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International audience; Endogeic earthworms live and burrow in the soil to find their food. They burrow by pushing the soil aside or ingesting it and are thus sensitive to soil compaction. However there is a scarcity of data regarding the effects of soil bulk density on the burrowing behavior and activity of endogeic earthworms. We carried out laboratory experiments using repacked soil cores with various levels of bulk density (from 1.18 to 1.38 g cm-3) in which individuals of Aporrectodea caliginosa or Allolobophora chlorotica were incubated for six weeks. The burrow systems inside the soil cores and the compaction around the burrows were then analyzed using X-ray tomography. Soil water infiltration measurements were also carried out. The increase in bulk density had a negative impact on all burrow system characteristics (length, volume, diameter, continuity, number of burrows). When bulk density increased from 1.18 to 1.38 g cm-3, volume, diameter, continuity and the number of burrows decreased on average by 77 %, 21 %, 81 % and 58 %, respectively. The increase in density due to compaction around the burrows was similar whatever the species and the bulk density. Increasing soil bulk density from 1.18 to 1.38 g cm-3 also greatly decreased water infiltration (-89 % for both species) and increased breakthrough time (10 and 25-fold for A. chlorotica and A. caliginosa respectively). However, compared to a control without earthworms, water infiltration in cores incubated with endogeic species was only increased significantly at 1.18 and 1.23 g cm-3. This illustrates that burrows made by endogeic earthworms moderately increase water infiltration and only when the soil bulk density is low. Data provided in this study could be used to refine simulation models of earthworm burrowing behavior where burrowing is assumed to be mainly governed by soil water content, temperature and soil bulk density.

International audience; 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-5-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) chlorpyrifosethyl-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 7d 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.

Interactions of earthworm species with their environment and in particular the soil they inhabit is poorly understood. In this study, we aimed to use mid infrared absorption (MIR) spectroscopy of earthworm bodies in combination with discriminant analysis of principal components (DAPC), in order to investigate if their biogeochemical composition is determined by their species belonging or the soil they inhabit. Our conceptual approach included the comparison of MIR spectra of four earthworm species (Lumbricus terrestris, Allolobophora chlorotica, Aporrectodea caliginosa and Aporrectodea icterica) sampled from four different agricultural fields in France. Moreover, we tested the influence of the habitat on the earthworm MIR spectra by exchanging individuals and soils in an incubation experiment. MIR spectra varied according to species and sampling site. The MIR spectra of earthworm bodies also changed during the incubation experiment, and for L. terrestris, A. chlorotica and A. icterica depended on the properties of the soil ingested. This suggested that the food source in different habitats impacted the biogeochemical composition of earthworm bodies. DAPC allowed for the identification of earthworm species ranging from 64 to 100% of accuracy (mean 78%). We thus conclude that MIR spectroscopy may be a suitable approach to identify earthworm species, but that their signature may also allow to investigate their ecology as determined by the soil they inhabit.

Accurate species identification is crucial in ecological research on sentinel species such as earthworms. Here, we developed a reliable and cost-efficient method for the rapid identification of taxa within the Allolobophora chlorotica complex of cryptic earthworm species. We combined high resolution melting (HRM) analysis with DNA barcoding (Bar-HRM) in a three-step approach: (i) selection of a mini-barcode in the 16S mitochondrial gene, (ii) test of the method on a panel of 16 reference individuals and (iii) identification at the species level of 24 unknown specimens. Thus, we proved the efficacy of Bar-HRM to discriminate between cryptic sister (or sibling) earthworm species and we provided a standardized method that can be easily adapted to other taxa.

