Earthworm Dendrobaena Veneta Research Articles

Metallomic mapping of gut and brain in heavy metal exposed earthworms: A novel paradigm in ecotoxicology

This study explored the uptake of lead in the epigeic earthworm Dendrobaena veneta exposed to 0, 1000, and 2500 μg Pb/g soil. The soil metal content was extracted using strong acid digestion and water leaching, and analysed by means of Inductively Coupled Plasma Mass Spectrometry (ICP-MS) to estimate absolute and bioavailable concentrations of metals in the soil. The guts and heads of lead-exposed earthworms were processed into formalin-fixed and paraffin embedded sections for high-resolution multi-element metallomic imaging via Laser Ablation ICP-MS (LA-ICP-MS). Metallomic maps of phosphorus, zinc, and lead were produced at 15-μm resolution in the head and gut of D. veneta. Additional 4-μm resolution metallomic maps of the earthworm brains were taken, revealing the detailed localisation of metals in the brain. The Pb bioaccumulated in the chloragogenous tissues of the earthworm in a dose-dependent manner, making it possible to track the extent of soil contamination. The bioaccumulation of P and Zn in earthworm tissues was independent of Pb exposure concentration. This approach demonstrates the utility of LA-ICP-MS as a powerful approach for ecotoxicology and environmental risk assessments.

Taxon-specific ability of saprophagous soil macrofauna to reintegrate carbon from agricultural waste into soil

Saprophagous soil macroinvertebrates may potentially degrade agricultural wastes. However, it is not known, to what extent and representatives of which taxa may help reintegrating carbon from crop residues back into soil without triggering massive carbon release into the atmosphere. To tackle this problem, we conducted a three-month-long microcosm experiment with 21 different species of macrofauna (each treatment replicated four times) belonging to 13 families to test their ability to degrade wheat straw. Simultaneously CO2 release from the microcosms was measured. Five species did not survive under experimental conditions. Among the remaining 16 species, three significantly increased wheat straw decomposition with Oryctes nasicornis larvae having inflicted the highest straw mass loss (64%) in comparison with the control, where no animals were added (29%). None of the tested species increased cumulative CO2 evolution from the microcosms, while two species significantly reduced it. The reduction of carbon loss with aerobic respiration was recorded for Cetonia aurata larvae and the earthworm Dendrobaena veneta (respectively 2.5 and 2-fold in relation to the control – 53.8±4.6 mg CO2-C g−1 soil dry weight during the entire experiment). The original integrative Carbon Sequestration Index by Macrofauna (CSIM) calculated for both of the measured parameters suggests that the woodlouse Armadillidium vulgare and to a smaller extent the earthworm D. veneta appear to be the most promising organisms for industrial climate-friendly organic waste recycling in terms of survival, straw processing and simultaneous reduction of CO2 emissions from soil. Our results proved that the engagement of saprophagous macrofauna in crop residue decomposition is a viable technique of carbon reincorporation into the soil. It is accompanied with CO2 release mitigation into the atmosphere.

Mechanisms of carbon dioxide detection in the earthworm Dendrobaena veneta

IntroductionCarbon dioxide (CO2) is a critical biological signal that is noxious to many animals at high concentrations. The earthworm Dendrobaena veneta lives in subterranean burrows containing high levels of CO2 and respires through its skin. Despite the ecological and agricultural importance of earthworms, relatively little is known about how they make decisions in their environment, including their response to elevated levels of CO2.MethodsTo examine CO2 detection in this species, we designed the exudate assay, in which we placed an earthworm in a sealed container, exposed it to varying concentrations of CO2 for one minute, and recorded the amount of exudate secreted. Because earthworms excrete exudate in response to noxious stimuli, we hypothesized that the amount of exudate produced was proportional to the amount of irritation. We repeated these experiments after treatment with several blockers for molecules with potential involvement in CO2 detection, including carbonic anhydrases, guanylate cyclase, TRPA1, ASICs, and OTOP channels. We also confirmed the presence of homologous transcripts for each of these gene families in an epithelial transcriptome for D. veneta. Additionally, since organisms often detect CO2 levels indirectly by monitoring the conversion to carbonic acid (a weak acid), we used the exudate assay to evaluate aversion to additional weak acids (formic acid, acetic acid, and propionic acid).ResultsEarthworms excreted significantly more exudate in response to CO2 in a dosage-dependent manner, and this response was muted by the general carbonic anhydrase inhibitor acetazolamide, the carbonic anhydrase IX/XII inhibitor indisulam, the calcium channel blocker ruthenium red, the sodium channel blocker amiloride, and the acid-sensing ion channel blocker diminazene aceturate.DiscussionThese data provide evidence of the role of carbonic anhydrase and epithelial sodium channels in earthworm CO2 detection, establish that, similar to other subterranean-dwelling animals, earthworms are extremely tolerant of CO2, and contribute to our understanding of the mechanisms used by earthworms to detect and react to weak acids in their environment.

