide exposure at 17 apple orchards using different pest protection strategies (organic, integrated pest management [IPM], conventional, and abandoned) located within a 300-km² subregion near Avignon in southeastern France). The most common earthworm species in the 17 orchards was Allolobophora chlorotica. We examined inherent variability in ChE activity that might be attributable to soil characteristics and found that differences in soil structure or type did not significantly influence ChE activity. Furthermore, there was no relation between ChE specific activity and earthworm weight, and thus activity does not require correction for weight. Ten earthworms were collected in two successive months (April and May 2003) from each of the 17 orchards. Compared to the activity in worms from the control abandoned orchards, ChE activity was significantly decreased in earthworms from half the IPM and conventional orchards in April and all these orchards in May. Notably, ChE activity was also lower in earthworms from three organic orchards during May. No relation was observed between ChE decrease and the number of treatments (total or only organophosphorous and carbamate pesticides). Cholinesterase activity in earthworms from abandoned orchards varied between the two collecting periods, illustrating the difficulty in obtaining reference values for the use of ChE as a biomarker in field studies. [ABSTRACT FROM AUTHOR]

Abstract: To validate cholinesterase activity as a biomarker of pesticide exposure, we characterized the tissue distribution (whole body, nervous tissue and crop/gizzard), activity at two seasons of cholinesterase in six different species of earthworms collected in an unpolluted field: Lumbricus terrestris, Lumbricus castaneus, Aporrectodea nocturna, Aporrectodea caliginosa, Allolobophora chlorotica and Aporrectodea rosea. The major part of total cholinesterase activity was found in the nervous tissue while activity in crop/gizzard was weak. The level of the total cholinesterase activity was stable for each species considered throughout the year (spring and autumn). Lumbricus species exhibited three-fold higher specific activity than the others (0.086±0.015 U mg−1 and 0.235±0.036 U mg−1 for Allolobophora or Aporrectodea, and Lumbricus species respectively). This stability of the base level makes cholinesterase activity a useful biomarker for monitoring effects of pesticide under natural conditions. Cholinesterase activity was characterized using different substrates and inhibitors. It seems likely that the cholinesterases are acetylcholinesterases in most species investigated as they preferentially hydrolyzed acetylthiocholine and were inhibited by eserine, but not by tetraisopropyl pyrophoramide (iso-OMPA). Characterization of cholinesterase from Allolobophora chlorotica is uncertain and it cannot be classified as a true AChE. [Copyright &y& Elsevier]

Earthworm species interact with each other in soils, but these interactions are poorly understood. Moreover, these key soil organisms are influenced by abiotic soil components such as organic matter. Here, we investigated the influence of Allolobophora chlorotica and Aporrectodea nocturna , two earthworm species from different ecological categories, on the incorporation of organic matter, reproduction and weight change of the endogeic Aporrectodea caliginosa. Two different types of organic matter i) a compost and ii) a fresh refined organic material, both from green waste, were used. Earthworm parameters were monitored during an 8-weeks laboratory experiment using a vineyard soil in order to identify positive and negative interactions between species. Irrespective of species, earthworms interacted preferentially with smaller particles, more decomposed and with a lower C/N ratio. For an equivalent earthworm biomass, similar amount of green compost was incorporated by A. caliginosa and A. nocturna. However, A. chlorotica did not bury this material. The green compost increased the reproduction rate of A. caliginosa when associated to A. chlorotica. Moreover, the association with the epi-anecic A. nocturna increased the reproduction rate of A. caliginosa with the addition of refined organic matter (fresh material). Furthermore, in both earthworm associations, the weight loss of A. caliginosa was reduced by the addition of green compost to the soil surface. These results highlight the importance of earthworm interactions in maintaining populations, and emphasized the need of field studies to confirm these interactions, particularly in the context of soil fertility where organic amendments are often applied. • A. nocturna increases A. caliginosa reproduction rate with fresh organic matter. • Green compost improves earthworm body mass and reproduction rate compared to control. • Earthworms incorporate more easily fine organic particles with low C/N ratio. • A. caliginosa incorporates green compost similarly to A. nocturna. [ABSTRACT FROM AUTHOR]

To assess the effects of agricultural management strategies on earthworm energy reserves (glycogen and lipids), 16 apple orchards under different strategies (organic, Integrated Pest Management (IPM), conventional and abandoned) were selected. Soil samples and 10 adults of the most common earthworm species ( Allolobophora chlorotica ) were sampled in each orchard. The glycogen and lipid concentrations in the earthworms did not correlate with any soil characteristics and no significant differences in earthworm weight were observed between strategies. However, significantly lower glycogen and lipid concentrations were found in earthworms inhabiting conventional orchards, with a decrease of 45 and 63% compared to organic and abandoned orchards respectively. Earthworms from IPM orchards had intermediate values. This suggests that pesticide usage leads to the observed decrease of energy reserves in A. chlorotica . Thus the reduced insecticide use in IPM compared to conventional strategies, albeit significant, appears to be too small to result in pronounced effects on energy reserves in this earthworm species. [ABSTRACT FROM AUTHOR]