Pesticides used to protect plants from pests, threat grievously non-target organisms such as earthworms. Due to their feeding and burrowing activities, earthworms are in direct contact with soil particles and microorganisms, as well as pollutants including pesticides. This work investigated (1) the effect of an organophosphate “ethyl-parathion” on the sensitivity of two endogeic earthworms’ species, Aporrectodea caliginosa and Allolobophora chlorotica; and (2) the role of the gut-microbiota, in synergy with the earthworm’s detoxification pathways, in pesticide tolerance or detoxification. In the first part, biochemical and behavioral responses showed that A. caliginosa is more sensitive to “ethyl-parathion” exposure than A. chlorotica. The endpoints measured ranged from physiological (weight), biochemical (AChE, CbEs, GST) to behavioral biomarkers (cast production and burrowing activity). Our findings showed that the sensitivity of A. caliginosa could be mainly due to the intrinsic sensitivity of its AChE to “ethyl-parathion”. The role of the carboxylesterases, acting as bioscavenger of OP, and the role of the detoxifying enzymes GST did not appear to be efficient mechanisms involved in A. chlorotica tolerance. In the second part, we aimed to characterize the microbiome within the ingested soil, the cast and the gut tissue of A. caliginosa and A. chlorotica in control or polluted soils. Our results showed differences in the microbial composition between these compartments. In this line, we suggested that these two earthworms’ species harbor a species-specific microbiome in their gut. In particular, our findings showed that the earthworm’s gut acts as a “biological filter” for ingested microbial communities during the gut passage. At the level of the gut, we identified four dominated genus within the gut of A. caliginosa versus two dominated genus in the gut of A. chlorotica. Notably, we identified a Rhodococcus strain, which is highly abundant in the gut of A. chlorotica. Previous studies reported Rhodococcus strains for their ability to degrade some group of pesticides. We suggest that the presence of this strain could contribute to the tolerance of A. chlorotica. Finally, we showed that the effect of ethyl-parathion on soil enzyme activities mainly depend on soil texture rather than the presence and/or the species of earthworms. According to our findings, it is of considerable importance to include more than one species to assess toxicity from organophosphorus insecticides, due to the interspecific differences that can occur within the same ecological category. Moreover, the identification and the functional analysis of the microorganisms found in the earthworm’s gut and able to intervene in pesticide detoxification could enhance our knowledge about the fate of the pesticide inside the organism, and could be an important tool for bioremediation program.; Les pesticides utilisés pour protéger les plantes des insectes nuisibles constituent une menace pour les organismes non cibles tels que les vers de terre. En raison de leur activité de bioturbation de sol, les vers de terre sont en contact direct avec les particules et les micro-organismes du sol, ainsi qu'avec les polluants, notamment les pesticides. L‘objectif de ce travail est d‘étudier (1) l‘effet d‘un organophosphoré (OP) «éthyl-parathion» sur la sensibilité de deux espèces de vers de terre endogés, Aporrectodea caliginosa et Allolobophora chlorotica; et (2) le rôle du microbiote intestinal, en synergie avec les voies de détoxification du ver de terre, dans la tolérance ou la détoxification des pesticides. Dans la première partie, les réponses biochimiques et comportementales ont montré que A. caliginosa est plus sensible à l'exposition à «l‘éthyl-parathion» que A. Chlorotica. Les résultats portent sur l‘analyse de biomarqueurs physiologiques (poids), biochimiques (AChE, CbEs, GST) et comportementaux (production de turricules et activité de creusement). Nous avons montré que la sensibilité de A. caliginosa semble liée à la sensibilité intrinsèque de l‘AChE à «l‘éthyl-parathion». De plus, le rôle des carboxylestérases, capables de piéger les insecticides OP, ainsi que le rôle de détoxification des GST notamment, ne semblaient pas être des mécanismes efficaces impliqués dans la tolérance de A. chlorotica. Dans la deuxième partie, nous avons caractérisé, en présence ou non d‘insecticide, le microbiote dans le sol ingéré, les turricules et les intestins des 2 vers de terre. Nos résultats ont montré des différences dans la composition microbienne de ces compartiments. A cet égard, nous avons suggéré que chacune de ces espèces hébergent un microbiote spécifique de l‘espèce dans leur intestin. Nos résultats ont notamment montré que l‘intestin du ver de terre agit comme un «filtre biologique» pour les communautés microbiennes ingérées lors du passage dans l‘intestin. A ce niveau, nous avons identifié, au niveau bactérien, quatre genres dominants dans l'intestin de A. caliginosa et deux genres dominants dans l'intestin de A. chlorotica. Nous avons notamment identifié une souche de Rhodococcus, très abondante dans l'intestin de A. chlorotica. Des études ont montré que des souches de Rhodococcus peuvent dégrader certains groupes de pesticides. Nous suggérons que la présence de cette souche pourrait contribuer à la tolérance de A. chlorotica. Enfin, nous avons montré que l‘effet de l‘éthyl-parathion sur les activités enzymatiques du sol dépend principalement de la texture du sol et non pas de la présence et/ou de l‘espèce de ver de terre.Selon nos conclusions, il est extrêmement important d'inclure plus d'une espèce pour évaluer la toxicité des insecticides organophosphorés, en raison des différences interspécifiques pouvant se produire au sein d'une même catégorie écologique. De plus, l'identification et l'analyse fonctionnelle des micro-organismes présents au niveau de l‘intestin et susceptibles d'intervenir dans la détoxication des pesticides permettraient d‘améliorer nos connaissances sur le devenir du pesticide dans l'organisme et pourraient constituer un outil important dans les programmes de bioremédiation.

