Salinity-associated differential gene expression in natural populations of the euryhaline killifish Aphanius iberus

Background Salinity is a major ecological driver in aquatic environments and strongly infl uences the physiology,distribution, and survival of fi sh species. While many fi shes are restricted to narrow salinity ranges,euryhaline species can tolerate large osmotic fl uctuations despite the substantial physiologicaladjustments required. The Spanish toothcarp, Aphanius iberus , an endemic Mediterranean killifi sh, is oneof such exceptional species capable of inhabiting environments ranging from freshwater to hypersalinesystems. However, despite this remarkable resilience, the molecular mechanisms underlying salinitytolerance and osmoregulatory plasticity in this species remain poorly understood. Results We analyzed using RNA sequencing transcriptomes from the gills and gastrointestinal tract ofindividuals from four wild populations spanning a natural salinity gradient from freshwater (0.75 PSU) tobrackish (5-10 PSU) and hypersaline (~50 PSU) habitats. Differential gene expression analyses revealedstrong tissue-specifi c patterns and environment-dependent responses. A substantially higher number ofdifferentially expressed genes was detected in the gills, where genes associated with ion transport,cytoskeletal remodeling, and energetic metabolism varied across salinity conditions, refl ecting theircentral role in osmoregulation. In the gastrointestinal tract, pathways related to lipid and carbohydratemetabolism were enriched, particularly in brackish populations. In addition, several genes associatedwith osmotic stress responses, including ATPases, histones, and transposable elements, showedsignifi cant expression differences across populations. Conclusions These fi ndings offer novel insights into the molecular architecture of salinity tolerance in euryhalinefi shes, highlighting coordinated transcriptional responses across tissues involved in ion regulation andmetabolic adjustment and improving our understanding of how euryhaline fi shes cope with extreme andfl uctuating osmotic environments. From a conservation perspective, identifying the molecular basis ofthis physiological plasticity contributes to the management of this highly threatened endemic speciesand the dynamic coastal habitats it inhabits.