Characterisation of the three-dimensional structure of earthworm burrow systems using image analysis and mathematical morphology

The aim of this work was to exemplify the specific contribution of both two- and three-dimensional (31)) 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 noctunia), 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.

EARTHWORM ACTIVITY IN FOREST AND SAVANNA SOILS NEAR BOA VISTA, RORAIMA, BRAZIL

From May to December, 1992 we studied the earthworm activity, as part of the ECOFIT Program at Surrão ( 30km from Boa Vista), verifying the duration of the activity and quantifying it through the casts deposited on the soil surface. Three transects were stablished: a 100m long in the savanna, a 40m in the transitional area and 100m long in the forest. Specimens were collected monthly along the transects according to the method proposed by TSBF and the casts were collected from the squares previously placed on the transects. Two earthworm species were found in the three areas: Pontoscolex roraìmensis and Pontoscolex corethrurus. The first species depositing its casts on the soil surface. Rainy season wich usually occurs from April to August, determined the duration of activity for the two species (however, 1992 was atypical in that rainy season, only started in May). P. roraìmensis produced casts in the three areas from May to August followed by aestivation. In the savanna, P. corethrurus started aestivation in the same period as P. roraìmensis but remained active until November in the two other areas. P. roraìmensiswas found up 50m from the forest border, being associated to the presence of Curatella americana and Byrsonima sp, two typical savanna bushes.

Earthworm assemblages in an ecotone between forest and arable field and their relations with soil properties

The objective of this work was to assess the effects of a forest-field ecotone on earthworm assemblages. Five sites (blocks) differing in the type of crop rotation used in the field were studied in Central Bohemia, Czech Republic. In each block, sampling was carried out in seven parallel rows perpendicular to a transect from a forest (oak or oak-pine) to the centre of a field, both in spring and autumn 2001-2003. Individual rows were located in the forest (5 m from the edge), in the forest edge, and in the field (at 5, 10, 25, 50 and 100 m distances from the forest edge). The density and biomass of earthworms were lowest in the forest, increased markedly in the forest edge, decreased again at 5 or 10 m distance from the forest edge and then continuously increased along the distance to the field boundary. The highest number of species was found in the forest edge and in the field boundary. Individual species differed in their distribution along the transect. Both density and biomass of earthworms were correlated with distance from forest edge, soil organic matter content, soil porosity, and water infiltration rate.

Earthworm communities in organic and conventional coffee cultivation

The objective of this work was to evaluate the effect of organic and conventional coffee crops on biomass, population density and diversity of earthworms, in Lerroville, district of Londrina County, Paraná state, Brazil. Earthworm communities were sampled in three areas with organic coffee cultivation (CO1, CO2 and CO3), two with conventional coffee (CC1 and CC2), and a native forest fragment (MT). The soil of the areas CO1, CC1, and MT was classified as Nitossolo Vermelho (Rhodic Kandiudox), while CO2, CO3, and CC2 were on Latossolo Vermelho (Rhodic Hapludox). Eight samples were taken in each area on two occasions, winter and summer, using the Tropical Soil Biology and Fertility (TSBF) method in the 0-20 cm soil layer. The earthworms were handsorted and preserved in 4% formaldehyde, and were later weighed, counted and identified. The highest earthworm biomass, both in winter and summer, occurred in the CO3 area. For population density, the higher numbers of individuals were found in CO1 and CO3. The highest number of species was identified in the organic cultivation. The adoption of organic practices in coffee cultivation favored the diversity, density and biomass of earthworm communities.

The earthworm fauna of regenerating forests and anthropogenic habitats in the coastal region of Paraná

The aim of this study was to evaluate abundance, biomass and diversity of earthworms in the southern coast region of the Mata Atlântica biodiversity hotspot. A total of 51 study sites in pastures, banana monocultures, mixed agroforestry systems, secondary forests in succession and old-growth forests near the coast of Paraná, Brazil, were evaluated. Each site was sampled once. Species richness of the earthworms was generally low and varied little between sites. At all sites except for one, the peregrine species Pontoscolex corethrurus (Glossoscolecidae) strongly dominated. Three other peregrine species, Amynthas corticis, Amynthas gracilis (Megascolecidae) and Ocnerodrilus occidentalis (Ocnerodrilidae), were frequent in moist sites. No autochthonous species were found. Abundance and biomass of earthworms varied strongly within and between sites (0-338 individuals m-2, 0-96 g m-2 fresh weight). Pastures had significantly lower abundance than all other sites. The forest sites had similar earthworm abundance and biomass, with a tendency to be higher in younger succession stages. The coastal plain region has been strongly altered by human activities. Reasons for the lack of any autochthonous species and the dominance of one peregrine species require further investigation.

