Salinization poses an increasing problem worldwide, threatening freshwater organisms and raising questions about ability to adapt. We explore the mechanisms enabling a planktonic crustacean to tolerate elevated salinity. By gradually raising water salinity in clonal cultures from 185 Daphnia magna populations, we showed that salt tolerance strongly correlates with native habitat salinity, indicating local adaptation. A GWAS revealed a major effect of the Alpha,alpha-trehalose-phosphate synthase ( TPS ) gene, suggesting that trehalose production facilitates salinity tolerance. We found a positive correlation between water salinity and trehalose concentrations in tolerant animals, while intolerant animals failed to produce trehalose. Using CRISPR/Cas9, a silenced TPS gene supported the role of trehalose under salt stress. Our study highlights how a keystone freshwater animal adapts to salinity stress using an evolutionarily conserved mechanism known in plants and bacteria, but not in metabolic-active animals. Highlights Salinity tolerance is a locally adapted trait in Daphnia magna The trehalose TPS gene shows a strong association with salinity tolerance Trehalose content increases with water salinity allowing survival of tolerant genotypes Non-functional TPS genes reduce salinity tolerance by preventing trehalose production
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