Field application of de novo transcriptomic analysis to evaluate the effects of sublethal freshwater salinization on Gasterosteus aculeatus in urban streams
Freshwater salinization poses global challenges for aquatic organisms inhabiting urban streams, impacting their physiology and ecology. However, current salinization research predominantly focuses on mortality endpoints in limited model species, overlooking the sublethal effects on a broader spectrum of organisms and the exploration of adaptive mechanisms and pathways under natural field conditions. To address these gaps, we conducted high-throughput sequencing transcriptomic analysis on the gill tissue of the euryhaline fish Gasterosteus aculeatus, investigating its molecular response to salinity stress in the highly urbanized river Boye, Germany. We found that in stream sections with sublethal concentrations of chloride costly osmoregulatory systems were activated, evidenced by the differential expression of genes related to osmoregulation. Our enrichment analysis revealed differentially expressed genes (DEGs) related to transmembrane transport and regulation of transport and other osmoregulation pathways, which aligns with the crucial role of these pathways in maintaining biological homeostasis. Notably, we identified candidate genes involved in increased osmoregulatory activity under salinity stress, including those responsible for moving ions across membranes: ion channels, ion pumps, and ion transporters. Particularly, genes from the solute carrier family SLC, aquaporin AQP1, chloride channel CLC7, ATP-binding cassette transporter ABCE1, and ATPases member ATAD2 exhibited prominent differential expression. These findings provide insights into the potential molecular mechanisms underlying the adaptive response of euryhaline fish to salinity stress and have implications for their conservation and management in the face of freshwater salinization. Overall design: To assess the effects of sublethal freshwater salinization on Gasterosteus aculeatus in urban streams, we selected three sampling stations along a chloride concentration gradient in two tributaries and the main channel of the River Boye (Germany). G. aculeatus individuals were collected using electrofishing equipment, euthanized with an overdose of tricaine methanesulfonate (MS222), and their gill tissue was promptly harvested, fixed in RNAprotect (QIAGEN), and stored at -20°C. Nine samples were obtained, comprising three from each of the control (Station B), mid-salinity (Station A), and high-salinity (Station C) stations. Subsequently, we conducted a differential expression analysis employing a Trinity de novo approach, comparing the expression profiles of three biological replicates from the control station (B) with those from the treatment stations (A and C).