AbstractOwing to their extraordinary niche diversity, the Crustacea are ideal for comprehending the evolution of osmoregulation. The processes that effect systemic hydro-electrolytic homeostasis maintain hemolymph ionic composition via membrane transporters located in highly specialized gill ionocytes. We evaluated physiological and molecular hyper- and hypo-osmoregulatory mechanisms in two phylogenetically distant, freshwater crustaceans, the crab Dilocarcinus pagei and the shrimp Macrobrachium jelskii, when osmotically challenged for up to 10 days. When in distilled water, hemolymph osmolality and [Cl−] increased briefly in D. pagei, stabilizing at initial values, while [Na+] decreased continually. Gill V(H+)-ATPase, Na+/K+-ATPase and Na+/K+/2Cl− gene expressions were unchanged. In M. jelskii, hemolymph osmolality, [Cl−] and [Na+] decreased continually for 12 h, the shrimps no longer surviving. Gill transporter gene expressions increased 2- to 5-fold. After 10-days exposure to brackish water, D. pagei was isosmotic, iso-chloremic and iso-natriuremic. Gill V(H+)-ATPase expression decreased while Na+/K+-ATPase and Na+/K+/2Cl− expressions were unchanged. In M. jelskii, the hemolymph was hypo-regulated, particularly [Cl−]. Transporter expressions initially increased 3- to 12-fold, declining to control values. Gill V(H+)-ATPase expression underlies the ability of D. pagei to survive in fresh water while Na+/K+-ATPase and Na+/K+/2Cl− expressions enable M. jelskii to deal with osmotic challenge. These findings reveal divergent responses in two unrelated crustaceans habiting a similar osmotic niche. While D. pagei has maintained the capacity to tolerate elevated cellular isosmoticity despite its inability to secrete salt, M. jelskii displays clear hypo-osmoregulatory ability. Each species has developed distinct strategies at the transcription and systemic levels during adaptation to fresh water.Summary statementDuring their evolutionary adaptation to fresh water, unrelated hololimnetic crustaceans have developed physiological strategies like tolerating elevated cellular isosmoticity or regulating hypo-osmoregulatory ability at the gene transcription level.