Unraveling the molecular mechanisms of fish physiological response to freshwater salinization: A comparative multi-tissue transcriptomic study in a river polluted by potash mining

Freshwater salinization is an escalating global environmental issue that threatens freshwater biodiversity, including fish populations. This study aims to uncover the molecular basis of salinity physiological responses in a non-native minnow species (Phoxinus septimaniae x P. dragarum) exposed to saline effluents from potash mines in the Llobregat River, Barcelona, Spain. Employing high-throughput mRNA sequencing and differential gene expression analyses, brain, gills, and liver tissues collected from fish at two stations (upstream and downstream of saline effluent discharge) were examined. Salinization markedly influenced global gene expression profiles, with the brain exhibiting the most differentially expressed genes, emphasizing its unique sensitivity to salinity fluctuations. Pathway analyses revealed the expected enrichment of ion transport and osmoregulation pathways across all tissues. Furthermore, tissue-specific pathways associated with stress, reproduction, growth, immune responses, methylation, and neurological development were identified in the context of salinization. Rigorous validation of RNA-seq data through quantitative PCR (qPCR) underscored the robustness and consistency of our findings across platforms. This investigation unveils intricate molecular mechanisms steering salinity physiological response in non-native minnows confronting diverse environmental stressors. This comprehensive analysis sheds light on the underlying genetic and physiological mechanisms governing fish physiological response in salinity-stressed environments, offering essential knowledge for the conservation and management of freshwater ecosystems facing salinization. Overall design: This study investigates the ecological consequences of freshwater salinization caused by potash mining activities near the Llobregat River in Barcelona, Spain, focusing on the molecular underpinnings of salinity adaptation in the invasive minnow (Phoxinus septimaniae x P. dragarum). The research aims to uncover tissue-specific responses in the brain, gills, and liver by employing high-throughput mRNA sequencing and differential gene expression analyses. Minnows were sampled from two stations along the Llobregat River: the "Salt" station near a potash mine, exhibiting extreme salinity (18.1 ppt), and a "reference station" upstream with significantly lower salinity. Six fish samples (three from each station) were immediately fixed and stored. RNA extraction and sequencing were performed on the brain, liver, and gill tissues. A de novo transcriptomic analysis assessed differential gene expression and gene ontology variation between the “salt” and “reference” tissues.