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Nowadays, when one of the most significant problems for mankind is saving the soil from pollution. It is well-known that one of the most important means of soil rehabilitation and remediation are soil inhabitants, their biodiversity and products of their life activity. Given the significant role of soil inhabitants in soil formation processes, it is important to consider their role in the processes of reprocessing and bioaccumulation of heavy metals. That especially concerns the earthworms, whose role in soil formation and maintenance of natural fertility is well-known and causes the interest of soil scientists and ecologists. The paper shows the degree of bioaccumulation of heavy metals (copper, zinc and lead) in the body of earthworms. Study involved three species of earthworms, which were collected in the vicinity of Tbilisi – Aporrectodea rosea (Savigny, 1826), Eisenia veneta (Rosa, 1886) and Allolobophora chlorotica (Savigny, 1826) – showed that earthworms of different species accumulate different amounts of heavy metals – copper, zinc and lead and, depending on the species, after being placed in heavy metals solutions, they demonstrate the different intensity of movement. The amount of heavy metals in the body of an earthworm depends on the structure of the body tissues and maybe on the structure of the skin-muscular sac.

Permanent grasslands provide a healthy and undisturbed environment. We investigated how mulching altered the soil physicochemical parameters, earthworm abundance, biomass, species composition and vegetation cover compared to grassland and cultivated alfalfa field. Microplots (2 × 2 m) were mulched with either weed control fabric (WF) alone or WF combined with straw (WF + S) on a grassland to grow tomato in Ősagárd (Nógrád county, Hungary) between 2018 and 2021. We had two other microhabitats: a conventionally cultivated alfalfa field (CA) and grassland (GR). We measured soil parameters (physical: soil moisture content (SMC), soil penetration resistance (SPR); chemical: pH, soil organic matter; and biological: earthworm abundance, biomass, species composition and vegetation). SMC was significantly higher on covered plots (WF; WF + S) compared to CA and GR. SPR values were the highest in CA and GR at 20 cm depth. The abundance and biomass of earthworms were the highest in GR and lowest in CA in all seasons. Plant abundance was highly influenced by season and habitat. Despite the higher compaction and lower SMC figures, grass vegetation still provided a more suitable environment for earthworms than mulched plots (WF, WF + S). Therefore, where there is agricultural production on grassland, we suggest leaving uncultivated and uncovered patches as biodiversity corridors. [ABSTRACT FROM AUTHOR]

Nowadays, when one of the most significant problems for mankind is saving the soil from pollution. It is well-known that one of the most important means of soil rehabilitation and remediation are soil inhabitants, their biodiversity and products of their life activity. Given the significant role of soil inhabitants in soil formation processes, it is important to consider their role in the processes of reprocessing and bioaccumulation of heavy metals. That especially concerns the earthworms, whose role in soil formation and maintenance of natural fertility is well-known and causes the interest of soil scientists and ecologists. The paper shows the degree of bioaccumulation of heavy metals (copper, zinc and lead) in the body of earthworms. Study involved three species of earthworms, which were collected in the vicinity of Tbilisi – Aporrectodea rosea (Savigny, 1826), Eisenia veneta (Rosa, 1886) and Allolobophora chlorotica (Savigny, 1826) – showed that earthworms of different species accumulate different amounts of heavy metals – copper, zinc and lead and, depending on the species, after being placed in heavy metals solutions, they demonstrate the different intensity of movement. The amount of heavy metals in the body of an earthworm depends on the structure of the body tissues and maybe on the structure of the skinmuscular sac. [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]

