Allolobophora Chlorotica Research Articles

Impacts of epigeic, anecic and endogeic earthworms on metal and metalloid mobility and availability

The introduction of earthworms into soils contaminated with metals and metalloids has been suggested to aid restoration practices. Eisenia veneta (epigeic), Lumbricus terrestris (anecic) and Allolobophora chlorotica (endogeic) earthworms were cultivated in columns containing 900 g soil with 1130, 345, 113 and 131 mg kg(-1) of As, Cu, Pb and Zn, respectively, for up to 112 days, in parallel with earthworm-free columns. Leachate was produced by pouring water on the soil surface to saturate the soil and generate downflow. Ryegrass was grown on the top of columns to assess metal uptake into biota. Different ecological groups affected metals in the same way by increasing concentrations and free ion activities in leachate, but anecic L. terrestris had the greatest effect by increasing leachate concentrations of As by 267%, Cu by 393%, Pb by 190%, and Zn by 429% compared to earthworm-free columns. Ryegrass grown in earthworm-bearing soil accumulated more metal and the soil microbial community exhibited greater stress. Results are consistent with earthworm enhanced degradation of organic matter leading to release of organically bound elements. The degradation of organic matter also releases organic acids which decrease the soil pH. The earthworms do not appear to carry out a unique process, but increase the rate of a process that is already occurring. The impact of earthworms on metal mobility and availability should therefore be considered when inoculating earthworms into contaminated soils as new pathways to receptors may be created or the flow of metals and metalloids to receptors may be elevated.

Metal-specific effects on metallothionein gene induction and riboflavin content in coelomocytes of Allolobophora chlorotica

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 (1 mM) 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.

The soil-dwelling earthworm Allolobophora chlorotica modifies its burrowing behaviour in response to carbendazim applications

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.

Prey choice by carabid beetles feeding on an earthworm community analysed using species- and lineage-specific PCR primers

The carabid beetle Pterostichus melanarius is a major natural enemy of pests, such as aphids and slugs in agricultural systems. Earthworms are a dominant non-pest component of the diet of P. melanarius which help sustain the beetles during periods when the pest population is low or absent. In this study we wanted to test whether this predator exercises prey choice among different earthworm species or ecological groups. High levels of genetic diversity within morphological species of earthworm necessitated the development of primers that were specific not just to species but lineages and sub-lineages within species as well. Gut samples from beetles were analysed using multiplex-PCR and fluorescent-labelled primers. Calibratory feeding trials were undertaken to calculate median detection times for prey DNA following ingestion. Extensive testing demonstrated that the primers were species-specific, that detection periods were negatively related to amplicon size and that meal size had a highly significant effect on detection periods. Monte Carlo simulations showed that, in general, worms were being predated in proportion to their densities in the field with little evidence of prey choice, other than probable avoidance of the larger, deep-living species. There was no evidence that epigeic species were being taken preferentially in comparison with endogeic species. There was also no evidence that defensive secretions by Allolobophora chlorotica reduced predation pressure on this species by P. melanarius. We concluded that any management system that increases earthworm densities generally, regardless of component species, is likely to be optimal for increasing numbers of this beneficial beetle predator.

Influence of texture and amendments on soil properties and earthworm communities

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 10–20 cm) and after two growth seasons. Compost-amended plots had higher pH, wet aggregates stability, total N and C, OM, K and Ca in the 0–10 cm 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 (0–10 cm) were significantly higher in clay loam, with a similar proportion in A. caliginosa (263 ind m −2) and A. chlorotica (228 ind m −2). In sandy loam, A. caliginosa was the most abundant (18 ind m −2), A. chlorotica was scarce (1.3 ind m −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 420 ind m −2 vs. 36 ind m −2 with mineral amendments, A. caliginosa 344 ind m −2 vs. 183 ind m −2 and epigeic earthworms 19 ind m −2 vs. 12 ind m −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.