Pesticides used to protect plants from pests, threat grievously non-target organisms such as earthworms. Due to their feeding and burrowing activities, earthworms are in direct contact with soil particles and microorganisms, as well as pollutants including pesticides. This work investigated (1) the effect of an organophosphate “ethyl-parathion” on the sensitivity of two endogeic earthworms’ species, Aporrectodea caliginosa and Allolobophora chlorotica; and (2) the role of the gut-microbiota, in synergy with the earthworm’s detoxification pathways, in pesticide tolerance or detoxification. In the first part, biochemical and behavioral responses showed that A. caliginosa is more sensitive to “ethyl-parathion” exposure than A. chlorotica. The endpoints measured ranged from physiological (weight), biochemical (AChE, CbEs, GST) to behavioral biomarkers (cast production and burrowing activity). Our findings showed that the sensitivity of A. caliginosa could be mainly due to the intrinsic sensitivity of its AChE to “ethyl-parathion”. The role of the carboxylesterases, acting as bioscavenger of OP, and the role of the detoxifying enzymes GST did not appear to be efficient mechanisms involved in A. chlorotica tolerance. In the second part, we aimed to characterize the microbiome within the ingested soil, the cast and the gut tissue of A. caliginosa and A. chlorotica in control or polluted soils. Our results showed differences in the microbial composition between these compartments. In this line, we suggested that these two earthworms’ species harbor a species-specific microbiome in their gut. In particular, our findings showed that the earthworm’s gut acts as a “biological filter” for ingested microbial communities during the gut passage. At the level of the gut, we identified four dominated genus within the gut of A. caliginosa versus two dominated genus in the gut of A. chlorotica. Notably, we identified a Rhodococcus strain, which is highly abundant in the gut of A. chlorotica. Previous studies reported Rhodococcus strains for their ability to degrade some group of pesticides. We suggest that the presence of this strain could contribute to the tolerance of A. chlorotica. Finally, we showed that the effect of ethyl-parathion on soil enzyme activities mainly depend on soil texture rather than the presence and/or the species of earthworms. According to our findings, it is of considerable importance to include more than one species to assess toxicity from organophosphorus insecticides, due to the interspecific differences that can occur within the same ecological category. Moreover, the identification and the functional analysis of the microorganisms found in the earthworm’s gut and able to intervene in pesticide detoxification could enhance our knowledge about the fate of the pesticide inside the organism, and could be an important tool for bioremediation program.; Les pesticides utilisés pour protéger les plantes des insectes nuisibles constituent une menace pour les organismes non cibles tels que les vers de terre. En raison de leur activité de bioturbation de sol, les vers de terre sont en contact direct avec les particules et les micro-organismes du sol, ainsi qu'avec les polluants, notamment les pesticides. L‘objectif de ce travail est d‘étudier (1) l‘effet d‘un organophosphoré (OP) «éthyl-parathion» sur la sensibilité de deux espèces de vers de terre endogés, Aporrectodea caliginosa et Allolobophora chlorotica; et (2) le rôle du microbiote intestinal, en synergie avec les voies de détoxification du ver de terre, dans la tolérance ou la détoxification des pesticides. Dans la première partie, les réponses biochimiques et comportementales ont montré que A. caliginosa est plus sensible à l'exposition à «l‘éthyl-parathion» que A. Chlorotica. Les résultats portent sur l‘analyse de biomarqueurs physiologiques (poids), biochimiques (AChE, CbEs, GST) et comportementaux (production de turricules et activité de creusement). Nous avons montré que la sensibilité de A. caliginosa semble liée à la sensibilité intrinsèque de l‘AChE à «l‘éthyl-parathion». De plus, le rôle des carboxylestérases, capables de piéger les insecticides OP, ainsi que le rôle de détoxification des GST notamment, ne semblaient pas être des mécanismes efficaces impliqués dans la tolérance de A. chlorotica. Dans la deuxième partie, nous avons caractérisé, en présence ou non d‘insecticide, le microbiote dans le sol ingéré, les turricules et les intestins des 2 vers de terre. Nos résultats ont montré des différences dans la composition microbienne de ces compartiments. A cet égard, nous avons suggéré que chacune de ces espèces hébergent un microbiote spécifique de l‘espèce dans leur intestin. Nos résultats ont notamment montré que l‘intestin du ver de terre agit comme un «filtre biologique» pour les communautés microbiennes ingérées lors du passage dans l‘intestin. A ce niveau, nous avons identifié, au niveau bactérien, quatre genres dominants dans l'intestin de A. caliginosa et deux genres dominants dans l'intestin de A. chlorotica. Nous avons notamment identifié une souche de Rhodococcus, très abondante dans l'intestin de A. chlorotica. Des études ont montré que des souches de Rhodococcus peuvent dégrader certains groupes de pesticides. Nous suggérons que la présence de cette souche pourrait contribuer à la tolérance de A. chlorotica. Enfin, nous avons montré que l‘effet de l‘éthyl-parathion sur les activités enzymatiques du sol dépend principalement de la texture du sol et non pas de la présence et/ou de l‘espèce de ver de terre.Selon nos conclusions, il est extrêmement important d'inclure plus d'une espèce pour évaluer la toxicité des insecticides organophosphorés, en raison des différences interspécifiques pouvant se produire au sein d'une même catégorie écologique. De plus, l'identification et l'analyse fonctionnelle des micro-organismes présents au niveau de l‘intestin et susceptibles d'intervenir dans la détoxication des pesticides permettraient d‘améliorer nos connaissances sur le devenir du pesticide dans l'organisme et pourraient constituer un outil important dans les programmes de bioremédiation.