Soil structure is closely linked to biological activities. However, identifying, describing and quantifying soil aggregates remain challenging. X‐ray micro computed tomography (X‐ray μCT) provides a detailed view of the physical structure at a spatial resolution of a few microns. It could be a useful tool to discriminate soil aggregates, their origin and their formation processes for a better comprehension of soil structure properties and genesis. Our study aims to (a) determine different X‐ray μCT‐based aggregate parameters for differentiating earthworm casts belowground (earthworm aggregates) from aggregates that are not formed by earthworms (non‐earthworm aggregates), and (b) to evaluate if these parameters can also serve as specific "tomographic signatures" for the studied earthworm species. For this purpose, we set up a microcosm experiment under controlled conditions during 8 weeks, including three species of earthworms tested separately: the epigeic Lumbricus rubellus, the anecic Lumbricus terrestris and the endogeic Allolobophora chlorotica. Our results show that X‐ray μCT analysis helps distinguish earthworm aggregates from non‐earthworm ones using (a) the relative volume of the components within aggregates and (b) the volumetric mass of aggregates and their global volume. In particular, the volume ratio of mineral grains within the aggregates is significantly different according to earthworm species. So, X‐ray μCT is a powerful and promising tool for studying the composition of earthworm casts and their formation. However, future research is needed to take into account the shapes and spatial distribution of the aggregates' components, in particular the different states of organic matter decomposition. Highlights: Can earthworm belowground casts be differentiated from other soil aggregates using X‐ray μCT?The use of X‐ray μCT for characterizing soil aggregates at a spatial resolution of a few microns.A combination of X‐ray μCT variables discriminates earthworm casts from non‐earthworm aggregates.X‐ray μCT, used alone, is relevant for defining species‐specific signatures of earthworm casts. [ABSTRACT FROM AUTHOR]

Permanent grasslands provide a healthy and undisturbed environment. We investigated how mulching altered the soil physicochemical parameters, earthworm abundance, biomass, species composition and vegetation cover compared to grassland and cultivated alfalfa field. Microplots (2 × 2 m) were mulched with either weed control fabric (WF) alone or WF combined with straw (WF + S) on a grassland to grow tomato in Ősagárd (Nógrád county, Hungary) between 2018 and 2021. We had two other microhabitats: a conventionally cultivated alfalfa field (CA) and grassland (GR). We measured soil parameters (physical: soil moisture content (SMC), soil penetration resistance (SPR); chemical: pH, soil organic matter; and biological: earthworm abundance, biomass, species composition and vegetation). SMC was significantly higher on covered plots (WF; WF + S) compared to CA and GR. SPR values were the highest in CA and GR at 20 cm depth. The abundance and biomass of earthworms were the highest in GR and lowest in CA in all seasons. Plant abundance was highly influenced by season and habitat. Despite the higher compaction and lower SMC figures, grass vegetation still provided a more suitable environment for earthworms than mulched plots (WF, WF + S). Therefore, where there is agricultural production on grassland, we suggest leaving uncultivated and uncovered patches as biodiversity corridors.

We carried out mesocosm experiments using either the anecic earthworm Lumbricus terrestris or the endogeic earthworm Allolobophora chlorotica and loam, silt loam and sandy loam soils to investigate the differing impact of these earthworm of different ecotypes on aggregate formation (percentage water stable aggregates, %WSA) and soil water holding capacity (WHC), two soil properties that underpin many of the ecosystem services provided by soils. Earthworms significantly increased %WSA (by 16–56% and 19–63% relative to earthworm-free controls for L. terrestris and A. chlorotica, respectively). For L. terrestris, this increase was significantly greater in the upper 6.5 cm of the soil where their casts were more obviously present. Allobophora chlorotica treatments significantly increased WHC by 7–16%. L. terrestris only caused a significant increase in WHC (of 11%) in the upper 6.5 cm of the sandy loam soil. Linear regression indicated a consistent relationship between increases in %WSA and WHC for both earthworm species. However, for a given %WSA, WHC was higher for A. chlorotica than L. terrestris likely due to the known differences in their burrow structure. Overall, earthworms increased soil %WSA and WHC but the significant species/ecotype differences need to be considered in discussions of the beneficial impacts of earthworms to soil properties. [ABSTRACT FROM AUTHOR]

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. [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]

