This study investigated ionoregulatory strategies used by freshwater (FW) nymphs of the mayfly Like other FW organisms, nymphs maintain hemolymph ion levels (in mmol l: Na ∼102; Cl ∼84; K ∼6; pH ∼7.35) far in excess of their surroundings. This appears to be accomplished by the combined actions of the alimentary canal, Malpighian tubules (MTs) and tracheal gills. The alimentary canal contributes in a region-specific manner, a view supported by: (1) spatial differences in the activity of basolateral Na/K-ATPase (NKA) and apical V-type H-ATPase (VA) and (2) region-specific Na and K flux rates. Both indicate a prominent role for the hindgut (rectum) in K reabsorption. MTs also exhibit region-specific differences in Na and K flux rates that are coupled with an organized but tortuous architecture. NKA and VA activities were highest in MTs versus all other organs examined. Tracheal gills were found to be sites of Na uptake, but no difference in Na uptake was found between gills taken from different regions of the abdomen or spatially along individual gills. This is likely because each gill exhibited a dense population of NKA and/or VA immunoreactive cells (putative ionocytes). Data provide new insight into how FW mayfly nymphs regulate salt and water balance using the alimentary canal, MTs and tracheal gills as well as the first direct evidence that tracheal gills acquire ions from FW.

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