Effects of Tebuconazole on the Earthworm Dendrobaena veneta: Full Life Cycle Approach

Tebuconazole (TEB), a widely used triazole fungicide, is effective against soil-borne and foliar fungal pathogens. Toxicants can exhibit varying effects depending on the life stage of organisms, although standard toxicity tests typically focus on adult individuals. This study aimed to assess TEB’s potential adverse effects on the earthworm Dendrobaena veneta throughout its life cycle. Effects were evaluated by exposing cocoons to varying TEB concentrations, monitoring hatching, newly hatched juvenile mass, and growth to adulthood. A reproduction test assessed impacts on adults, offering insights into how these results compared with cocoon exposure findings. Results revealed that TEB delayed hatching at concentrations of 25, 50, and 100 mg/kg by 6, 8, and 15.5 days, respectively. Newly hatched juveniles exhibited a 15.96% (50 mg/kg) and 27.37% (100 mg/kg) reduction in body mass compared to controls, with no subsequent compensation during growth. Results from the reproduction tests showed no adverse effects on adult survival, but the effects are observed on juveniles, indicating a higher sensitivity of this developmental stage. While several adverse effects were observed, it is important to note that these occurred at concentrations exceeding recommended application rates. TEB appears safe for earthworms when used correctly, but the presence of multiple contaminants and stressors warrants consideration.

Autophagy of Candida albicans cells after the action of earthworm Venetin-1 nanoparticle with protease inhibitor activity

The present studies show the effect of the Venetin-1 protein-polysaccharide complex obtained from the coelomic fluid of the earthworm Dendrobaena veneta on Candida albicans cells. They are a continuation of research on the mechanisms of action, cellular targets, and modes of cell death. After the action of Venetin-1, a reduced survival rate of the yeast cells was noted. The cells were observed to be enlarged compared to the controls and deformed. In addition, an increase in the number of cells with clearly enlarged vacuoles was noted. The detected autophagy process was confirmed using differential interference contrast, fluorescence microscopy, and transmission electron microscopy. Autophagic vesicles were best visible after incubation of fungus cells with the Venetin-1 complex at a concentration of 50 and 100 µg mL−1. The changes in the vacuoles were accompanied by changes in the size of mitochondria, which is probably related to the previously documented oxidative stress. The aggregation properties of Venetin-1 were characterized. Based on the results of the zeta potential at the Venetin-1/KCl interface, the pHiep = 4 point was determined, i.e. the zeta potential becomes positive above pH = 4 and is negative below this value, which may affect the electrostatic interactions with other particles surrounding Venetin-1.

Effects of olive mill waste (OMW) contaminated soil on biochemical biomarkers and reproduction of Dendrobaena veneta.

Olive oil industry is economically important in Mediterranean countries. Disposal of olive mill waste (OMW) presents an environmental concern in those countries due to its high salinity and its high level of polyphenols. In order to reuse OMW, those properties have to change either through the filtration process and addition of adsorbents or by composting. One of the most important organisms in composting of organic wastes is earthworms. However, data on the effects of OMW on earthworms are scarce. The main aim of our study was to investigate whether OMW contaminated soil (OMW CS) causes adverse effects on molecular and organism level in epigeic earthworm Dendrobaena veneta and on microbiological activity. Changes of measured biochemical biomarkers (AChE, CAT, GST, lipids, MDA) varied depending on the quantity of added OMW CS and the exposure duration. Oxidative stress occurred after 7days of exposure, while in most cases enzyme activity recovered after 28days. At the highest ratio of contaminated soil (50%), reproduction was completely inhibited. The second aim was to investigate the impact of earthworms on phenol degradation and microbial activity, indicating an important role in the bioremediation of contaminated soils. Our results show that above a certain quantity an OMW CS has an adverse effect on earthworms, while the impact of earthworms on soil microbial activity was positive but transient. Yet, as the results also imply that earthworms have an impact on phenol degradation, they can be used to help remediation of OMW CS and its subsequent usage in agriculture. However, the quantity of OMW CS that can be safely added should be determined first.

