Identification of the role of Rh protein in ammonia excretion of the swimming crab Portunus trituberculatus

In Portunus trituberculatus, a full-length cDNA of Rhesus-like glycoprotein (Rh protein), encoding the entire 478 amino acid protein, has been identified in gills, and plays an essential role in ammonia (NH3/NH4+) excretion. Phylogenetic analysis of Rh-like proteins from crabs was clustered, showing high conservation of the ammonium transporter domain and transmembrane segments essential to the function of Rh protein. Rh protein of P. trituberculatus (PtRh) was detected in all tested tissues, and showed the highest expression in the gills. To further characterize the role of PtRh in ammonia metabolism and excretion, double-stranded RNA-mediated RNA interference of PtRh was employed. Knockdown of PtRh upregulated mRNA expression of ammonia excretion-related genes encoding aquaporin (AQP), K+ channels and vesicle-associated membrane protein (VAMP), increased the activity of Na+/K+-ATPase (NKA) and V-type H+-ATPase (V-ATPase), and initially reduced then elevated the expression of the Na+/H+-exchanger (NHE). dsRNA-mediated reduction in PtRh significantly reduced ammonia excretion rate and increased ammonia and glutamine (Gln) levels in the hemolymph, together with an increase of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) activity, indicating a central role for PtRh in ammonia excretion and detoxification mechanisms. Taken together, we conclude that Rh protein is a primary contributor to ammonia excretion of P. trituberculatus, which may be the basis of their ability to inhabit benthic water with high ammonia levels.