Gene expression profiles in earthworms exposed to ZnO and ZnO:Mn nanomaterials

Metal containing nanomaterials applied for various purposes such as cosmetic products may be released into the environment during their use. These nanomaterials, which are studied within the EU project DIAGONAL, can be taken up by various organisms, then bioaccumulate and potentially exert toxic effects. Uptake of metal nanomaterials in organisms such as earthworms was previously demonstrated to increase through dissolution in water in the soil ecosystem. However, modes of action, gene regulation and metabolism in earthworms exposed to metal oxide nanomaterials and metal ions are largely unexplored. The aim of our study is to quantify gene expression in earthworms (Eisenia fetida) in response to ZnO:Mn nanomaterial and MnCl₂ exposure using bulk RNA-seq analysis.

In a first experiment, earthworms were either exposed to clean reference soil or to soil contaminated with the ZnO nanomaterial. Earthworms were harvested after 2, 3 and 7 days of exposure. In addition, a second experiment was performed in which earthworms were exposed to the ZnO:Mn nanomaterial and to a MnCl₂ positive control treatment and their negative control treatments. Whole earthworms from all these experiments were ground after which the ground tissue after which a transcriptomics analysis was performed. 

Depending on the experiment, extracted and sequenced RNA was assembled and annotated de novo or using a reference database, which resulted in read count number per gene and sampled earthworms. Reads counts were normalized and analyzed for differential gene expression along with potential time-dependent effects in the third experiment. Functional enrichment of genes found differentially expressed in the obtained RNA-seq data identifies how these genes are involved in regulating the response to ZnO and ZnO:Mn nanomaterials and MnCl₂ in earthworms.