Candida albicans cell wall as a target of action for the protein\u2013carbohydrate fraction from coelomic fluid of Dendrobaena veneta

The protein–polysaccharide fraction (AAF) isolated from the coelomic fluid of the earthworm Dendrobaena veneta destroys C. albicans cells by changing their morphology, disrupting cell division, and leading to cell death. Morphological changes in C. albicans cells induced by treatment with AAF were documented using DIC, SEM, and AFM. Congo Red staining showed that the fungal wall structure was changed after incubation with AAF. The effect on C. albicans cell walls was shown by AFM analysis of the surface roughness of fungal cell walls and changes in the wall thickness were visualized using Cryo-SEM. The FTIR analysis of C. albicans cells incubated with AAF indicated attachment of protein or peptide compounds to the fungal walls. The intact LC–ESI–MS analysis allowed accurate determination of the masses of molecules present in AAF. As shown by the chromatographic study, the fraction does not cross biological membranes. The Cryo-TEM analysis of AAF demonstrated the ability of smaller subunits to combine into larger agglomerates. AAF is thermally stable, which was confirmed by Raman spectroscopy. AAF can be considered as a potential antifungal antibiotic with activity against clinical C. albicans strains.

Properties of Vermicomposts Derived from Cameroon Sheep Dung

Due to a need for sustainability in agriculture, waste products ought to be utilized in the most appropriate way. A study was undertaken relating to the vermicomposting of Cameroon sheep dung (CSD) by the earthworm Dendrobaena veneta. Processing of this waste was investigated using unadulterated CSD and in a 1:1 mixture with unpalatable (waste) hay (CSDH). Results demonstrated that these materials were actively processed by D. veneta with vermicomposts obtained which can be characterized by a higher amount of total nitrogen, phosphorus and potassium (average, respectively, 17.0, 10.5, 13.2 g kg−1 d.m.), as well as lower total carbon and magnesium content (respectively, 340 and 3.2 g kg−1 d.m.), compared with the initial waste material. No significant differences were found between CSD and CSDH vermicomposts with respect to chemistry. Levels of selected trace elements (average: Cu 17.5–18.8, Cr 5.7–5.8, Pb 13.5–14.4, Ni < 3, Cd < 0.4 mg kg−1 d.m.) in both vermicomposts did not exclude their application to agricultural soil as a fertilizer.

Pro-apoptotic action of protein-carbohydrate fraction isolated from coelomic fluid of the earthworm Dendrobaena veneta against human colon adenocarcinoma cells

Earthworm coelomic fluid (CF) is known as a rich source of various bioactive compounds with promising anticancer features. However, it has been demonstrated that CF affects functionality of both, cancer and normal cells. This non-selective activity causes a major problem for medical application of CF.In this study, we present the anticancer activity of the active protein-carbohydrate fraction (AF) isolated from thermally treated CF of earthworm Dendrobaena veneta. The in vitro effect of the AF was examined in human colon model including normal human colon epithelium (CCD 841 CoTr) and human colon adenocarcinoma (HT-29 and LS180) cell lines. We investigated the impact of AF on cell viability neutral red and lactate dehydrogenase assays, morphology May-Grünwald-Giemsa staining assay proliferation MTT tetrazolium salt and BrdU incorporation assays as well as cell cycle progression propidium iodide/RNase staining and the activity of human 20S proteasome the hydrolysis of AMC from a Suc-LLVY-AMC peptide substrate. Additionally, the influence of AF on apoptosis was examined in HT-29 cells by Annexin V/PI, Hoechst 33342 staining and active caspase-3 assays.Our investigation demonstrated that AF at the tested concentration range does not affect the viability and morphology of CCD 841 CoTr cells. Simultaneously, AF inhibits human 20S proteasome activity as well as significantly decreases mitochondrial metabolism, disturbs cell cycle and induces apoptosis via activation of procaspase-3 in HT-29 cancer cells. Obtained results demonstrate the antiproliferative and proapoptotic activity of AF that can be useful in developing therapeutic strategies to treat human colon cancer.

Antitumor activity and apoptotic action of coelomic fluid from the earthworm Dendrobaena veneta against A549 human lung cancer cells.