International audience; Landscape features are known to alter the spatial genetic variation of aboveground organisms. Here, we tested the hypothesis that the genetic structure of belowground organisms also responds to landscape structure. Microsatellite markers were used to carry out a landscape genetic study of two endogeic earthworm species, Allolobophora chlorotica (N = 440, eight microsatellites) and Aporrectodea icterica (N = 519, seven microsatellites), in an agricultural landscape in the North of France, where landscape features were characterized with high accuracy. We found that habitat fragmentation impacted genetic variation of earthworm populations at the local scale. A significant relationship was observed between genetic diversity (He, Ar) and several landscape features in A. icterica populations and A. chlorotica. Moreover, a strong genetic differentiation between sites was observed in both species, with a low degree of genetic admixture and high Fst values. The landscape connectivity analysis at the regional scale, including isolation by distance, least-cost path and cost-weighted distance approaches, showed that genetic distances were linked to landscape connectivity in A. chlorotica. This indicates that the fragmentation of natural habitats has shaped their dispersal patterns and local effective population sizes. Landscape connectivity analysis confirmed that a priori favourable habitats such as grasslands may constitute dispersal corridors for these species.

Earthworms are crucial for production and maintenance of soil structure and their activities can strongly impact soil functioning ( e.g. water regulation, nutrient dynamics). This laboratory study investigated the bioturbation activity of three endogeic species, Allolobophora chlorotica , Allolobophora icterica and Aporrectodea caliginosa , as affected by different locations of organic matter (OM) in the soil profile: OM scattered on the soil surface (surface-OM) or homogeneously mixed into the soil (mixed-OM). Microcosms, each containing a combination of one species (three individuals) and one OM location, were subjected to controlled environmental conditions (temperature, humidity and day/night cycle) for 60 days. At the end of the experiment, microcosms were cut into multiple horizontal cross-sections every centimetre and bioturbation activities were analyzed based on the number of burrows, the burrowed area and the percentage of burrowed area totally refilled with casts. Results showed that regardless of species, there was significantly fewer burrows and a greater percentage of burrowed area refilled with casts under mixed-OM than under surface-OM. A. chlorotica and A. caliginosa had a significantly greater burrowed area under mixed-OM than under surface-OM. Regardless of OM location, as depth increased, burrow number and area decreased for A. chlorotica and generally increased for A. icterica . In contrast, burrowing activity of A. caliginosa was affected by OM location as depth increased: under mixed-OM, burrow number decreased but burrowed area remained constant, whereas under surface-OM, burrow number remained constant and burrowed area increased. These results improve understanding of effects of endogeic species on soil structure and highlight effects of OM location on earthworm bioturbation. Especially this study gives information about the burrowing activity of A. icterica which has so far been little documented, and also informs about refilled burrows which is a major parameter for soil functioning.

