Effects of high environmental ammonia on branchial ammonia excretion rates and tissue Rh-protein mRNA expression levels in seawater acclimated Dungeness crab Metacarcinus magister

Abstract

In the present study of the marine Dungeness crabs Metacarcinus magister, the long term effects of high environmental ammonia (HEA) on hemolymph ammonia and urea concentrations, branchial ammonia excretion rates and mRNA expression levels of the crustacean Rh-like ammonia transporter (RhMM), H+-ATPase (subunit B), Na+/K+-ATPase (α-subunit) and Na+/H+-exchanger (NHE) were investigated. Under control conditions, the crabs’ hemolymph exhibited a total ammonia concentration of 179.3±14.5μmolL−1, while urea accounted for 467.2±33.5μmolL−1, respectively. Both anterior and posterior gills were capable of excreting ammonia against a 16-fold inwardly directed gradient. Under control conditions, mRNA expression levels of RhMM were high in the gills in contrast to very low expression levels in all other tissues investigated, including the antennal gland, hepatopancreas, and skeletal muscle. After exposure to 1mmolL−1 NH4Cl, hemolymph ammonia increased within the first 12h to ca. 500µmolL−1 and crabs were able the keep this hemolymph ammonia level for at least 4days. During this initial period, branchial RhMM and H+-ATPase (subunit B) mRNA expression levels roughly doubled. After 14days of HEA exposure, hemolymph ammonia raised up to environmental levels, whereas urea levels increased by ca. 30%. At the same time, whole animal ammonia and urea excretion vanished. Additionally, branchial RhMM, H+-ATPase, Na+/K+-ATPase and NHE mRNA levels decreased significantly after long term HEA exposure, whereas expression levels of RhMM in the internal tissues increased substantially. Interestingly, crabs acclimated to HEA showed no mortality even after 4weeks of HEA exposure. This suggests that M. magister possesses a highly adaptive mechanism to cope with elevated ammonia concentrations in its body fluids, including an up-regulation of an Rh-like ammonia transporter in the internal tissues and excretion or storage of waste nitrogen in a so far unknown form.