Short-term gene expression dynamics in earthworms exposed to ZnO nanomaterial

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 environmental organisms such as earthworms was previously demonstrated to depend on the properties of the soil (e.g. Lahive et al., 2023). However, modes of action, gene regulation and metabolism in earthworms exposed to metal nanomaterials are largely unexplored. The aim of our study is to quantify gene expression in earthworms (Eisenia fetida and Eisenia andrei) in response to ZnO nanomaterial exposure using bulk RNA-seq analysis. Earthworms were either exposed to clean reference soil or to soil contaminated with the ZnO nanomaterial. Earthworms were harvested after 2, 3, 4 and 7 days of exposure to control or ZnO contaminated soil. Whole earthworms were ground in liquid nitrogen after which the ground tissue was divided into two equal amounts enabling both transcriptomics and metabolomics analysis. RNA was extracted using a TRIzol kit after which libraries were prepared and then sequenced using an Illumina Novaseq platform. Assembled reads were mapped to genes using the reference genomes with accession numbers GCA_003999395.1 and GWHACBE00000000 for E. fetida and E. andrei, respectively. Reads counts were normalized using the relative log expression method. Thereafter differential gene expression analysis was performing to identify any effect induced by the ZnO nanomaterial along with potential time-dependent effects. Functional enrichment of genes found differentially expressed in the obtained RNA-seq data will identify how these genes are involved in regulating the response to ZnO nanomaterials in earthworms with respect to time. The present study is unique in assessing the gene expression responses in earthworms over days 0, 2, 3, 4 and 7 from the start of the exposure to ZnO. In addition, elucidating this response aims to cover potential mechanisms towards exposure to ZnO. Finally, the response assesses to what extent applications of ZnO nanomaterials such as cosmetic products and sunscreens can be considered non-toxic for environmental organisms in the soil.