It is known that earthworm coelomic fluid (CF) can affect not only cancer but also normal cells. The study demonstrated that the CF of the earthworm Dendrobaena veneta exhibited cytotoxicity against A549 lung cancer cells but did not toward the bronchial epithelial cell line BEAS-2B. The selective effect on the tumor cells was achieved after a short-term CF heat pre-treatment at 70°C. The cytotoxic effect of the CF was time- and concentration-dependent. The CF noticeably decreased the viability and affected the morphology of the A549 cells. Scanning electron microscopy revealed a different degree of destruction of the nucleus and cytoplasm of A549 cells. As determined by atomic force microscopy, the cell surface roughness increased while the cell stiffness was reduced upon the CF treatment. A twofold increase in the caspase 3, 4, 5, and 10 levels was observed in the A549 cells after the incubation with the CF. The results obtained by flow cytometry using Annexin V confirmed the proapoptotic effect of the earthworm CF on A549 lung cancer cells. The D.veneta CF and active fraction obtained with cytotoxicity toward A549 lung cancer is an interesting and promising preparation for further biological, chemical, and biomedical research.

Anti-Candida albicans effect of the protein-carbohydrate fraction obtained from the coelomic fluid of earthworm Dendrobaena veneta.

An antifungal active fraction (AAF) from the coelomic fluid (CF) of the earthworm Dendrobaena veneta was isolated. The aim of the study was to analyze the antifungal activity of the AAF and to carry out chemical characterization of the fraction. The active fraction showed antifungal activity against a clinical C. albicans isolate, C. albicans ATCC 10231, and C. krusei ATCC 6258. It effectively reduced the metabolic activity of C. albicans cells and influenced their morphology after 48 hours of incubation. Scanning electron microscopy (SEM) images revealed loss of integrity of the cell wall induced by the active fraction. Calcofluor White staining showed changes in the structure of the C. albicans cell wall induced by the AAF. The fungal cells died via apoptosis and necrosis after the treatment with the studied fraction. Electrophoresis under native conditions revealed the presence of two compounds in the AAF, while SDS/PAGE gel electrophoresis showed several protein and carbohydrate compounds. The active fraction was analyzed using Raman spectroscopy, MALDI TOF/TOF, and ESI LC-MS. The Raman analysis confirmed the presence of proteins and determined their secondary structure. The MALDI TOF/TOF analysis facilitated detection of four main compounds with a mass of 7694.9 m/z, 12292.3 m/z, 21628.3 m/z, and 42923.2 m/z in the analyzed fraction. The presence of carbohydrate compounds in the preparation was confirmed by nuclear magnetic resonance (NMR) and gas chromatography (GC-MS). The ATR-FTIR spectrum of the AAF exhibited high similarity to the spectrum of egg white lysozyme. The AAF showed no endotoxicity and cytotoxicity towards normal skin fibroblasts (HSF); therefore, it can be used for the treatment of skin and mucous membrane candidiasis in the future. Given its efficient and selective action, the fraction seems to be a promising preparation with antifungal activity against C. albicans.

Acute and subchronic effects of three herbicides on biomarkers and reproduction in earthworm Dendrobaena veneta

Earthworms are exposed to herbicides both through their skin and digestive system. Herbicides can influence earthworms' survival, physiology and reproduction. However, there is a lack of data on herbicide effects on earthworms as they are often regarded as low or non-toxic. The aim of our study was to investigate whether widely used commercial formulations of glyphosate (GLF), tembotrione (TBT) and nicosulfuron (NCS) each applied at three environmentally relevant concentrations have adverse effects on various biomarkers and reproduction in epigeic earthworm Dendrobaena veneta. The activities of measured biomarkers varied depending on the herbicide used and the exposure duration and suggest that oxidative stress plays an important role in the toxicity of tested herbicides. Namely, GLF caused an acetylcholinesterase (AChE) activity induction after seven days, and NCS after 28 days, while TBT caused an inhibition up to 47% (6.6 μg kgdw soil−1) after seven days. Only TBT caused a significant change (H2 = 13.96, p = 0.002) to catalase (CAT) after seven days of exposure. Malondialdehyde concentrations (MDA) were increased all the time after NCS exposure, but only after seven days in GLF and 28 days in TBT treatments, respectively. The tested herbicides did not have a significant effect on reproduction success, expect of NCS which increased the number of juveniles (p < 0.05).

The earthworm Dendrobaena veneta (Annelida): A new experimental-organism for photobiomodulation and wound healing.

