Low levels of Cd induce persisting epigenetic modifications and acclimation mechanisms in the earthworm Lumbricus terrestris

Abstract

Toxic effects of cadmium (Cd), a common soil pollutant, are still not very well understood, particularly in regard to its epigenetic impact. Therefore, the aim of this study was to assess DNA methylation changes and their persistence in the earthworm Lumbricus terrestris upon chronic low dose Cd exposure using methylation sensitive amplification polymorphism (MSAP). Moreover, the biomarker response and fitness of the earthworms, as well as the expression of detoxification-related genes (metallothionein (MT) and phytochelatin synthase (PCS)) was evaluated. Low levels of Cd caused an increase in genome-wide DNA methylation, which remained partly modified, even after several months of recovery in unpolluted soil. Increased cellular stress seemed to decrease after two weeks of exposure whereas fitness parameters remained unaffected by Cd, probably as a result from the activation of detoxification mechanisms like the expression of MTs. Interestingly, even though the level of Cd exposure was very low, MT expression levels indicate the development of acclimation mechanisms. Taken together, this study demonstrates that acclimation, as well as epigenetic modifications can occur already in moderately polluted environments. In addition, these effects can have long-lasting impacts on key species of soil invertebrates and might persist long after the actual heavy metal challenge has passed.

Fig 1. Cd accumulation in tissue of L. terrestris in control and Cadmium (Cd)-exposed individuals over 19 weeks including three exposure periods.

1: Earthworms spent 12 weeks in clean soil (grey line) or soil spiked with 10 mg/kg CdCl₂ (black line). 2: All earthworms were kept in clean soil for five weeks (recovery). 3: Both the control- and Cd-derived group were split up into two groups, with one being transferred to clean soil (grey lines) and one being transferred to soil spiked with 60 mg/kg CdCl₂ (thick black lines) for two weeks. The expressions "Control- and Cd-derived group" refer to the respective treatment during exposure period 1. Stars indicate significant differences to the controls (p≤0.05).

Fig 2. Biomarker response in L. terrestris control and Cadmium (Cd)-exposed individuals over 19 weeks including three different exposure periods.

1: Earthworms spent 12 weeks in clean soil (grey lines) or soil spiked with 10 mg/kg CdCl₂ (black lines). 2: All earthworms were kept in clean soil for five weeks (recovery). 3: Both the control-and Cd-derived group were each split up into two groups, with one being transferred to clean soil (grey lines) and one being transferred to soil spiked with 60 mg/kg CdCl₂ (thick black lines) for two weeks. The expressions "Control- and Cd-derived group" refer to the respective treatment during exposure period 1. (a) DNA damage measured in coelomocytes using comet assay. (b) Catalase (CAT) activity measured in tissue samples. (c) Malondialdehyde (MDA) concentrations in tissue samples. Stars indicate significant differences to the controls (p≤0.05).

Fig 3. Metallothionein 2 (MT2) mRNA copy numbers in L. terrestris tissue of control and Cadmium (Cd)-exposed individuals over 19 weeks including three exposure periods.

1: Earthworms spent 12 weeks in clean soil (grey lines) or soil spiked with 10 mg/kg CdCl₂ (black lines). 2: All earthworms were kept in clean soil for five weeks (recovery). 3: Both the control- and Cd-derived group were each split up into two groups, with one being transferred to clean soil (grey lines) and one being transferred to soil spiked with 60 mg/kg CdCl₂ (thick black lines) for two weeks. The expressions "Control- and Cd-derived group" refer to the respective treatment during exposure period 1. Stars indicate significant differences to the controls (p≤0.05).

Fig 4. Phytochelatin synthase (PCS) mRNA copy numbers in L. terrestris tissue of control and Cadmium (Cd)-exposed individuals over 19 weeks including three exposure periods.

1: Earthworms spent 12 weeks in clean soil (grey lines) or soil spiked with 10 mg/kg CdCl₂ (black lines). 2: All earthworms were kept in clean soil for five weeks (recovery). 3: Both the control- and Cd-derived group were each split up into two groups, with one being transferred to clean soil (grey lines) and one being transferred to soil spiked with 60 mg/kg CdCl₂ (thick black lines) for two weeks. The expressions "Control- and Cd-derived group" refer to the respective treatment during exposure period 1. Stars indicate significant differences to the controls (p≤0.05).

Fig 5. Methylation changes in L. terrestris coelomocytes in controls (C) (grey lines) and individuals exposed to soil spiked with 10 mg/kg CdCl₂ (Cd) (black lines).

Left and right panels refer to replica I and II, respectively. Only fragments where methylation did not change in the control treatment were considered for analysis. (a) Percentage of methylated fragments among all methylation states in MSL fragments in replica I (407 MSL) and replica II (463 MSL), between control and Cd-treated earthworms. (b) Principal co-ordinate analysis was performed using the data on the epigenetic distance of differentially methylated MSL between treatment groups in replica I (76 MSL) and replica II (94 MSL). (c) Type of DNA methylation change among differentially methylated MSL in replica I (76 MSL) and replica II (94 MSL). The percentages of the left pie chart indicate fragments which showed the same methylation changes after 7 months in clean soil. ↑ indicates an increase and ↓ a decrease in methylation compared to Cd 0 (earthworms used for Cd exposure sampled before the exposure start),—indicates that methylation returned to the Cd 0 level; Cd 4/Cd 12: earthworms sampled after 4 or after 12 weeks of exposure to 10 mg/kg CdCl₂.