Transcriptomics reveals the action mechanisms and cellular targets of citrate-coated silver nanoparticles in a ubiquitous aquatic fungus

Silver nanoparticles (AgNPs) are among the major groups of contaminants of emerging concern for aquatic ecosystems. The massive application of AgNPs relies on the antimicrobial properties of Ag, raising concerns about their potential risk to ecologically important freshwater microbes and the processes they drive. Moreover, it is still uncertain whether the effects of AgNPs are driven by the same mechanisms underlying those of Ag ions (Ag⁺). We employed transcriptomics to better understand AgNP toxicity and disentangle the role of Ag⁺ in the overall toxicity towards aquatic fungi. To that end, the worldwide-distributed aquatic fungus Articulospora tetracladia, that plays a central role in organic matter turnover in freshwaters, was selected and exposed for 3 days to citrate-coated AgNPs (∼20 nm) and Ag⁺ at concentrations inhibiting 20% of growth (EC₂₀). Responses revealed 258 up- and 162 down-regulated genes upon exposure to AgNPs and 448 up- and 84 down-regulated genes under exposure to Ag⁺. Different gene expression patterns were found after exposure to each silver form, suggesting distinct mechanisms of action. Gene ontology (GO) analyses showed that the major cellular targets likely affected by both silver forms were the biological membranes. GO-based biological processes indicated that AgNPs up-regulated the genes involved in transport, nucleobase metabolism and energy production, but down-regulated those associated with redox and carbohydrate metabolism. Ag⁺ up-regulated the genes involved in carbohydrate and steroid metabolism, whereas genes involved in localization and transport were down-regulated. Our results showed, for the first time, distinct profiles of gene expression in aquatic fungi exposed to AgNPs and Ag⁺, supporting different modes of toxicity of each silver form. Also, our results suggest that Ag⁺ had a negligible role in the toxicity induced by AgNPs. Finally, our study highlights the power of transcriptomics in portraying the stress induced by different silver forms in organisms.