Identification of the main generator source of longitudinal muscle contraction in the earthworm ventral nerve cord

The main generator source of a longitudinal muscle contraction was identified as an M (mechanical-stimulus-sensitive) circuit composed of a presynaptic M-1 neuron and a postsynaptic M-2 neuron in the ventral nerve cord of the earthworm, Amynthas hawayanus, by simultaneous intracellular response recording and Lucifer Yellow-CH injection with two microelectrodes. Five-peaked responses were evoked in both neurons by a mechanical, but not by an electrical, stimulus to the mechanoreceptor in the shaft of a seta at the opposite side of an epidermis-muscle-nerve-cord preparation. This response was correlated to 84% of the amplitude, 73% of the rising rate and 81% of the duration of a longitudinal muscle contraction recorded by a mechano-electrical transducer after eliminating the other possible generator sources by partitioning the epidermis-muscle piece of this preparation. The pre- and postsynaptic relationship between these two neurons was determined by alternately stimulating and recording with two microelectrodes. Images of the Lucifer Yellow-CH-filled M-1 and M-2 neurons showed that both of them are composed of bundles of longitudinal processes situated on the side of the nerve cord opposite to stimulation. The M-1 neuron has an afferent process (A1) in the first nerve at the stimulated side of this preparation and the M-2 neuron has two efferent processes (E1 and E3) in the first and third nerves at the recording side where their effector muscle cell was identified by a third microelectrode.

Facilitation of the main generator source of earthworm muscle contraction by a peripheral neuron

A constant facilitation of responses evoked in the earthworm muscle contraction generator neurons by responses evoked in the neurons of its peripheral nervous system was demonstrated. It is based on the proposal that these two responses are bifurcations of an afferent response evoked by the same peripheral mechanical stimulus but converging again on this central neuron. A single-peaked generator response without facilitation was demonstrated by sectioning the afferent route of the peripheral facilitatory modulatory response, or conditioning response (CR). The multipeaked response could be restored by restimulating the sectioned modulatory neuron with an intracellular substitutive conditioning stimulus (SCS). These multi-peaked responses were proposed to be the result of reverberating the original single peaked unconditioned response (UR) through a parallel (P) neuronal circuit which receives the facilitation of the peripheral modulatory neuron. This peripheral modulatory neuron was named "Peri-Kästchen" (PK) neuron because it has about 20 peripheral processes distributed on the surface of a Kästchen of longitudinal muscle cells on the body wall of this preparation as revealed by the Lucifer Yellow-CH-filling method.

Persistent attenuation and enhancement of the earthworm main muscle contraction generator response induced by repeated stimulation of a peripheral neuron

Responses evoked in the earthworm, Amynthas hawayanus, main muscle contraction generator M-2 (postsynaptic mechanical-stimulus-sensitive) neuron by threshold mechanical stimuli in 2-s intertrial intervals (ITI) were used as the control or unconditioned responses (UR). Their attenuation induced by decreasing these intervals in non-associative conditioning and their enhancement induced by associating the unconditioned stimuli (US) to a train of short (0.1 s) hyperpolarizing electrical substitutive conditioning stimuli (SCS) in the Peri-Kästchen (PK) neuron were measured in four parameters, i.e., peak numbers (N) and amplitude ()averaged from 120 responses, sum of these amplitudes (SAMP) and the highest peak amplitude (V) over a period of 4 min. Persistent attenuation similar to habituation was induced by decreasing the control ITI to 0.5 s and 2.0 s in non-associative conditioning within less than 4 min. Dishabituation was induced by randomly pairing one of these habituated US to an electrical stimulus in the PK neuron. All four parameters of the UR were enhanced by forward (SCS-US), but not backward (US-SCS), association of the US with 25, 100 and 250-Hz trains of SCS with 40-ms interstimulus intervals (ISI) for 4 min and persisted for another 4 min after turning off the SCS. The enhancement of these parameters was proportional to the SCS frequencies in the train. No UR was evoked by the SCS when the US was turned off after 4 min of classical conditioning.

Accumulated metal speciation in earthworm populations with multi-generational exposure to metalliferous soils: cell fractionation and high energy synchrotron analyses

Predicting metal bioaccumulation and toxicity in soil organisms is complicated by site-specific biotic and abiotic parameters. In this study we exploited tissue fractionation and digestion techniques, combined with X-ray absorption spectroscopy (XAS), to investigate the whole-body and subcellular distributions, ligand affinities, and coordination chemistry of accumulated Pb and Zn in field populations of the epigeic earthworm Lumbricus rubellus inhabiting three contrasting metalliferous and two unpolluted soils. Our main findings were (i) earthworms were resident in soils with concentrations of Pb and Zn ranging from 1200 to 27 000 mg kg(-1) and 200 to 34 000 mg kg(-1), respectively; (ii) Pb and Zn primarily accumulated in the posterior alimentary canal in nonsoluble subcellular fractions of earthworms; (iii) site-specific differences in the tissue and subcellular partitioning profiles of populations were observed, with earthworms from a calcareous site partitioning proportionally more Pb to their anterior body segments and Zn to the chloragosome-rich subcellular fraction than their acidic-soil inhabiting counterparts; (iv) XAS indicated that the interpopulation differences in metal partitioning between organs were not accompanied by qualitative differences in ligand-binding speciation, because crystalline phosphate-containing pyromorphite was a predominant chemical species in the whole-worm tissues of all mine soil residents. Differences in metal (Pb, Zn) partitioning at both organ and cellular levels displayed by field populations with protracted histories of metal exposures may reflect their innate ecophysiological responses to essential edaphic variables, such as Ca2+ status. These observations are highly significant in the challenging exercise of interpreting holistic biomarker data delivered by "omic" technologies.

