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Effects of ammonia-N (0.05, 2, 10 and 20 mg l −1 ) on the neuroendocrine regulation of ammonia transport were investigated in Litopenaeus vannamei. The results showed that corticotrophinreleasing hormone, adrenocorticotropic hormone, dopamine, noradrenaline and 5-hydroxytryptamine concentrations in all ammonia-N groups increased significantly between 3 and 12 h. Cortisol increased significantly between 3 and 24 h. All hormones except crustacean hyperglycemic hormone were reduced to control levels. mRNA abundance of guanylyl cyclase increased significantly during the experiment. Dopamine receptor D 4 and α 2 adrenergic receptor mRNA abundance in treatments decreased significantly at the beginning, and eventually returned to the control level, whereas mRNA abundance of the 5-HT 7 receptor increased significantly only within the first 12 h. Changes in protein kinase (PKA, PKG) mRNA abundance were similar to the patterns of biogenic amines and crustacean hyperglycemic hormone, peaking at 6 and 12 h, respectively, whereas PKC mRNA abundance decreased within 24 h. 14-3-3 protein, FXYD2 and cAMP-response element binding protein mRNA abundance increased significantly and peaked at 6 h. β-catenin and T-cell factor mRNA abundance increased significantly throughout the experiment and peaked at 12 h. The upregulation of Rh protein, K + channel, Na + /K + -ATPase, V-type H + -ATPase and vesicle associated membrane protein (VAMP) mRNA, together with downregulation of Na + /K + /2Cl − cotransporter mRNA, indicated an adjustment of general branchial ion-/ammonia-regulatory mechanisms. Meanwhile, hemolymph ammonia concentration was significantly increased in most ammonia-N exposure groups. Histological investigation revealed the hepatopancreatic damage caused by ammonia-N. Results suggest that hormones, biogenic amines and Wnt/β-catenin play a principal role in adapting to ammonia-N exposure and facilitating ammonia transport.

In , 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 (NH/NH ) 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 (Rh) was detected in all tested tissues, and showed the highest expression in the gills. To further characterize the role of Rh in ammonia metabolism and excretion, double-stranded RNA-mediated RNA interference ofRh was employed. Knockdown of Rh 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 Rh 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 forRh in ammonia excretion and detoxification mechanisms. Taken together, we conclude that Rh protein is a primary contributor to ammonia excretion of , which may be the basis of their ability to inhabit benthic water with high ammonia levels.

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