Abstract
The aim of this work was to identify in Eisenia fetida genes whose expression are regulated following exposure to a complex mixture of metallic trace elements (MTE) representative of a highly polluted smelter soil. Suppression subtractive hybridization (SSH) was used to construct cDNA libraries enriched in up- or down-regulated transcripts in the immunecirculating cells of the coelomic cavities, namely coelomocytes, from worms exposed to metallic pollution. Among 1536 SSH-derived cDNA clones sequenced, we identified 764 unique ESTs of which we selected 18 candidates on the basis of their redundancy. These selected candidates were subjected to a two-step validation procedure based on the study of their expression level by real-time PCR. The first step consisted in measuring the expression of the 18 candidates in worms exposed to artificial contaminated soil. The second step consisted in measuring the expression in animals exposed to a ‘‘naturally'' contaminated soil sampled close to a smelter. Both steps allowed us to highlight 3 candidates that are strongly induced in worms exposed to a smelter polluted soil. These candidates are: the well-known MTE-induced Cd–metallothionein and 2 original biomarkers, lysenin, and a transcript, which cloning of the complete coding sequence identified as the coactosin-like protein