Impact of roots, mycorrhizas and earthworms on soil physical properties as assessed by shrinkage analysis

Summary Soil biota such as earthworms, arbuscular mycorrhizal fungi (AMF) and plant roots are known to play a major role in engineering the belowground part of the terrestrial ecosystems, thus strongly influencing the water budget and quality on earth. However, the effect of soil organisms and their interactions on the numerous soil physical properties to be considered are still poorly understood. Shrinkage analysis allows quantifying a large spectrum of soil properties in a single experiment, with small standard errors. The objectives of the present study were, therefore, to assess the ability of the method to quantify changes in soil properties as induced by single or combined effects of leek roots ( Allium porrum ), AMF ( Glomus intraradices ) and earthworms ( Allolobophora chlorotica ). The study was performed on homogenised soil microcosms and the experiments lasted 35 weeks. The volume of the root network and the external fungal hyphae was measured at the end, and undisturbed soil cores were collected. Shrinkage analysis allowed calculating the changes in soil hydro-structural stability, soil plasma and structural pore volumes, soil bulk density and plant available water, and structural pore size distributions. Data analysis revealed different impacts of the experimented soil biota on the soil physical properties. At any water content, the presence of A. chlorotica resulted in a decrease of the specific bulk volume and the hydro-structural stability around 25%, and in a significant increase in the bulk soil density. These changes went with a decrease of the structural pore volumes at any pore size, a disappearing of the thinnest structural pores, a decrease in plant available water, and a hardening of the plasma. On the contrary, leek roots decreased the bulk soil density up to 1.23 g cm −3 despite an initial bulk density of 1.15 g cm −3 . This increase in volume was accompanied with a enhanced hydro-structural stability, a larger structural pore volume at any pore size, smaller structural pore radii and an increase in plant available water. Interestingly, a synergistic effect of leek roots and AMF in the absence of the earthworms was highlighted, and this synergistic effect was not observed in presence of earthworms. The structural pore volume generated by root and AMF growth was several orders of magnitude larger than the volume of the organisms. Root exudates as well as other AMF secretion have served as carbon source for bacteria that in turn would enhance soil aggregation and porosity, thus supporting the idea of a self-organization of the soil–plant–microbe complex previously described.

Impact of five different tillage systems on soil organic carbon content and the density, biomass, and community composition of earthworms after a ten year period

To assess the impact of different types of soil tillage on the density, biomass, and community composition of earthworms, a long-term field study was performed in which soils were tilled in different ways for ten years. This study included five different types of tillage: (i) plough, (ii) grubber, (iii) disc harrow, (iv) mulch sowing, and (v) direct sowing. At the end of the experiment the earthworm density, biomass, and community composition, and the SOC (soil organic carbon) content were determined. The results show that density, biomass, and community composition of earthworm populations varied in relation to the type of soil tillage used. The density of anecic earthworm species decreased when soils were managed by conventional ploughing, relative to reduced tillage practices, whereas conversely the density of endogeic species increased. Additionally, the varying types of soil tillage influenced the abundance and biomass of different earthworm species in different ways. The density of Aporrectodea caliginosa was positively influenced by ploughing, whereas Aporrectodea longa, Lumbricus castaneus, and Satchellius mammalis showed a positive relationship to the grubber and Allolobophora chlorotica to direct sowing. We attribute these changes to modifications in the vertical distribution of SOC and varying potentials for mechanical damage of earthworms by tillage. A decrease in tillage intensity modified the vertical SOC distribution in the topsoil and consequently revealed positive effects on earthworm biodiversity, thus sustaining soil functioning.

Fishing for Worms

EVOLUTION The earthworm Allolobophora chlorotica has two color morphs: pink and green. The facts that these morphs are found in different habitats and crosses between them are sterile have suggested that they represent distinct species. King et al. have examined two mitochondrial genes of A.

Opening a can of worms: unprecedented sympatric cryptic diversity within British lumbricid earthworms