Does speciation impact on Cu uptake by, and toxicity to, the earthworm Eisenia fetida?

Eisenia fetida were exposed to different concentrations (0.009, 0.049 and 0. 125 mg L-1) of Cu in an aqueous medium. Cu speciation was manipulated through the addition of different concentrations (0. 15, 0.35 and 50 mg L-1) of EDTA. Cu speciation (as labile and non-labile pools) was determined using Differential Pulse Anodic Stripping Voltammetry. Labile Cu includes free Cu ions together with weak inorganic Cu complexes and a small fraction of easily dissociable organic complexes. Metal uptake and earthworm mortality increased with total Cu concentration in the EDTA free solutions. In the presence of EDTA both metal uptake and mortality decreased. These experiments demonstrate that Cu speciation governs uptake and consequent toxicity of Cu to E. fetida. (c) 2007 Elsevier Masson SAS. All rights reserved.

Effect of organic complexation on the toxicity of Cu to the earthworm Eisenia fetida

The relationship between Cu speciation in solution and mortality and tissue Cu concentrations in Eisenia fetida was investigated. E. fetida were exposed to solutions containing 0.009, 0.049 and 0.125 mg Cu L-1 and 0, 0.15, 0.35 and 50 mg EDTA L-1. Mortalities of 100, 60, 50 and 25% were recorded in the 0.125 mg Cu L-1 solutions containing 0, 0.15, 0.35 and 50 mg EDTA L-1, respectively. Similarly tissue body burden decreased with increasing EDTA concentration. Complexation capacity of the solution increased with EDTA concentration. In the 0.125 mg Cu L-1 solution labile Cu concentration decreased with increasing EDTA concentration. These trends are attributed to complexation of free Cu ions with EDTA molecules, and the non-bioavailable nature of the resultant Cu-EDTA complex. (C) 2007 Elsevier Ltd. All rights reserved.

A Cu tolerant population of the earthworm Dendrodrilus rubidus (Savigny, 1862) at Coniston Copper Mines, Cumbria, UK

Dendrodrilus rubidus were sampled from a mine spoil soil at Coniston Copper Mine, an abandoned Cu mine in Cumbria, UK and a Cu-free control site. Earthworms were maintained for 14 d in both Kettering loam and a Moorland soil amended with Cu nitrate. Mortality, condition index, weight change and tissue concentration were determined. In both soils D. rubidus native to the mine site were able to tolerate significantly higher soil Cu concentrations (MWRT, p <= 0.001), and exhibited significantly less change in weight (t-test, p <= 0.001) and a lower loss in condition (t-test, p <= 0.001) than control earthworms. For a given soil Cu concentration tissue Cu concentrations were greater in the mine site earthworms. Low cocoon production and viability from the mine site population prevented the determination of toxicity parameters on the F1 generation and may be an indicator of the cost of tolerance to the population. (c) 2007 Elsevier Ltd. All rights reserved.

Liming upland grassland: the effects on earthworm communities and the chemical characteristics of carbon in casts

Different earthworm species have different tolerances of acid soil conditions, and the application of lime to upland grassland to improve the grazing quality may therefore alter the size and diversity of the earthworm community. Altering soil properties may also affect the chemical characteristics of organic C in earthworm casts. We surveyed the earthworm community of an upland grassland in southern Scotland at the outset of annual lime applications, and after 3 years, and used C-13 nuclear magnetic resonance (NMR) spectroscopy to assess the distribution of C between different functional groups in the organic matter. In addition, soil was incubated for 8 weeks with several earthworm species in the presence or absence of lime, and the earthworm casts were subsequently analysed by C-13 NMR spectroscopy. Liming did not significantly affect earthworm abundance or species diversity, but it did affect the chemical composition of the casts. Casts from earthworms incubated in unlimed soil had greater ratios of alkyl-C to O-alkyl-C, indicative of more decomposed, recalcitrant C, and spectra from litter-feeding species had the greatest intensities of O-alkyl-C signals. In limed soil, the largest O-alkyl-C signal intensities were not restricted to litter-feeding species, indicating an increase in the quality of organic matter ingested by geophagous species.

Earthworm induced mineral weathering: preliminary results

The role of earthworms in mineral weathering was investigated. The minerals anorthite, biotite, olivine, smectite and kaolinite were mixed with a sterilized manure substrate. Two treatments were used: minerals with earthworms and minerals without earthworms. The earthworms were established in mesocosms and left to process the substrates for 1, 2, 4 and 6 months. Four sacrificial replicates were used per time period. Changes in mineralogy were analysed using X-ray diffraction. Weathering of anorthite, biotite, smectite and kaolinite appears to be accelerated by the earthworms. There was evidence for the transformation of smectite to illite and the formation of a new mineral phase from kaolinite. Olivine appears not to be weathered by earthworms. Different minerals also appear to weather at different rates. (c) 2007 Elsevier Masson SAS. All rights reserved.