Increasing commercial application of silver nanoparticles (Ag NP) and subsequent presence in wastewater and sewage sludge has raised concerns regarding their effects in the aquatic and terrestrial environment. Several studies have employed standardised acute and chronic earthworm-based tests to establish the toxicological effects of Ag NP within soil. These studies have relied heavily on the use of epigiec earthworm species which may have limited ecological relevance in mineral soil. This study assessed the influence of Ag NP (uncoated 80 nm powder) and AgNO 3 on survival, change in biomass and avoidance behaviour in a soil dwelling (endogiec) species, Allolobophora chlorotica . Earthworms were exposed for 14 days to soils spiked with Ag NP or AgNO 3 at 0, 12.5, 25, 50 and 100 mg kg −1 either separately for survival and biomass measurement, or combined within a linear gradient to assess avoidance. Avoidance behaviour was shown to provide the most sensitive endpoint with an observable effect at an Ag NP/AgNO 3 concentration of 12.5 mg kg −1 compared with 50 mg kg −1 for biomass change and 100 mg kg −1 for survival. Greater mortality was observed in AgNO 3 (66.7%) compared with Ag NP-spiked soils (12.5%) at 100 mg kg −1 , attributed to increased presence of silver ions. Although comparison of results with studies employing Eisenia fetida and Eisenia andrei suggest that the A. chlorotica response to Ag NP is more sensitive, further research employing both epigeic and endogeic earthworms under similar experimental conditions is required to confirm this observation. [ABSTRACT FROM AUTHOR]

• Earthworms enhanced wheat growth under watered and drought conditions. • Soil extractions indicated no impact of earthworms on nutrient availability. • Earthworm-treatment microbiomes had fewer pathogens and more beneficial microbes. • Increase plant growth in earthworm treatments was due to changes in the microbiome. Earthworms enhance plant growth but the precise mechanism by which this occurs is not known. An understanding of the mechanism could potentially support changes in agricultural management reducing fertiliser usage and therefore costs and the carbon footprint of agriculture. We conducted a factorial experiment in which 5 strains of wheat were grown in the presence and absence of earthworms under regular watering and droughted conditions. The different wheat strains all responded in a similar fashion. Plant biomass was greater in the presence of earthworms and under regular watering. The presence of earthworms reduced the impact of drought on plant biomass and also slowed down the rate of drying of the droughted soils. Plant nutrient content (N, P, Si) showed no consistent pattern with treatments but plant total N, P and Si mirrored plant biomass and decreased in the order earthworm-present watered > earthworm-present droughted > earthworm-absent watered > earthworm-absent droughted. Nutrient availability in the soil, as assessed by chemical extractions showed no consistent pattern with treatments. Differential gene expression of plants was greater between watering treatments than between earthworm treatments. Genes that were differentially expressed between the earthworm treatments predominantly related to plant defences, abiotic stress and control of plant growth though a couple were linked to both nitrogen cycling and stress responses. The soil microbiome of the earthworm-present treatments was more associated with nutrient-rich environments, the promotion of plant growth and the suppression of plant pathogens whilst that of the earthworm-absent treatments included a variety of plant pathogens. Our data are consistent with enhanced plant growth being due to changes in the microbiome brought about by earthworm processing of the soil rather than changes in nutrient availability directly due to earthworm activity. [ABSTRACT FROM AUTHOR]