International audience; Earthworm burrow systems are generally described based on postulated behaviours associated with the three ecological types. In this study, we used X-ray tomography to obtain 3D information on the burrowing behaviour of six very common anecic (Aporrectodea nocturna and Lumbricus terrestris) and endogeic (Aporrectodea rosea, Allolobophora chlorotica, Aporrectodea caliginosa, Aporrectodea icterica) earthworm species, introduced into repacked soil cores for 6 weeks. A simple water infiltration test, the Beerkan method, was also used to assess some functional properties of these burrow systems. Endogeic worms make larger burrow systems, which are more highly branched, less continuous and of smaller diameter, than those of anecic worms. Among the anecic species, L. terrestris burrow systems are shorter (9.2 vs 21.2 m) with a higher number (14.5 vs 23.5) of less branched burrows (12.2 vs 20.2 branches m(-1)), which are also wider (7.78 vs 5.16 mm) than those of A. nocturna. In comparison, the burrow systems made by endogeic species appeared similar to each other. However, A. rosea burrows were short and narrow, whereas A. icterica had a longer burrow system (15.7 m), more intense bioturbation intensity (refilled macropores or soil lateral compaction around them) and thus a greater number of burrows. Regarding water infiltration, anecic burrow systems were far more efficient due to open burrows linking the top and bottom of the cores. For endogeic species, we observed a linear relationship between burrow length and the water infiltration rate (R (2) = 0.49, p < 0.01). Overall, the three main characteristics significantly influencing water infiltration were burrow length, burrow number and bioturbation volume. This last characteristic highlighted the effect of burrow refilling by casts

International audience; Although the role of earthworms in soil functioning is often emphasised, many important aspects of earthworm behaviour are still poorly understood. In this study we propose a simple and cost-effective method for estimating burrow system area and continuity, as well as a new and often neglected parameter, the percentage of burrow refilling by the earthworms own casts. This novel parameter is likely to have a huge influence on the transfer properties of the burrow system. The method uses standard repacked soil cores in PVC cylinders and takes advantages of clay shrinkage and the fact that earthworms were previously shown to prefer to burrow at the PVC/soil interface. In this way, after removing the PVC cylinders off dry cores, the external section of the burrow system made by earthworms along the soil walls could be easily described. We applied this method to characterise the burrow systems of four earthworms species: two anecics (Aporrectodea caliginosa nocturna and Aporrectodea caliginosa meridionalis) and two endogeics (Aporrectodea caliginosa icaliginosa and Allolobophora chlorotica). After one month the burrow's area generated by both anecic species were much larger (about 40 cm(2)) than the endogeic burrow's area (about 15 cm(2)). A. noctuma burrow system continuity was higher than that of A. meridionalis and both anecic burrow systems were more continuous than those made by the endogeic earthworms. This was partly explained by the far larger proportion of the burrow area that was refilled with casts: approximately 40% and 50% for Al. chlorotica and A. caliginosa, respectively compared with approximately 20% for the anecic burrows. We discuss whether these estimates could be used in future models simulating the dynamics of earthworm burrow systems by taking into account both burrow creation and destruction by earthworms. (C) 2013 Elsevier B.V. All rights reserved.