Photobiomodulation (PBM) is a manipulation of cellular behavior using non-ablative low intensity light sources. This manipulation triggers a cascade of metabolic effects and physiological changes resulting in improved tissue repair, of benefit in the treatment of tissue injury, degenerative or autoimmune diseases. PBM has witnessed an exponential increase in both clinical instrument technology and applications. It is therefore of benefit to find reliable experimental models to test the burgeoning laser technology for medical applications. In our work, we proposed the earthworm Dendrobaena veneta for the study of nonablative laser-light effects on wound healing. In our preliminary work, D. veneta has been shown to be positively affected by PBM. New tests using D. veneta were set up to evaluate the effectiveness of a chosen 808 nm-64 J/cm2–1W-CW laser therapy using the AB2799 hand-piece with flat-top bean profile, on the wound healing process of the earthworm. Effective outcome was assimilated through examining the macroscopic, histological, and molecular changes on the irradiated posterior-segment of excised-earthworms with respect to controls. Three successive treatments, one every 24 hours, were concluded as sufficient to promote the wound healing, by effects on muscular and blood vessel contraction, decrement of bacteria load, reduction of inflammatory processes and tissue degeneration. D. veneta was demonstrated to be a reliable experimental organism that meets well the 3Rs principles and the National Science Foundation statement. Through their genetic and evolutionary peculiarity, comparable to those of scientifically accredited models, D. veneta allows the effect of laser therapies by multidisciplinary methods, at various degree of complexity and costs to be investigated.

Uptake and elimination kinetics of the biocide triclosan and the synthetic musks galaxolide and tonalide in the earthworm Dendrobaena veneta when exposed to sewage sludge.

Sewage sludge is an important amendment that enriches soils with organic matter and provides plants with nutrients such as nitrogen and phosphorus. However, knowledge on the fate and effects of organic pollutants present in the sludge on soil organisms is limited. In the present study, the uptake of triclosan, galaxolide, and tonalide in the earthworm Dendrobaena veneta was measured 1 wk after amendment of agricultural soil with sewage sludge, while elimination kinetics were assessed over a 21-d period after transferring worms to clean soil. After 1-wk exposure, earthworms had accumulated 2.6 ± 0.6 μg g-1 galaxolide, 0.04 ± 0.02 μg g-1 tonalide, and 0.6 ± 0.2 μg g-1 triclosan. Both synthetic musks were efficiently excreted and below the limit of quantification after 3 and 14 d of depuration for tonalide and galaxolide, respectively. Triclosan concentrations, on the other hand, did not decrease significantly over the depuration period, which may lead to the transfer of triclosan in the food web. Environ Toxicol Chem 2017;36:2068-2073. © 2017 SETAC.

The photobiomodulation effect of higher-fluence 808-nm laser therapy with a flat-top handpiece on the wound healing of the earthworm Dendrobaena veneta: a brief report.

The photobiomodulation effect of higher-fluence 808-nm laser therapy with a flat-top handpiece on the wound healing of the earthworm Dendrobaena veneta: a brief report.

Assessment of Sediment Heavy Metals Pollution Using Screening Methods (XRF, TGA/MS, XRPD and Earthworms Bioassay)

The aim of this study is focused on the use of screening methods (TG/DTA coupled with MS, XRF, AAS, XRPD and earthworm bioassay) for sediments pollution assessing by heavy metals (Cu, Zn, Pb, Hg) coming from the former mining workloads in the central Spis, Eastern Slovakia. The screening methods (XRF, AAS) indicated pollution of studied sediments by Cu, Zn, Pb, Hg. The earthworms Dendrobaena veneta caused in some studied samples decrease of heavy metals concentration after their 7 days’ exposure in sediments. The other screening methods such as thermal analysis and XRPD analysis, does not confirm the specifically changes in physicochemical properties comparing the properties before and after 7 days’ earthworm's exposure.

Analysis of the cytoskeleton organization and its possible functions in male earthworm germ-line cysts equipped with a cytophore.

We studied the organization of F-actin and the microtubular cytoskeleton in male germ-line cysts in the seminal vesicles of the earthworm Dendrobaena veneta using light, fluorescent and electron microscopy along with both chemically fixed tissue and life cell imaging. Additionally, in order to follow the functioning of the cytoskeleton, we incubated the cysts in colchicine, nocodazole, cytochalasin D and latrunculin A. The male germ-line cells of D. veneta are interconnected via stable intercellular bridges (IB), and form syncytial cysts. Each germ cell has only one IB that connects it to the anuclear central cytoplasmic mass, the cytophore. During the studies, we analyzed the cytoskeleton in spermatogonial, spermatocytic and spermatid cysts. F-actin was detected in the cortical cytoplasm and forms distinct rings in the IBs. The arrangement of the microtubules changed dynamically during spermatogenesis. The microtubules are distributed evenly in whole spermatogonial and spermatocytic cysts; however, they primarily accumulate within the IBs in spermatogonia. In early spermatids, microtubules pass through the IBs and are present in whole cysts. During spermatid elongation, the microtubules form a manchette while they are absent in the cytophore and in the IBs. Use of cytoskeletal drugs did not alter the general morphology of the cysts. Detectable effects—the occurrence of nuclei in the late spermatids and manchette fragments in the cytophore—were observed only after incubation in nocodazole. Our results suggest that the microtubules are responsible for cytoplasmic/organelle transfer between the germ cells and the cytophore during spermatogenesis and for the positioning of the spermatid nuclei.Electronic supplementary materialThe online version of this article (doi:10.1007/s00441-016-2398-6) contains supplementary material, which is available to authorized users.

