# Soil Hg Contamination Impact on Earthworms'
## 4 Discussion
### Effect of Hg in Earthworm, Cast and Soil Microbiome  
We hypothesized that the diversity and community structure of the earthworm gut microbiome, as well as the microbiome of earthworm cast and the surrounding soil, would be affected by Hg-contamination (first hypothesis). This pattern was clearly observed in experiment 2, where the richness and Shannon diversity index of both the soil in the pots and the earthworm gut bacterial communities decreased in the Hg-contaminated treatments. However, in experiment 1, in which the Hg contamination was milder and consisted of only a single Hg-contaminated soil layer, the bacterial and fungal alpha diversity increased in Hg-contaminated earthworm samples. A similar increase in bacterial alpha diversity was previously observed along the soil Hg-contamination gradient at the same site where the soil was collected for our study [24]. Moreover, this increase in diversity might support the rise of rare or weaker competitor OTUs under intermediate stress, as proposed by the disturbance hypothesis in community ecology [50]. Hg-tolerant OTUs might therefore be favored under Hg-contaminated treatments, as their resistance capabilities to Hg boost their competitive abilities over dominant taxa under natural soil conditions. Furthermore, soil bacterial diversity was found to be positively correlated with total Hg and MeHg content [21]. The strong shift of the bacterial and fungal community structure in the Hg-contaminated earthworm samples is potentially a direct consequence of the high proportion of accumulated THg and MeHg in the earthworm tissues. Such a large change within the microbial community structure was not observed between the Hg-contaminated and uncontaminated soil and cast samples. The effect of the Hg-contaminated treatments was also more pronounced for the bacterial than the fungal communities in both experiments, suggesting a higher resistance of fungi to Hg contamination in the earthworm gut, similar to observations made in Hg-contaminated soils [5; 13]. The drastic decrease in the relative abundance of the Mollicutes (phylum Firmicutes), largely dominating the uncontaminated earthworm bacterial community, is a direct cause of shift in the bacterial community structure. Interestingly, Firmicutes have been reported to dominate the gut microbiome of earthworms fed with organic compost, but their relative abundance decreased when the earthworms were fed with mineral soil [51]. However, to our knowledge, a sensitivity of the Firmicutes to heavy metal accumulation has not been reported elsewhere. The dominant bacterial phyla observed in the Hg-contaminated earthworm samples, i.e., Chloroflexi, Acidobacteria, Actinobacteria, Planctomycetes, Proteobacteria, Bacteroidetes and Verrucomicrobia, are phyla that are rather ubiquitous in the soil and have been found previously in Hg-contaminated soils [24; 47; 52]. In accordance with our second hypothesis, we observed a number of tolerant bacterial and fungal taxa. These Hg-tolerant taxa were phylogenetically very diverse but grouped mostly within the phyla dominating the Hg-treated samples. This notwithstanding, taxonomically related taxa within the same phylum showed very different responses to Hg contamination; some relatively abundant taxa were clearly sensitive to Hg, whereas other taxa from the same classes were positively associated with the Hg-contaminated treatment. This was the case for taxa within the Beta- and Gammaproteobacteria, Flavobacteria and Verrucomicrobia classes. Taxa within the Proteobacteria and Verrucomicrobia were reported to be Hg-tolerant in Hg-contaminated soils [24; 52], along with Gemmonatimanedetes [52], Burkholderia [5; 47], and Rhodobiaceae and Chtoniobacter [24], which were not identified as Hg-tolerant taxa in earthworm samples in the present study. These differences might stem from the complexity of microbial communities in natural soil systems, as many environmental factors other than Hg also potentially shape the microbial community composition.