Earthworms are considered as key actors of soil processes at different spatial and temporal scales and provide essential ecosystem services linked to climate regulation or primary production. However, little is known about their basic functional roles (e.g. organic matter decomposition, soil structuring processes) in perturbed systems such as urban or alluvial soils. Alluvial soils are characterized by regular physical perturbation through flooding and associated erosion/sedimentation processes which are rather similar to perturbations (e.g. temporal instability, spatial heterogeneity) affecting urban soils. Due to their close soil characteristics, we hypothesized that in both cases, soil functioning is similar with respect to soil fauna activity. Under controlled conditions, our objective was to investigate the effects of two endogeic earthworm species, Allolobophora chlorotica (pink morph) and Aporrectodea rosea (the two most abundant species found in the studied urban site), on soil organic matter (SOM) dynamics and soil structure (network of earthworm burrows) comparing an urban and an alluvial soil. We investigated the growth of individuals (weight gain and reproduction success) and assessed their effects on SOM decomposition (cumulative C–CO 2 emission, nitrogen and phosphorus mineralization) and soil structure (macroporosity, total length and connectivity of segments) after one and three months of incubation. Our results showed higher growth of A. rosea in the alluvial soil compared to the urban soil. However, the total length of burrows, carbon and nitrogen mineralization were often higher in the urban soil especially when the two species were combined. This trend can be mainly explained by lower organic matter content found in the urban soil which may influence positively the burrowing activity and negatively the growth of earthworms. Endogeic earthworms appear a key feature of the soil functioning in the urban context through their roles on organic matter transformation, the formation and maintenance of the soil structure. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Earthworms are recognized to increase soil porosity, reorganize soil structure, and stimulate soil microflora and nutrient mineralization. The properties of earthworm casts should depend both on earthworm species or ecological group and on soil properties. Interactions between earthworm species and soil types have been suggested, but only poorly demonstrated. In order to better understand those interactions, two hypotheses led our study: (1) Soil type has a greater influence on cast properties than earthworm; (2) Earthworms from different species influence cast properties differently; (3) The intensity and direction of the impact of each earthworm species on cast properties vary with soil properties. Fifteen physical and chemical variables (N–NH 4 + , N–NO 3 − , total organic C and N, C/N ratio, CaCO 3 , pH, P, K + , Mg 2+ , Mn 2+ , Na + , CEC, moisture, wettability) were measured in casts of three earthworm species ( Lumbricus terrestris , Allolobophora chlorotica and Aporrectodea rosea ) produced in three temperate soils. Univariate and multivariate analyses showed that earthworm species and soil types significantly impacted cast properties. pH, N t , K and Mg contents were interactively altered by both factors. Multivariate analysis showed that a difference of soil type had a major impact on casts properties (62%) compared to the impact of a difference of earthworm species (10%). Cast properties were most impacted by L . terrestris , then by A . chlorotica and last by A . rosea . The response ratio (ratio of the properties of the casts to the properties of the bulk soil) was used to quantify the effect of earthworm species compared to the control soil. It showed a higher response of variables in casts in nutrient-rich soils, especially in casts of L . terrestris . The interactions between earthworm species and soil types on cast properties were discussed with regards to earthworm ecology, properties of the soil, and earthworm modifications of cast microflora. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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 5.5mm 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 10mm 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. [Copyright &y& Elsevier]

Metal pollution affects earthworm coelomocytes, including their differential counts, riboflavin content and metallothioneins (MT) involved in metal homoeostasis and detoxification. The present work shows effects of Ni, Cu, Zn, Cd, and Pb at the same molarity (1mM) on coelomocytes of Allolobophora chlorotica after 2-day worm dermal exposure to metal chlorides. Numbers of coelomocytes/eleocytes extruded by electric shock and amounts of riboflavin in coelomocyte lysates were significantly decreased in Cu-exposed worms, less diminished in response to Ni, Zn, Cd, and unaffected by Pb. In sharp contrast, real-time PCR revealed a very strong (272 fold) MT-mRNA induction in response to Cd only. The induction was very low in response to Zn, Cu, Pb, and Ni ions (2.6, 2.1, 1.4, and 1.3-fold, respectively). In conclusion, decreased cell counts and riboflavin content are molecular biomarkers of Cu exposure while induction of MT-mRNA is a molecular biomarker of worm Cd exposure. [Copyright &y& Elsevier]

