## 3 Responses to Hg Exposure at Different Levels of Biological Organization in _Elodea nuttallii_
### Gene Level Responses  
In recent years, ecotoxicological studies using omics have increased because of the availability of new sequencing methods that can be applied to non-model species of interest (e.g., RNAseq). Transcriptomic compares gene expression in chosen conditions vs. a control condition (reference). Until our studies in _E. nuttallii_ (Table 1), transcriptomic had not been applied to the analyses effects of Hg in aquatic plants.  
In _E. nuttallii_, we first performed the analysis of effects of 0.04, 4 and 40\(\cdot\)10\({}^{-7}\) M IHg for 24 h through differential gene analysis by illumina RNAseq in _E. nuttallii_[20]. Treatments of up-regulated genes coding for protein of stress such as chaperones, a regulation of genes found in energy metabolism pathways (e.g., sugar catabolism), attributed to an inhibition of energy reserves production by photosynthesis [20]. Concomitantly, we measured the down-regulation of genes related to homeostasis, such as metal transporters, for reducing and controlling Hg accumulation [20]. Indeed, the down-regulation of the gene _EtCOPT1_, with increasing concentrations of Hg, was observed. The data supported the uptake of Hg through high affinity Cu transporters [20]. Transcriptomic analysis also pointed towards IHg toxicity to trigger an anti-oxidative response and alter protein structure. In _E. nuttallii_, IHg (24 h 4\(\cdot\)10\({}^{-7}\) M) also up-regulated genes involved in N-metabolism and the biosynthesis of lipids [35]. We also compared transcriptomes upon 2 h exposure to increasing IHg and MMHg concentrations (10\({}^{-11}\) to 10\({}^{-8}\) M) [25]. The significantly regulated contigs are involved in similar gene ontology (GO) categories, including amino acid, energy and secondary metabolisms, as well as development, and transport (Figure 2a). The main difference was that MMHg regulated up to 20 times more contigs at similar intracellular concentration, evidencing that MMHg had a higher molecular impact than IHg (Figure 2b). A recent study in the Olt River (Romania), on in situ caged _E. nuttallii_ downstream a chlor-alkali plant, revealed a strong gene regulation after 2 h exposure, in line with the expected impact at much higher Hg concentrations (Figure 2), despite 1.2\(\cdot\)10\({}^{-11}\) M Hg concentration in water and a DOC of 3 mg\(\cdot\)L\({}^{-1}\) resulting in nonsignificant uptake [10]. It has to be noted that, among GO terms of significantly regulated contigs, a predominant part in all treatments show an unknown function, indicating some level of uncertainty in the analysis of responses, as well as a high potential for discoveries in the biology of Hg stress.  
Figure 2: (**a**) proportion of regulated genes among gene ontology terms (Mapman terminology) and (**b**) number of deregulated genes (DEGs) as a function of THg bioaccumulated in the intracellularly in _E. nuttallii_ exposed for 2 h with IHg, MMHg in the laboratory or in the Babeni Reservoir (Romania) [10; 25].  
The use of gene expression has shown promising results for the characterization of the precise early cellular toxicity pathways of Hg as well as for the definition of new biomarker of exposure. One strong interest of omics is its high sensitivity, allowing the measurement of significant responses at environmental concentrations. In _E. nuttallii_, the analysis of the expression of selected genes directly on RNA could be realized, with the nCounter method developed by [PERSON] [20; 25]. Statistical analysis (hierarchical clustering) of treatments using global gene expression signatures discriminated pM from nM concentrations of IHg or other stress exposure for concentrations as low as 1:10\({}^{-9}\) M THg [20; 25]. Transcriptomics could thus be used to derive more sensitive biomarkers for ecotoxicological testing than higher organization level endpoints.