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Additional file 2.

Additional file 3.

Additional file 5.

Additional file 1.

Earthworms are ecologically important soil dwelling organisms which are affected by anthropogenic pollution through agriculture and industry. Cd is one of the main soil pollutants with a high carcinogenic and toxicogenic potential. Lumbricus terrestris, the common earthworm, has therefore been used in lab exposures to low doses of environmentally relevant Cd concentrations over three months with a subsequent recovery period in control soil followed by an exposure to high levels of Cd. Quantitative RealTime PCR, comet assay, and enzyme activity assays have been applied to determine metallothionein (MT) and phytochelatin synthase (PCS) levels as well as DNA damage and oxidative stress in earthworms. MTs are probably able to act as the exclusive mechanism to detoxify low doses of Cd over several months and to prevent the organism from Cd-induced damages and oxidative stress. Control earthworms exposed to high Cd concentrations revealed an increase of MT gene expression levels. Low dose Cd-exposed earthworms which face a high Cd concentration are able to induce MT expression to a much higher degree. The transcriptional regulation mechanism of MTs is therefore suggested to be responsive to the exposure history. Therefore, it seems that highly flexible coping strategies have evolved in earthworms facing environmental stress like Cd pollution.

Emerging evidence suggests that soil heavy metal pollution can alter epigenetic marks in soil fauna, with DNA methylation as one of the underlying mechanisms - a process which can be persistent and even heritable. The aim of this study was to assess the consequences of chronic heavy metal exposure on genome wide DNA methylation in the earthworm Lumbricus terrestris. Earthworms were exposed to low (10 mg/kg) environmentally relevant concentration of cadmium (Cd) for a period of 3 months. Through the exposure period, the overall health status of the earthworms was monitored at the cellular (DNA damage, oxidative stress, lipid damage) and organismic level (fitness, reproductive success). Genome wide DNA methylation status was assessed by methylation sensitive amplification polymorphism (MSAP) at the beginning of the exposure and after 1 and 3 months exposure. During the exposure, earthworm fitness and cellular mechanisms remained unaffected by Cd. However, on the epigenetic level, already after 1 month of exposure, Cd induced significant increase in DNA methylation, which remained constant until the end of the exposure. The results of this study demonstrate that low levels of environmental pollution can lead to the occurrence of subtle epigenetic modifications, even when no impacts on the cellular and organismic level can be observed.

Emerging evidence suggests that soil heavy metal pollution can alter epigenetic marks in soil fauna, with DNA methylation as one of the underlying mechanisms - a process which can be persistent and even heritable. The aim of this study was to assess the consequences of chronic heavy metal exposure on genome wide DNA methylation in the earthworm Lumbricus terrestris. Earthworms were exposed to low (10 mg/kg) environmentally relevant concentration of cadmium (Cd) for a period of 3 months. Through the exposure period, the biomarker response and health status of the earthworms was monitored at the cellular (DNA damage, oxidative stress, lipid damage) and organismic level (fitness, reproductive success) and their detoxification capacity was assessed by monitoring the expression of metallothionein (MT) gene. Genome wide DNA methylation status was assessed by methylation sensitive amplification polymorphism (MSAP) at the beginning of the exposure and after 1 and 3 months of exposure. During the exposure, earthworm fitness remained unaffected by Cd. Cellular stress was evident in the first 2 weeks of the exposure but afterwards it returned to the control level which corresponded well with the MT expression results. On the epigenetic level, already after 1 month of exposure, Cd induced significant increase in DNA methylation, which remained increased until the end of the exposure. The results of this study demonstrate that low levels of environmental pollution can lead to the occurrence of subtle epigenetic modifications, even when no or very low impact on the cellular and organismic level can be observed. These findings reinforce the application of epigenetic tools as predictors of toxicological response and suggest their use in environmental toxicology assessment.

A variety of environmental stressors induce the expression of Metallothioneins (MTs), which mainly act as radical scavengers in oxidative stress response and detoxification of heavy metals. Earthworm MTs are widely used as biomarkers for metal pollution, however, the mechanism of MT gene activation is, however, largely unknown in most invertebrate species. Therefore, the present study aims to characterize the MT induction behavior upon Cd exposure and physical injury in two earthworm species (Lumbricus rubellus and L. terrestris). A basal MT transcription with no circadian pattern could be detected and a significant increase of MT gene expression was observed upon Cd exposure in a dose and time dependent manner. Interestingly, tip-cut earthworms did not show any MT transcription in controls or Cd exposed individuals. MT promoter studies revealed a member of the CRE binding protein family as putative transcriptional MT activator most likely in both controls and in heavy metal exposed earthworms. Additionally, MRE binding sites have been suggested to play a role in MT gene activation, although the typical MRE binding protein, MTF-1, is lacking in earthworms. The present study discloses novel information on MT gene transcription and its putative activation mechanisms. Moreover, we hypothesize that there might be a link between detoxification- and tissue repair mechanisms in earthworms.