Carbendazim-amended soil was placed above or below unamended soil. Control tests comprised two layers of unamended soil. Allolobophora chlorotica earthworms were added to either the upper or the unamended soil. After 72h vertical distributions of earthworms were compared between control and carbendazim-amended experiments. Earthworm distributions in the carbendazim-amended test containers differed significantly from the ‘normal’ distribution observed in the control tests. In the majority of the experiments, earthworms significantly altered their burrowing behaviour to avoid carbendazim. However, when earthworms were added to an upper layer of carbendazim-amended soil they remained in this layer. This non-avoidance is attributed to (1) the earthworms’ inability to sense the lower layer of unamended soil and (2) the toxic effect of carbendazim inhibiting burrowing. Earthworms modified their burrowing behaviour in response to carbendazim in the soil. This may explain anomalous results observed in pesticide field trials when carbendazim is used as a control substance. [Copyright &y& Elsevier]

Abstract: In Canada, the influence of earthworms on soil is considered particularly important in the Southern regions where agriculture has developed. A field experiment was duplicated on a clay loam and a sandy loam soil in the Saint-Lawrence Valley. Both sites were enriched with urban compost or mineral amendment. The effects of amendments on soil properties and the earthworm community were evaluated by uni- and multivariate analyses at two soil depths (0–10 and 10–20cm) and after two growth seasons. Compost-amended plots had higher pH, wet aggregates stability, total N and C, OM, K and Ca in the 0–10cm soil layer than plots receiving mineral fertilizer. Two endogeic species, Aporrectodea caliginosa and Allolobophora chlorotica were dominant in both sites. Other species were all epigeic and were rare. Abundance and biomass of earthworms (0–10cm) were significantly higher in clay loam, with a similar proportion in A. caliginosa (263indm−2) and A. chlorotica (228indm−2). In sandy loam, A. caliginosa was the most abundant (18indm−2), A. chlorotica was scarce (1.3indm−2) and epigeic species were absent. The density and biomass of earthworms were significantly higher with compost amendment, up to 12 times in clay loam: A. chlorotica reached a mean density of 420indm−2 vs. 36indm−2 with mineral amendments, A. caliginosa 344indm−2 vs. 183indm−2 and epigeic earthworms 19indm−2 vs. 12indm−2. In co-inertia analysis, the main factors explaining data heterogeneity were soil texture (axis 1) and organic matter content (axis 2). A. chlorotica was associated with OM, while A. caliginosa was associated with silt and clay contents. Additionally, soil wet aggregate stability was the variable that best correlated with endogeic species. The overall earthworm population was low, which is common for tilled agricultural soils, but clearly benefited from the OM input from the urban compost, providing further evidence that fertilization based on organic residues, as implemented in organic agriculture, is highly beneficial for earthworms and soil quality. [Copyright &y& Elsevier]

Summary: Breeding experiments have questioned the status of the green (g) and pink (p) colour morphs of Allolobophora chlorotica. Should they become regarded as separate species, the name Allolobophora virescens (Savigny, 1826) is available for the pink form of this earthworm. Breeding experiments were established to investigate the status of this species utilising previously unmated laboratory-reared adults. Pairs were set up for 10 days to facilitate mating, separated and then maintained in isolation for 28 days to obtain individual reproduction results. Cocoon production and viability were first determined from intra-morphic (p×p and g×g) and inter-morphic (p×g) pairings. All p×p and g×g pairings bred true with mean cocoon production in g×g (4.6 cocoons/worm/28 days) significantly greater (P<0.05) than in p×p (3.2 cocoons/worm/28 days). In p×g pairings, both pink and green individuals produced cocoons (3.9 and 5.9 cocoons/worm/28 days, respectively). However here, viability of cocoons produced by g was very low at 6%, compared with 59% for p. In a second experiment, previously unmated offspring from inter-morphic pairings (pg) were paired with like individuals (pg×pg) and also paired with pure bred pink (p1) and green (g1) individuals (p1×pg and g1×pg, respectively). All crosses produced cocoons. All cocoons produced by pg×pg crosses were non-viable. In p1×pg and g1×pg crosses the pg parent produced viable cocoons (64% and 79% viability, respectively) but cocoons produced by p1 and by g1 parents from these matings were 100% non-viable. It is suggested that severely restricted viability of cocoons produced by the green morph in p×g pairings provides evidence for genetic divergence of the 2 morphs. Furthermore, back-crossing of hybrids (pg) with pure bred morphs has indicated that hybrids are male sterile. [Copyright &y& Elsevier]