Immune system participates in brain regeneration and restoration of reproduction in the earthworm Dendrobaena veneta

Earthworm decerebration causes temporary inhibition of reproduction which is mediated by certain brain-derived neurohormones; thus, cocoon production is an apposite supravital marker of neurosecretory center functional recovery during brain regeneration. The core aim of the present study was to investigate aspects of the interactions of nervous and immune systems during brain regeneration in adult Dendrobaena veneta (Annelida; Oligochaeta). Surgical brain extirpation was combined, either with (i) maintenance of immune-competent coelomic cells (coelomocytes) achieved by surgery on prilocaine-anesthetized worms or (ii) prior extrusion of fluid-suspended coelomocytes by electrostimulation. Both brain renewal and cocoon output recovery were significantly faster in earthworms with relatively undisturbed coelomocyte counts compared with individuals where coelomocyte counts had been experimentally depleted. These observations provide empirical evidence that coelomocytes and/or coelomocyte-derived factors (e.g. riboflavin) participate in brain regeneration and, by implication, that there is close functional synergy between earthworm neural and immune systems.

THE USE OF SELECTED ANAESTHETIC DRUGS IN SEARCH OF A METHOD FOR IMPROVING EARTHWORMS’ WELFARE

This paper describes selected effects of body contact of earthworms Dendrobaena veneta Rosa with local anaesthetic (LA) drugs used for human anesthesia (lidocaine and prilocaine) and anaesthetics for aquatic animals (MS-222). The findings showed safe and effective immobilization of earthworms with prilocaine at a concentration of 0.25-1%. At the applied concentrations lidocaine was safe, but less effective. On the other hand, MS-222, at the applied concentrations had a strongly irritating effect for earthworms and induced convulsive body movements connected with a discharge of coelomic fluid. The results may be relevant both for improving the welfare of earth worms during experiments and for the organization of research involving testing drugs on invertebrates. In this case, by using earthworms as an experimental model and by applying the method for measuring their mobility after contact with anaesthetics, which has been described in this article, it might be possible to replace experiments on guinea pigs, rabbits, rats and mice, which are expensive and require an approval of an ethics committee, with laboratory tests on earthworms.

Soil amendment with municipal sewage sludge affects the immune system of earthworms Dendrobaena veneta

Dendrobaena veneta is an earthworm species capable of consuming a wide range of organic wastes which may be used as a field indicator of municipal sewage sludge applied to land. The aim of the present 8-week laboratory experiment was to check viability, reproduction and the immune system of D. veneta maintained in soil without food additions (control 0s group) or in soil amended with 25% or 50% municipal sewage sludge (25s and 50s groups, respectively). Reproduction and immunity are important physiological functions whose detailed study can provide information on the effects of pollutants. After the 8-week exposure period, earthworm mortality (2 out 20 individuals) was recorded only in the 50s group. Reproduction was high in the 25s group (44 cocoons and 41 juveniles) whereas reproduction was almost completely inhibited both in the food-deprived control 0s group (1 cocoon, 3 juveniles) and in the 50s group containing a high amount of sludge (2 cocoons). Significantly increased numbers of non-invasively extruded coelomocytes were recorded 3 weeks after the start of the experiment in the 50s group, but they dropped to the food-deprived control level by the end of 8 weeks likely due to exhaustion of the immune system coping with sludge-derived microbes and/or toxins. In contrast, numbers of coelomocytes in the 25s group increased gradually reaching the maximum at the end of the experiments. In conclusion, high amounts of municipal sewage sludge are detrimental to worms, inhibiting reproduction and inflicting mortality. A moderate amount of municipal sewage sludge provides a good source of nutrients for D. veneta, supporting their growth and reproduction for at least 8 weeks. Immunological parameters might serve as useful indicators of earthworm exposure to sewage sludge.