Methylation of mercury in earthworms and the effect of mercury on the associated bacterial communities

Abstract

Methylmercury compounds are very toxic for most organisms. Here, we investigated the potential of earthworms to methylate inorganic-Hg. We hypothesized that the anaerobic and nutrient-rich conditions in the digestive tracts of earthworm's promote the methylation of Hg through the action of their gut bacteria. Earthworms were either grown in sterile soils treated with an inorganic (HgCl2) or organic (CH3HgCl) Hg source, or were left untreated. After 30 days of incubation, the total-Hg and methyl-Hg concentrations in the soils, earthworms, and their casts were analyzed. The impact of Hg on the bacterial community compositions in earthworms was also studied. Tissue concentrations of methyl-Hg in earthworms grown in soils treated with inorganic-Hg were about six times higher than in earthworms grown in soils without Hg. Concentrations of methyl-Hg in the soils and earthworm casts remained at significantly lower levels suggesting that Hg was mainly methylated in the earthworms. Bacterial communities in earthworms were mostly affected by methyl-Hg treatment. Terminal-restriction fragments (T-RFs) affiliated to Firmicutes were sensitive to inorganic and methyl-Hg, whereas T-RFs related to Betaproteobacteria were tolerant to the Hg treatments. Sulphate-reducing bacteria were detected in earthworms but not in soils.

Figure 1. Experimental design.

Two experimental assays (A and B) were performed: In the main experiment (A) sterile soil was either treated with inorganic Hg (+inorganic−Hg), methyl-Hg (+methyl−Hg) or without Hg (−Hg) (i). The soils were incubated abiotically, with earthworms or with a earthworm rinsing suspension for 30 days in the dark at 15°C (ii). At the end of incubation, the earthworms were removed from the soil and the Hg and methyl-Hg concentrations in the soil and earthworms were determined (iii). In a separate experiment (B), casts (excreted feces) were used as controls to study a possible methylation of Hg by organisms introduced into the soil by earthworms. Earthworms were incubated for one week in soils treated with inorganic-Hg (i) before they were placed into new boxes containing soils treated with Hg (ii). After three days, the earthworms were removed (iii). Immediately after removing the earthworms a cast sampling period has been started for 28 days (iv).

Figure 2. PCoA of bacterial T-RFLP profiles in earthworms.

The influence of the Hg treatment (symbols) was estimated with Permutational MANOVA.

Figure 3. Percentage of particular bacterial T-RFs compared to the total abundance of T-RFs in earthworms.

The T-RFs shown were chosen because they were (i) relatively abundant or (ii) sensitive or tolerant to Hg and methyl-Hg amendment. (*) indicates the T-RFs differed significantly (p<0.05) between the soil Hg treatments. The relationships between the corresponding clone T-RFs and the environmental sample T-RFs are shown in the Figure 4.

Figure 4. Phylogram with the phylogenetic relationships of 16S rRNA gene sequences.

The phylogenetic tree shows the bacterial strains and environmental clones most closely to the T-RFs in Figure 3. Whenever possible, closest strains were used for the calculation of the tree but when no strain was available (e.g. many species of Mollicutes could not be isolated so far) the closest clone was used. The tree was calculated by Baysian inference using sequences of 898 bp lengths and shows the affiliation between the clones and closest related sequences of NCBI. The clones of our study are bold marked. Only bootstrap values above 0.9 are given. The scale bar represents 0.1 (10%) of sequence divergence.