Christophe Mazzia

B-type esterases in the snail Xeropicta derbentina : An enzymological analysis to evaluate their use as biomarkers of pesticide exposure

The study was prompted to characterize the B-type esterase activities in the terrestrial snail Xeropicta derbentina and to evaluate its sensitivity to organophosphorus and carbamate pesticides. Specific cholinesterase and carboxylesterase activities were mainly obtained with acetylthiocholine (K(m)=77.2 mM; V(max)=38.2 mU/mg protein) and 1-naphthyl acetate (K(m)=222 mM, V(max)=1095 mU/mg protein) substrates, respectively. Acetylcholinesterase activity was concentration-dependently inhibited by chlorpyrifos-oxon, dichlorvos, carbaryl and carbofuran (IC50=1.35x10(-5)-3.80x10(-8) M). The organophosphate-inhibited acetylcholinesterase activity was reactivated in the presence of pyridine-2-aldoxime methochloride. Carboxylesterase activity was inhibited by organophosphorus insecticides (IC50=1.20x10(-5)-2.98x10(-8) M) but not by carbamates. B-esterase-specific differences in the inhibition by organophosphates and carbamates are discussed with respect to the buffering capacity of the carboxylesterase to reduce pesticide toxicity. These results suggest that B-type esterases in X. derbentina are suitable biomarkers of pesticide exposure and that this snail could be used as sentinel species in field monitoring of Mediterranean climate regions.

Earthworm behaviour as a biomarker – a case study using imidacloprid

To determine whether earthworm behaviour can be used as a biomarker for pollutant effects, an experiment was set up using ®Confidor (imidacloprid) as a model pesticide. The burrowing behaviour of two earthworm species ofdifferent ecological types (the anecic Aporrectodea nocturna and the endogeic Allolobophora icterica) was studied using 2D terraria. The activity of two classical biochemical markers (AchE and GST activities) was also measured. Imidacloprid had no effect on the biochemical markers in either earthworm species whatever the concentration (0.01, 0.1 and 1 ppm). In contrast, earthworm behaviour changed dramatically in the presence of imidacloprid. Following one week of exposure to the pesticide at two different concentrations (0.5 and 1 ppm), burrow length, the rate of burrow reuse and the distance covered decreased for both species. We conclude that earthworm behaviour is a sensitive biomarker that can be studied with cheap and simple apparatus such as 2D terraria.

Assessing ecotoxicity and uptake of metals and metalloids in relation to two different earthworm species (Eiseina hortensis and Lumbricus terrestris)

Due to diffuse atmospheric fallouts of process particles enriched by metals and metalloids, polluted soils concern large areas at the global scale. Useful tools to assess ecotoxicity induced by these polluted soils are therefore needed. Earthworms are currently used as biotest, however the influence of specie and earthworm behaviour, soil characteristics are poorly highlighted. Our aim was therefore to assess the toxicity of various polluted soils with process particles enriches by metals and metalloids (Pb, Cd, Cu, Zn, As and Sb) collected from a lead recycling facility on two earthworm species belonging to different ecological types and thus likely to have contrasted behavioural responses (Eiseina hortensis and Lumbricus terrestris). The combination of behavioural factors measurements (cast production and biomass) and physico-chemical parameters such as metal absorption, bioaccumulation by earthworms and their localization in invertebrate tissues provided a valuable indication of pollutant bioavailability and ecotoxicity. Soil characteristics influenced ecotoxicity and metal uptake by earthworms, as well as their soil bioturbation.

Snail phenotypic variation and stress proteins: do different heat response strategies contribute to Waddington's widget in field populations?

On the basis of studies with laboratory strains of Drosophila and Arabidopsis, it has been hypothesized that potential buffers to the expression of phenotypic morphological variation, such as Hsp90 and possibly Hsp70, represent important components of Waddingtons widget, which may confer capacitive evolution. As studies on field populations of living organisms to test this hypothesis are lacking, we tested whether a heat response strategy involving high stress protein levels is associated with low morphological variation and vice versa, using four natural populations of Mediterranean pulmonate snails. In response to 8 hr of elevated temperatures, a population of Xeropicta derbentina with uniform shell pigmentation pattern showed remarkably high Hsp70 but low Hsp90 levels. In contrast, a highly variable population of Cernuella virgata kept both Hsp90 and Hsp70 levels low when held at diverse though environmentally relevant temperatures. Two other populations (Theba pisana and another X. derbentina population) with intermediate variation in shell pigmentation pattern were also intermediate in inducing Hsp70, though Hsp90 was maintained at a low level. The observed correlation of stress protein levels and coloration pattern variation provide the first indirect evidence for an association of stress proteins with Waddingtons widget under natural conditions.

Neuronal location of 5-hydroxytryptamine3 receptor-like immunoreactivity in the rat colon.