Abstract: The objective of this study was to determine the impact of earthworm bioturbation on the distribution and availability of zinc in the soil profile. Experiments were carried out with Allolobophora chlorotica and Aporrectodea caliginosa in 24 perspex columns (∅ 10cm), filled with 20–23cm non-polluted soil (OM 2%, clay 2.9%, pH 0.01M CaCl2 6.4), that was covered by a 3–5cm layer of aged zinc spiked soil (500mg Zn/kg dry soil) and another 2cm non-polluted soil on top. After 80 and 175 days, columns were sacrificed and each cm from the top down to a depth of 15cm was sampled. Earthworm casts, placed on top of the soil, were collected. Each sample was analyzed for total and CaCl2-exchangeable zinc concentrations. Effects of earthworm bioturbation were most pronounced after 175 days. For A. chlorotica, total and CaCl2-exchangeable zinc concentrations in the polluted layers were lower with than without earthworms. Total zinc concentrations in the non-polluted layers were higher in columns with earthworms. Casts of A. chlorotica collected on the soil surface showed slightly higher total zinc concentrations than non-polluted soil. Casts were found throughout the whole column. For A. caliginosa there were no differences in total zinc concentration between columns with and without earthworms. CaCl2-exchangeable zinc concentrations in the polluted layers were lower for columns with earthworms. Casts were mainly placed on top of the soil and contained total zinc concentrations intermediate between those in non-polluted and polluted soil layers. This study shows that different endogeic earthworm species have different effects on zinc distribution and availability in soils. A. chlorotica transfers soil throughout the whole column, effectively mixing it, while A. caliginosa decreases metal availability and transfers polluted soil to the soil surface. [Copyright &y& Elsevier]

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Surface cast production was investigated for several species of earthworms, including some not normally considered to produce surface casts. In single-species culture, the amount of cast material deposited on the surface by different species varied, with Allolobophora chlorotica < Lumbricus rubellus < Aporrectodea caliginosa < Aporrectodea longa. In field communities, results indicated that a single species dominated surface casting activity. The order of species dominance was consistent with the above findings from single-species cultures, if Lumbricus terrestris was considered to be equivalent to Ap. longa. Surface casting is an essential function within earthworm communities which maintains their living space. However, it involves an energy cost and carries a risk of predation. Therefore, there are disadvantages associated with this activity. All of the species studied produced surface casts unless other, usually larger, species were present. [ABSTRACT FROM AUTHOR]

Earthworm burrow systems have been simulated by modelling, from field observations, five main characteristics of burrow units: density (number of burrows per unit volume), mean length, angular orientation, middle coordinates and diameter. The functional properties of the burrow systems have been analysed using structural parameters, i. e. number of full perforations, mean available burrow surface and minimal burrow spacing, and by estimating the shortest pathway through the burrow system. The simulations are used to study the effects of each of the main characteristics on structural parameters and the shortest pathway. This work reveals that the specific role of density is to lead to a threshold by determining an optimal horizontal distance, and that the mean length governs the vertical critical dimension of the system. These simulations may be applied to explain the nature of field burrow systems. [ABSTRACT FROM AUTHOR]

• The effects of increasing bulk density on endogeic earthworm burrowing behaviour was assessed. • All the characteristics of the burrow systems were significantly impacted by an increase in bulk density. • Endogeics increased water infiltration only at low bulk density. • The endogeics compacted the soil around their burrows the same way irrespectively of the soil bulk density. 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. [ABSTRACT FROM AUTHOR]

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]

• Development of a method to identify cryptic earthworm species without any sequencing step. • A method combining HRM with DNA barcoding allowed to identify species within the Allolobophora chlorotica species aggregate. • The Bar-HRM method could be easily adapted to any complex of earthworm species. 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. [ABSTRACT FROM AUTHOR]