The growing usage of plant protection products in agroecosystems questions their unintentional deleterious effects on non-target organisms such as earthworms and their related ecological function. The aim of this study was to investigate the turnover of soil microbiota during the gut transit of two endogeic earthworms (Aporrectodea caliginosa and Allolobophora chlorotica) and how it is possibly affected by the organophosphorus insecticide ethyl-parathion (Pt). We measured prokaryotic and micro-eukaryotic community composition and abundances using two different soil types in the continuum of bulk soil, gut and casts samples collected during and after one week exposure to Pt. The presence of earthworm and Pt had limited effects on the soil microbiota, although Pt altered temporarily soil microbial abundances in the presence of earthworms, not in their absence. The earthworms' gut harbored a remarkably low prokaryotic diversity, dominated by two molecular Operational Taxonomic Units (mOTUs) affiliated to Rhodococcus and Pseudarthrobacter. Contrastingly, gut micro-eukaryotic communities were maintained at diversity levels similar to that of the soil, yet Pt augmented their diversity and changed their composition. Fresh casts collected out of the soil revealed evident variation of abundance, diversity and community composition according to the soil origin and the earthworm species. In particular, following gut transit casts were enriched with Bacteroidia and γ-Proteobacteria while depleted of Acidobacteria. Exposure to Pt increased the diversity and relative abundance of depleted mOTUs, which paves the way for future investigations on the role of Pt in rising microbial consumption due to an over-excitation of gastrointestinal motility. • The earthworms' gut microbiome shows restricted bacterial and diversified micro-eukaryotic compositions. • The stimulation of specific micro-eukaryotes in casts depends on the earthworm species. • Microbial taxa depleted from soil during digestion reveal the earthworms' opportunistic feeding strategy. • Ethyl-parathion mostly affects the soil and casts microbiota and marginally micro-eukaryotes of the gut microbiota. • Ethyl-parathion modifies the digestive capacities of earthworms by enhancing depletion of microbes from soil. [ABSTRACT FROM AUTHOR]

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 4 individuals/kg wet soil) and the time of exposure (1, 2, 6, 12, and 18 wk) could affect chlorpyrifos persistence in soil initially treated with 20 mg active ingredient kg − 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 30.3 d (control soils) and 44.5 d (low earthworm density) or 36.7 d (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 6 wk 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. [ABSTRACT FROM AUTHOR]

Pesticides exert important effects on the soil fauna and health. However, little is known about the interactions of soil, microorganisms and earthworms in the presence of pesticides and about their respective roles in the soil biological activity. The aim of this study was to evaluate the effect of the soil type on enzyme activities, measured in bulk soil and in casts of two earthworm species, after exposure to the organophosphorus pesticide parathion. To this aim, two endogeic earthworm species (Apporectodea caliginosa and Allolobophora chlorotica) were cross-acclimated in two different soil textures (each representing the most favorable soil environment for that species). Enzyme activities were measured as a soil quality indicator in samples of bulk soil (collected at day 4 and day 7 of exposure to parathion) and in earthworm casts (collected at day 7). A short exposure (4 days) to parathion significantly (ANOVA, p < 0.001) inhibited carboxylesterases (25–43 % of inhibition) and alkaline phosphatase (∼23 % of inhibition). At day 7 of exposure, parathion impact on the overall soil enzyme activities mainly depended on the soil texture. Indeed, activity inhibition was higher (ANOVA, p < 0.001) in silt-clay soil (decrease by 37 %) than in silt-loamy soil (decrease by ∼18 %). Conversely, parathion effect was not influenced by earthworm presence/absence and earthworm species. However, after soil exposure to parathion, earthworms (both species) improved enzyme activity restoration in their casts. • Parathion exposure induced a decrease in bulk soil enzyme activities. • Bulk soil enzyme activity changes were influenced by the soil type. • After parathion exposure, soil enzyme activities were restored in earthworm casts. • Earthworm species did not influence enzyme activities in control and polluted soils. [ABSTRACT FROM AUTHOR]

Abstract: 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 (α-naphthyl acetate [α-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 (α-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 (IC50) was comparable between tissue (IC50s range=4.01–9.67nM dichlorvos and 8480–6880nM paraoxon) and soil (IC50s 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. [Copyright &y& Elsevier]

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. • Species-specific tissue distribution of B-esterases was assessed. • Glutathione-S-transferase is exclusively cytosolic in both earthworm species. • Similar efficiency and OP sensitivity of carboxylesterase was found in both species. • Species differences in acetylcholinesterase inhibition depend on OP structure. • A. chlorotica was less sensitive to OP after in vivo compared to in vitro exposure. [ABSTRACT FROM AUTHOR]