International audience; By creating burrows, earthworms influence the transfer properties of soils. The effects of endogeic species on soil transfer properties, however, are not yet well understood because these earthworms generally create burrows that are refilled by casts and have no preferential vertical orientation. Thirty soil cores were incubated for various periods (1–3 or 4 weeks) at different earthworm densities (70, 210, 345 or 480 individuals m −2). The cores were then scanned using X-ray tomography and the burrow systems were characterised by measuring the total burrow volume, bioturbation volume (refilled burrows and lateral compaction around the burrows), the number of branches, tortuosity and continuity (assessed by computing the number of burrows with a vertical extension greater than 15, 20 and 25% of the core). We also computed the mean geodesic distance, i.e. the mean distance from the bottom to the top of the core assuming that distances inside burrows are null. Rainfall simulations were carried out on 17 cores chosen to encompass the variations observed in the burrow systems. The water transfer efficiency of each core was estimated by measuring two parameters: breakthrough volume and the percentage of water transmitted after 1 h of rain. Burrow and bioturbation volume increased significantly and steadily with time and earthworm density. We estimated that on average Allolobophora chlorotica burrowed 22 cm per week. All other burrow system characteristics also increased with time and earthworm density except the mean geodesic distance, which decreased significantly. This suggests that intraspecific interactions had no significant effect on burrow system geometry. Univariate PLS regressions were used to understand which burrow system characteristics had the strongest influence on water transfer. These regressions showed that the mean geodesic distance was the most important parameter. This means that in addition to individual burrow characteristics, the spatial arrangement of the whole burrow system also had a major effect on transfer properties.

International audience; X-ray tomography is used increasingly to study the macroporosity resulting from earthworm activity. However, macropores are not the only features visible on the images; other zones resulting from bioturbation by earthworms can be detected due to differences in greylevels. Four different soil cores were incubated with two earthworm species at two different densities (4 or 8 adults of the endogeic species Allolobophora chlorotica or 2 or 4 adults of the anecic species Aporrectodea nocturna). A fifth core without earthworms was used as a control. After six weeks, the cores were analysed by X-ray tomography using a medical scanner. The 3D earthworm burrow systems were reconstructed and a new and specific algorithm was used to determine other bioturbated zones (BZ) that were physically influenced by the earthworms. Expected differences in the structure of the burrow systems between the endogeic and anecic species were observed: the A. chlorotica burrows were narrower and more numerous, more branched and less vertical. When the earthworm density doubled, the volume and length of the A. chlorotica burrow system increased whereas no increase was observed for A. nocturna. The BZ, which were located in the upper section of the cores, represented almost the same volume as the macropores. These zones tended to be located further from the burrows in the A. chlorotica cores: 50% of the voxels corresponding to BZ were at a distance greater than 4 and 5.5 mm from the closest macropore for A. nocturna and A. chlorotica, respectively. Three processes may have contributed to form these zones, which are characterised by increased soil density: (i) soil compaction around the burrows during burrow creation, (ii) cast deposition in the burrows (burrow backfilling) and (iii) crushing of casts on the burrow walls (so called cutanes). The longer distance between BZ and macropores in A. chlorotica cores suggests that the proportion of burrow backfilling is higher for the endogeic species compared to the anecic species. If we assume that BZ located further than 10 mm from any burrow are actually burrows backfilled with casts, the volume of burrow backfilled in our study ranged from 14 to 18% for A. chlorotica and from to 8 to 10% for A. nocturna.

International audience; The taxonomic status of Allolobophora chlorotica is still the subject of considerable discussion. After the recent experimental demonstration that the two regularly observed colour morphs (pink and green) were not fully interfertile and likely represent two distinct species, molecular analyses revealed a more complex picture with the occurrence of five mitochondrial lineages (named L1 to L5) in A. chlorotica, two within the green morph and three within the pink morph. Although nuclear markers (AFLPs) confirmed that the pink morph might consist of three different taxa, AFLPs data suggested that the green morph may be a single taxon. In order to further characterize the population genetic structure within A. chlorotica in addition to test for reproductive isolation between lineages, eight polymorphic microsatellite loci were developed using a microsatellite-enriched genomic library from an individual belonging to lineage L2. The number of alleles per locus varied from 9 to 29 in a set of individuals belonging to L2. Considerable sub-structure was observed within L2 populations suggesting a low dispersal capability in this species and a distribution of the individuals in patches that could function as panmictic units. These markers were transferable to the other lineages but only five loci could be amplified consistently in four of the lineages (L1, L2, L3 and L4). Microsatellite data confirmed that the green morph represents a single taxon. Although the integrity of the three previously documented lineages within the pink morph was also generally supported by the data, microsatellites provided evidence for hybridization between lineages and between morphs in the field. Moreover, mitochondrial data revealed the existence of two additional mitochondrial lineages within the pink morph. The taxonomic status of the pink morph remains thus unclear, requiring a thorough and comprehensive study.