Earthworms play a major role in many aspects of soil fertility, food web ecology and ecosystem functioning, and hence are frequently the subjects of, for example, ecological and toxicological research. Our aim was to examine the genetic structure of common earthworm species, to identify cryptic lineages or species that may be distinct ecotypes or biotypes (and hence confound current research based upon morphotypes) and to try to explain the massive cryptic diversity that eventually emerged. We demonstrated that species such as Allolobophora chlorotica, Aporrectodea longa, Aporrectodea rosea and Lumbricus rubellus all comprise highly divergent lineages with species-level divergence at the mitochondrial cytochrome oxidase I (COI) gene. In Allo. chlorotica alone, we found 55 haplotypes for COI, with 35 of these being found in pink and 20 in green morph worms. There were no cases of the two colour morphs sharing COI haplotypes. Phylogenetic analyses of mitochondrial COI and 16S genes showed the presence of five highly divergent lineages, suggesting the presence of multiple cryptic species within Allo. chlorotica. There was no clear geographical pattern to lineage distribution and many populations were polymorphic for both mitochondrial DNA lineage and colour morph. Amplified fragment length polymorphism results, based on two primer combinations, were broadly congruent with mitochondrial DNA results with one significant exception. Despite showing over 14% divergence at COI, amplified fragment length polymorphism markers showed that the two green morph lineages may be interbreeding and therefore represent a single taxon. The cryptic diversity revealed by these results has profound consequences for all areas of earthworm research.

Dynamics of acetylcholinesterase activity recovery in two earthworm species following exposure to ethyl-parathion

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.

Root, mycorrhiza and earthworm interactions: their effects on soil structuring processes, plant and soil nutrient concentration and plant biomass

Earthworms, arbuscular mycorrhiza fungi (AMF) and roots are important components of the belowground part of terrestrial ecosystem. However, their interacting effects on soil properties and plant growth are still poorly understood. A compartmental experimental design was used in a climate chamber in order to investigate, without phosphorus (P) addition, the single and combined effects of earthworms (Allolobophora chlorotica), AMF (Glomus intraradices) and roots (Allium porrum) on soil structure, nutrient concentration and plant growth. In our experimental conditions, plant roots improved soil structure stability (at the level of macroaggregates) whereas earthworms decreased it. AMF had no effect on soil structure stability but increased P transfer from the soil to the plant and significantly increased plant biomass. Earthworms had no direct influence on P uptake or plant biomass, and the N/P ratio measured in the shoots indicated that P was limiting. Interactions between AMF and earthworms were also observed on total C and N content in the soil and on total root biomass. Their effects varied temporally and between the different soil compartments (bulk soil, rhizosphere and drilosphere). After comparison with other similar studies, we suggest that effects of earthworms and AMF on plant production may depend on the limiting factors in the soil, mainly N or P. Our experiment highlights the importance of measuring physical and chemical soil parameters when studying soil organism interactions and their influence on plant performance.

Direct and indirect effects of endogeic earthworms on plant seeds

Summary The soil seed bank functions to escape unfavourable environmental conditions and seed predation. Anecic earthworms are increasingly recognised as important dispersers and predators of plant seeds. In contrast, the role of the usually more abundant endogeic earthworms which live and feed in the soil on plant seeds is largely unknown. We tested whether (A) endogeic earthworms ( Aporrectodea rosea , Allolobophora chlorotica , Octolasion tyrtaeum , Aporrectodea caliginosa ) ingest and digest grassland plant seeds ( Phleum pratense , Bellis perennis , Trifolium repens , Poa trivialis , Plantago lanceolata , Medicago varia ), (B) the passage of seeds through the gut of endogeic earthworms modifies seed germination, and (C) excreta (mucus and casts) of endogeic earthworms modify seed germination. As a reference effects of the well-studied anecic species Lumbricus terrestris were determined. Endogeic earthworms ingested and digested all of the studied plant seeds; however, both ingestion and digestion were earthworm and plant species specific. Moreover, passage through the gut of endogeic earthworms and their excreta modified plant seed germination (gut passage: B. perennis ; excreta: Ph. pratense and Pl. lanceolata ). The results indicate that endogeic earthworms may strongly impact the composition of the soil seed bank and, consequently, plant community assembly via direct and indirect effects on plant seeds. Since post-dispersal seed predation is a key factor for the structure of plant communities, with the effect on seed survival potentially exceeding that of pre-dispersal predation, seed predation and changes in germination of seeds by endogeic earthworm species is likely to drive plant community composition.

