Human activities and climate change have significantly increased humic substances in freshwater ecosystems over the last few decades. This increase is particularly concerning during seasonal changes or after heavy rainfall, when concentrations can easily increase up to tenfold. This phenomenon, known as "browning," has unknown consequences for aquatic organisms. This study is the first to determine the effects of increasing humic substance concentrations on the transcriptomic and structural responses in the gills of rainbow trout (Oncorhynchus mykiss). Overall, 27 genes mainly involved in xenobiotic metabolism (cyp1a3, cyp1b1, pik3r6), immune response (rgs2, dll1, ccl19, acod1), and mucosal glycoprotein expression (muc2, prg4) were upregulated. No significant alterations were noted in gill morphology, although the molecular data strongly indicated a proinflammatory response. Our results highlight the risks posed by increasing humic substance concentrations for fish and aquatic ecosystems and emphasize the urgent need to implement effective monitoring and resource management strategies to address browning waters.

Leibniz Institute of Freshwater Ecology and Inland Fisheries Berlin Germany; University of South Bohemia in České Budějovice Faculty of Fisheries and Protection of Waters South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses Institute of Aquaculture and Protection of Waters Na Sádkách 1780 370 05 České Budějovice Czech Republic

Research Institute for Farm Animal Biology Dummerstorf Germany

University of Veterinary Medicine Hannover Fish Disease Research Unit Hannover Germany

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