Abstract: Earthworm immune-competent cells (coelomocytes) can be adversely affected by soil metal contamination. The aim of the present paper was to perform comparative studies on the coelomocytes of four lumbricid species dermally exposed for 2days to filter paper soaked with Ni, Cu, Zn, Cd, or Pb chlorides (1mM). Coelomocytes were expelled by electro-stimulation, counted, and assayed ex vivo by flow cytometry and spectrofluorometry. The coelomocytes of Allolobophora chlorotica and Dendrobaena veneta contain both amoebocytes and riboflavin-storing eleocytes; consequently, they were subjected to assays based on flow cytometric quantification of autofluorescent eleocytes and spectrofluorometric measurement of riboflavin content in coelomocyte lysates. In Al. chlorotica, the number of coelomocytes, the percentage of eleocytes, and the amount of riboflavin were significantly lower in Cu-exposed worms although these cytometric parameters were less affected by Ni, Zn, Cd exposure, and entirely unaffected by Pb exposure. In D. veneta, such cellular effects were also only observed in Cu-exposed worms. The coelomic fluids of Aporrectodea caliginosa and Lumbricus rubellus are constitutionally impoverished of eleocytes; therefore, these species could not be subjected to assays measuring the riboflavin content of these granular cells. Rather, the amoebocytes of Ap. caliginosa and L. rubellus were subjected to flow cytometric measurements of in vitro neutral red uptake (NR). However, the NR uptake assay was technically demanding, requiring a strictly normalized incubation period over all samples to yield useful comparative data. In contrast, the riboflavin content in the coelomocyte lysates of eleocyte-rich species appears to be a robust and convenient immune-function biomarker of environmental stress. [Copyright &y& Elsevier]

Abstract: The riboflavin content in extruded coelomocyte lysates derived from Dendrodrilus rubidus may serve as a sensitive bioindicator of soil metal pollution: the vitamin (B2) content has previously been found to be high in worms from unpolluted soil but low in worms inhabiting Zn/Pb mine soils, aerially deposited Ni-contaminated soil, and in worms experimentally transferred from clean soil to the metalliferous field soils. The aim of the present work was to extend these observations by comparing the number and riboflavin composition of coelomocytes retrieved from three lumbricid species (Allolobophora chlorotica, Dendrobaena veneta, Eisenia andrei) after 4-week exposures to an unpolluted commercial soil, two geochemically contrasting unpolluted field soils, and two different Zn/Pb/Cd-polluted soils from the Bukowno district in South Poland. Whilst eco-physiologically contrasting, these three earthworm species share the trait of possessing relatively high numbers of eleocytes, a category of immune-competent coelomocyte rich in autofluorescent riboflavin. Spectroflurometric analysis indicated that coelomocyte riboflavin content in worms maintained in strongly metalliferous soils or in unpolluted sandy-clay and loamy-sand soils was increased in coleomocytes from epigeic D. veneta and E. andrei species, whilst was decreased in endogeic A. chlorotica. In conclusion, the riboflavin content of earthworm coelomocytes is affected in species-specific ways by edaphic variables, including organic matter and metal pollution. [Copyright &y& Elsevier]

Abstract: Coelomic fluid and cells (coelomocytes) were extruded by the electric shock (1min, 4.5V) from field-collected individuals of Allolobophora chlorotica (Sav.). Coelomocytes were subjected to bright field and fluorescence light as well as electron microscopy (transmission and scanning). Results of all methods combined, revealed two distinct populations of circulating cells in the coelomic fluid: (1) autofluorescent eleocytes/chloragocytes filled with numerous large granules (chloragosomes) containing fluorophores, mainly riboflavin; and (2) amoebocytes, which can be differentiated into: (a) hyaline amoebocytes, firmly spreading in form of crinoline-like membranes; (b) granular amoebocytes with numerous dark granules spreading in form of fine pseudopodia. Flow cytometry confirmed the presence of highly complex autofluorescent cells (eleocytes/chloragocytes) and two cohorts of not-autofluorescence much less complex amoebocytes. [Copyright &y& Elsevier]