Distribution of earthworms and influence of soil properties across a successional sand dune ecosystem in NW England

An investigation of earthworms across a sand dune system in NW England examined species distribution and abundance with respect to soil physico-chemical conditions and management. Replicated 0.1 m 2 quadrats were examined every 50 m along 700 m transects across areas of vegetation succession and samples were then taken every 10 m where earthworms were first encountered. A translocation of Aporrectodea longa assessed the ability of this species to live in soils with a high sand content and laboratory work examined growth, maturation and survival of this species in soils with increasing proportions of sand (0–100%). Nine earthworm species were found on the dunes, but none in yellow dunes where organic matter (OM) content was <1%. Dendrobaena octaedra and Lumbricus rubellus were located 300 m from the strand line in grey dunes with an OM content of 3.9%. Allolobophora chlorotica and Lumbricus castaneus occurred within a wet dune slack at 340 m (OM content 11%). In areas of human disturbance (dune car park), A. longa, Aporrectodea caliginosa and Lumbricus terrestris were present, and in soil below pine trees L. castaneus and L. rubellus were present in litter. Laboratory growth of A. longa demonstrated significant ( p < 0.05) increases in growth (2–2.75 g) with 25 and 50% sand compared with 0, 75 and 100% over 24 weeks and a greater rate of maturation. Initial results show earthworm colonisation to be influenced by dune successional stage. Laboratory findings suggested A. longa could exist in higher sand content areas but experimental design needs development. The translocation was unsuccessful. Future investigations could examine soil properties more closely and undertake monitoring on site throughout the year.

Allolobophora chlorotica (Savigny, 1826): Evidence for classification as two separate species

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.

Enhancing the understanding of earthworm feeding behaviour via the use of fatty acidδ13C values determined by gas chromatography‐combustion‐isotope ratio mass spectrometry

Litter-dwelling (epigeic) Lumbricus rubellus and soil-dwelling (endogeic) Allolobophora chlorotica earthworms were observed aggregating under C(3) (delta(13)C = -31.3 per thousand; delta(15)N = 10.7 per thousand) and C(4) (delta(13)C = -12.6 per thousand; delta(15)N = 7.5 per thousand) synthetic dung pats applied to a temperate grassland (delta(13)C = -30.3 per thousand; delta(15)N = 5.7 per thousand) in an experiment carried out for 372 days. Bulk delta(13)C values of earthworms collected from beneath either C(3) or C(4) dung after 28, 56, 112 and 372 days demonstrated that (i) L. rubellus beneath C(4) dung were significantly (13)C-enriched after 56 days (delta(13)C = -23.8 per thousand) and 112 days (delta(13)C = -22.4 per thousand) compared with those from C(3) dung treatments (56 days, delta(13)C = -26.5 per thousand; 112 days, delta(13)C = -27.0 per thousand), and (ii) A. chlorotica were 2.1 per thousand (13)C-enriched (delta(13)C = -24.2 per thousand) relative to those from C(3) dung (delta(13)C = -26.3 per thousand) treatments after 372 days. Bulk delta(15)N values did not suggest significant uptake of dung N by either species beneath C(3) or C(4) dung, but showed that the endogeic species (total mean delta(15)N = 3.3 per thousand) had higher delta(15)N values than the epigeic species (total mean delta(15)N = 5.4 per thousand). Although the two species exhibited similar fatty acid profiles, individual fatty acid delta(13)C values revealed extensive routing of dietary C into body tissue of L. rubellus, but minor incorporation into A. chlorotica. In particular, the direct incorporation of microbial biomarker fatty acids (iC(17:0), aC(17:0)) from (13)C-labelled dung in situ, the routing of dung C into de novo synthesised compounds (iC(20:4)(omega)(6),C(20:5)(omega)(3), and the assimilation of essential fatty acids ((C(18:1)(omega)(9), C(18:1)(omega(7), C(18:2)(omega(6), C(18:3)(omega)(3)) derived from dung, were determined.