The presence of 5-hydroxytryptamine(3) receptors on enteric neurons is known from pharmacological data that date back more than 40 years. However, an adequate account of which neurons bear these receptors has not been made because suitable antisera have not been available. We have found that the majority of antisera that have been raised against sequences from the 5-hydroxytryptamine(3) receptor also recognize pre-prosomatostatin. We report that this source of false labeling can be eliminated by pre-incubating the antisera with a peptide designed for this purpose. We have used the pre-absorbed antiserum to localize 5-hydroxytryptamine(3) receptors in the rat colon. Immunoreactive nerve cell bodies occurred in the myenteric and submucosal ganglia. The majority had smooth cell bodies and long, smooth processes, that is, Dogiel type II morphology. The initial segments of the long processes of the Dogiel type II neurons were strongly immunoreactive. About 12% of immunoreactive myenteric nerve cells were of the same or smaller size, and had multiple short filamentous processes. Some of the immunoreactive Dogiel type II neurons were also immunoreactive for calretinin in both plexuses, and the majority were immunoreactive for calbindin in submucosal ganglia. Specific immunoreactivity occurred in non-varicose, but not in varicose, fibers in the myenteric and submucosal ganglia, and in fiber bundles that traversed the longitudinal and circular muscle layers. Immunoreactive varicose fibers were observed only in the mucosa. It is concluded that 5-hydroxytryptamine(3) receptors occur on intrinsic sensory neurons in the rat colon, and on extrinsic sensory nerve fibers that innervate the colon.

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.

Cholinesterase activity as a biomarker of pesticide exposure in Allolobophora chlorotica earthworms living in apple orchards under different management strategies

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-km(2) 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.

Inhibition, recovery and oxime-induced reactivation of muscle esterases following chlorpyrifos exposure in the earthworm Lumbricus terrestris.

Assessment of wildlife exposure to organophosphorus (OP) pesticides generally involves the measurement of cholinesterase (ChE) inhibition, and complementary biomarkers (or related endpoints) are rarely included. Herein, we investigated the time course inhibition and recovery of ChE and carboxylesterase (CE) activities in the earthworm Lumbricus terrestris exposed to chlorpyrifos, and the ability of oximes to reactivate the phosphorylated ChE activity. Results indicated that these esterase activities are a suitable multibiomarker scheme for monitoring OP exposure due to their high sensitivity to OP inhibition and slow recovery to full activity levels following pesticide exposure. Moreover, oximes reactivated the inhibited ChE activity of the earthworms exposed to 12 and 48 mg kg(-1) chlorpyrifos during the first week following pesticide exposure. This methodology is useful for providing evidence for OP-mediated ChE inhibition in individuals with a short history of OP exposure (< or = 1 week); resulting a valuable approach for assessing multiple OP exposure episodes in the field.

Solar radiation stress in climbing snails: behavioural and intrinsic features define the Hsp70 level in natural populations of Xeropicta derbentina (Pulmonata)

Ectotherms from sunny and hot environments need to cope with solar radiation. Mediterranean land snails of the superfamily Helicoidea feature a behavioural strategy to escape from solar radiation-induced excessive soil heating by climbing up vertical objects. The height of climbing, and also other parameters like shell colouration pattern, shell orientation, shell size, body mass, actual internal and shell surface temperature, and the interactions between those factors may be expected to modulate proteotoxic effects in snails exposed to solar radiation and, thus, their stress response. Focussing on natural populations of Xeropicta derbentina, we conducted a ‘snapshot’ field study using the individual Hsp70 level as a proxy for proteotoxic stress. In addition to correlation analyses, an IT-model selection approach based on Akaike’s Information Criterion was applied to evaluate a set of models with respect to their explanatory power and to assess the relevance of each of the above-mentioned parameters for individual stress, by model averaging and parameter estimation. The analysis revealed particular importance of the individuals’ shell size, height above ground, the shell colouration pattern and the interaction height × orientation. Our study showed that a distinct set of behavioural traits and intrinsic characters define the Hsp70 level and that environmental factors and individual features strongly interact.

Effects of historic metal(loid) pollution on earthworm communities

The effects of metal(loid)s (Pb, Cd, Cu, Zn, As and Sb) from atmospheric fallout on earthworm communities were investigated in a fallow meadow located close to a 60-year-old lead recycling factory. We examined abundance and species diversity as well as the ratio of adult-to-juvenile earthworms, along five 140 m parallel transects. The influence of soil pollution on the earthworm community at the plot scale was put in context by measuring some physico-chemical soil characteristics (OM content, N content, pH), as well as total and bioavailable metal(loid) concentrations. Earthworms were absent in the highly polluted area (concentration from 30,000 to 5000 mg Pb·kg(-1) of dried soil), just near the factory (0-30 m area). A clear and almost linear relationship was observed between the proportion of juvenile versus mature earthworms and the pollution gradient, with a greater proportion of adults in the most polluted zones (only adult earthworms were observed from 30 to 50 m). Apporectodea longa was the main species present just near the smelter (80% of the earthworms were A. longa from 30 to 50 m). The earthworm density was found to increase progressively from five individuals·m(-2) at 30 m to 135 individuals·m(-2) at 140 m from the factory. On average, metal(loid) accumulation in earthworm tissues decreased linearly with distance from the factory. The concentration of exchangeable metal(loid)s in earthworm surface casts was higher than that of the overall soil. Finally, our field study clearly demonstrated that metal(loid) pollution has a direct impact on earthworm communities (abundance, diversity and proportion of juveniles) especially when Pb concentrations in soil were higher than 2050 mg·kg(-1).