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 (1 mg kg−1) and a 10 fold higher dose (10 mg kg−1), on two earthworm species (Allolobophora chlorotica and Aporrectodea caliginosa). ChE activity and weight were measured every week for a 14 day period of exposure to parathion and then for 8 weeks in uncontaminated soil. After 3 days 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 28 days 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 3 days, for either dose, and reached 80–90% after 7 days exposure. While A. chlorotica exhibited the same pattern of inhibition for 10 mg kg−1 of parathion, the inhibition process was slower for the recommended dose with 50% inhibition after 7 days of exposure and 70% after 14 days. ChE activity recovery, after transfer to uncontaminated soil, also followed a different pattern for the two species. After exposure to 1 mg kg−1 parathion, ChE activity from A. chlorotica underwent a slow but constant recovery process to regain the control value after 8 weeks 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.

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.

Diplôme : Master Recherche

The aim of this work was to exemplify the specific contribution of both two- and three-dimensional (3D) X-ray computed tomography to characterise earthworm burrow systems. To achieve this purpose we used 3D mathematical morphology operators to characterise burrow systems resulting from the activity of an anecic ( Aporrectodea nocturna), and an endogeic species ( Allolobophora chlorotica), when both species were introduced either separately or together into artificial soil cores. Images of these soil cores were obtained using a medical X-ray tomography scanner. Three-dimensional reconstructions of burrow systems were obtained using a specifically developed segmentation algorithm. To study the differences between burrow systems, a set of classical tools of mathematical morphology (granulometries) were used. So-called granulometries based on different structuring elements clearly separated the different burrow systems. They enabled us to show that burrows made by the anecic species were fatter, longer, more vertical, more continuous but less sinuous than burrows of the endogeic species. The granulometry transform of the soil matrix showed that burrows made by A. nocturna were more evenly distributed than those of A. chlorotica. Although a good discrimination was possible when only one species was introduced into the soil cores, it was not possible to separate burrows of the two species from each other in cases where species were introduced into the same soil core. This limitation, partly due to the insufficient spatial resolution of the medical scanner, precluded the use of the morphological operators to study putative interactions between the two species

To study intraspecific and interspecific interactions between different ecological types of earthworm, the burrowing behaviour of two earthworm species (the anecic earthworm Aporrectodea nocturna and the endogeic earthworm Allolobophora chlorotica) was observed in a microcosm. Earthworms were either alone in the microcosm, together with a conspecific earthworm, or with an earthworm of the other species. Observations under red light, including those of the position of the animals and the burrow dug, were recorded 4 times a day for 8 days and provided the data needed to reconstruct the probable trajectories of each earthworm. Differences in movement and burrowing behaviour were observed. Comparisons between the two species confirmed the expected behaviour of each ecological type: A. nocturna reused its burrow system regularly, whereas A. chlorotica rarely did. Moreover, it was shown that A. chlorotica burrowed less and explored a smaller surface when in the presence of A. nocturna. Besides, A. nocturna burrowed less and explored a smaller surface when in the presence of another A. nocturna. If these interactions occur under natural conditions, they could affect the structure of the burrow systems of the earthworm species examined.

An experimental study of the consequences of intensive cattle trampling was carried out pure white clover and on a perennial rye-grass-white clover association. Direct and indirect effects of trampling on earthworm and plant communities and on soil structure were found. Trampling led to destruction of a large portion of the aerial system, stolons and roots, with removal of some of the vegetation cover (soil at least 50% bare). From a general view, trampling in experimental plots induced a large decrease (from 70 to 86%) of earthworm density and biomass. The functional structure was modified for the benefit of large size species. Small size species were more sensitive: some species, such as Lumbricus castaneus and Allolobophora chlorotica chlorotica typica disappeared. An increase of the proportion of adults could be related to larger sensitiveness of juvenile individuals in the same population and explained a significant increase of the individual mean weight. Different responses existed in function of the grassland type related to depth.