Structure and functioning of earthworm communities in woodland flooding systems used for drinking water production

Earthworms are known to influence water infiltration in soils, but most of the existing knowledge relates to grasslands and arable systems; little is known on the role of earthworms for water infiltration in forests. We studied earthworm populations and water infiltration rates in woodland flooding sites used for groundwater recharge and the production of drinking water. Intensive flooding may detrimentally affect earthworm populations and simultaneously result in clogging of the topsoil, with the latter being a common problem in groundwater recharge systems. However, clogging does not occur at our study site, the “Lange Erlen” (Basel, Switzerland) and total earthworm numbers and biomass in flooded sites exceeded those of non-flooded sites (+51% and +71%, respectively). Total earthworm numbers ( r = 0.85***), numbers of endogeic ( r = 0.64*) and epigeic ( r = 0.81**) earthworms and numbers of two species ( Lumbricus rubellus, r = 0.62* and Allolobophora chlorotica, r = 0.77**) significantly correlated with water infiltration rates. The results suggest that short-term flooding (max. 10 days) interrupted by longer recovery periods favor earthworm populations which likely contribute to the long-term (ca. 100 years) sustainability of the studied forest groundwater recharge system and thereby to effective and cost efficient drinking water production.

Relationships between earthworm populations and management intensity in cattle-grazed pastures in Ireland

Earthworm (Lumbricidae) populations were assessed in three replicated, field experiments with different management intensities conducted on grassland research farms on medium-textured mineral soils, located at Solohead (Co. Tipperary), Grange (Co. Meath) and Johnstown Castle (Co. Wexford), Ireland. The experiment at Solohead involved four levels of mineral fertilizer application (80, 175, 225 and 350 kg N ha −1), and the plots were rotationally grazed by dairy cows. Three levels of mineral fertilizer application were compared at Johnstown Castle (0, 225 and 390 kg N ha −1) and two at Grange (100 and 225 kg N ha −1), and the plots at these sites were rotationally grazed by suckler cows and followers or dry cattle. Between 10 and 15 earthworm species were recorded per site, with Allolobophora chlorotica and Aporrectodea caliginosa being dominant. Other abundant species included Aporrectodea rosea, Aporrectodea longa, Lumbricus terrestris, Lumbricus friendi, Lumbricus festivus, Lumbricus rubellus and Satchellius mammalis. Mean earthworm population densities per treatment ranged from 203 to 324 individuals m −2 and biomass from 60 to 176 g m −2. ANCOVA revealed significant overall positive relationships between management intensity (as indicated by N application rate) and earthworm abundance ( F = 4.47, p < 0.05) and biomass ( F = 17.32, p < 0.001). While there was considerable variation in earthworm response to treatments both within and between sites, populations were not adversely affected by intensive management in any of the sites. It appears that any adverse effect of trampling at higher stocking rates was more than compensated for by increased food supply in the form of dung and plant litter. The presence of high earthworm populations is potentially an important factor in counteracting adverse impacts of intensive grazing on pasture soils, especially on heavy soils liable to poaching.

Cholinesterase activity as a biomarker of pesticide exposure inAllolobophora chloroticaearthworms 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.

Flow cytometric measurement of neutral red accumulation in earthworm coelomocytes: Novel assay for studies on heavy metal exposure

Neutral red (NR) is a vital stain accumulating in lysosomes of the cells, exemplified here by the coelomocytes non-invasively extruded from the earthworm coelomic cavity. Flow cytometry allows to distinguish two main populations of earthworm coelomocytes, namely hyaline and granular amoebocytes (A), and eleocytes/chloragocytes (E), the latter exhibiting riboflavin-derived autofluorescence. Coelomocytes of Dendrobaena veneta, Eisenia fetida, Allolobophora chlorotica, and Lumbricus sp. were incubated in vitro with copper chloride at 0 (controls) to 88 μg/ml. Both amoebocytes and eleocytes accumulated neutral red, as evidenced by light and confocal microscopy and by flow cytometry. Flow cytometry allowed quantitative analysis of the adverse effects of an in vitro copper exposure on the intracellular neutral red accumulation in coelomocytes, phenomenon being completely prevented in the presence of metal-chelating agent, ethylenediaminetetraacetic acid (EDTA). In conclusion, flow cytometry may be used as an objective and fast tool for the measurement of NR accumulation in invertebrate cells, and the neutral red retention in the cells subjected to various stressors, including environmental pollution.

Characteristics of coelomocytes of the stubby earthworm, Allolobophora chlorotica (Sav.)

Coelomic fluid and cells (coelomocytes) were extruded by the electric shock (1 min, 4.5 V) 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.