Study On Nitrogenous Wastes Excretion Of The Swimming Crab Portunus Trituberculatus Exposed To Ammonia

Ammonia-N, which represents a major physiological challenge for crab culture, increased rapidly with time elapsed in an intensive culture system, and the gill is a key organ for crabs to exchange materials with the outside. In the present study, the gene expression profile of the gills in Portunus trituberculatus in response to 5mg/L NH4Cl for 48 h were detected by using a digital gene expression (DGE) technology approach, gene expression levels of nitrogenous wastes excretion associated channel proteins and uric acid and urea synthesis related enzymes were analyzed, nitrogenous wastes content in each tissue were measured, which deeply studied the physiological adaptation mechanism, detoxification mechanism and nitrogenous wastes excretion mechanism of the swimming crab P. trituberculatus under ammonia exposure.1 Digital Gene Expression analysis in gills of the swimming crab P. trituberculatus exposed to elevated ambient ammonia-NA total of 58,336 unigenes with average length 689bp were generated. Functional categorization of the differentially expressed genes revealed that amino acid metabolic, nucleobase metabolism, regulation of mitochondrial membrane potential, reactive oxygen species metabolic, antioxidation and immune response were the differentially regulated processes occurring during ammonia-N exposure. Seven genes related to the antioxidant system, mitochondrial electron transport, protein digestion and absorption and immune system were selected for validating the results by real-time quantitative PCR. This present study provides valuable gene information for a better understanding of molecular responses of P. trituberculatus under ammonia-N exposure.2 Effects of ammonia exposure on ammonia excretion in the swimming crab P. trituberculatusIn the present study of the swimming crab P. trituberculatus exposed to 0,1, and 5mg/L NH4Cl, the effects of ammonia exposure on ammonia and urea content in hemolymph; activity of H+-ATPase (subunit A) and Na+/K+-ATPase (a-subunit) (NKA) in gills; mRNA expression levels of the crustacean Rh-like ammonia transporter (Rh), K+ Channel, Na+/K+/2C1- co-transporter (NKCC), Na+/H+ -exchanger (NHE), Urea transporter (UT) and Vesicle associated membrane protein (VAMP) in gills were investigated. Ultrastructure of gills was also evaluated. All these results in this study showed a dose-dependent effect with ammonia exposure concentration. The data displayed a significant increase in hemolymph ammonia under ammonia exposure. Gill NKA and H+-ATPase activity showed a peak change during the experiment period and reached the maximum at 6h. Gill Rh protein and K+-channel gene expression were significantly up-regulated under ammonia stress and showed a peak change, and the maximum was occurred at 12h. NKCC and NHE gene expression levels in lmg/L and 5mg/L NH4C1 groups were significantly suppressed and reached the minimum at 6h and 3h respectively, then increased, but still remained below the level of the control group. NHE gene expression level was significantly reduced within the first 6h, but this decrease returned to the control level at 12h and significantly decreased again after 48h of exposure. It was concluded that the up-regulation of Rh mRNA together with up-regulation of K+-Channel mRNA, NKA activity, down-regulation of NKCC and NHE mRNA suggested a coordinated protective response to maintain a relatively low ammonia concentration in the body fluids during ambient ammonia exposure. The up-regulation of VAMP, H+-ATPase activity along with the ultrastructure of gills suggests a mechanism of exocytotic ammonia excretion that may exit in the gill of P. trituberculatus.3 Effect of ammonia exposure on metabolic pathways and tissue distribution of uric acid and urea in P. trituberculatusThe object of this study was to evaluate the effects of ammonia on the metabolic pathways and tissue distribution of nitrogenous wastes in the swimming crab Portunus trituberculatus. The results indicated that ammonia exposure had a significant effect on mRNA expression levels of xanthine oxidase (XO), carbamoyl phosphate synthetaseⅢ (CPSⅢ), arginase (ARG) and uricase (URI) in tissues, content of uric acid and urea in tissues, mRNA expression levels of urea transporter (UT) in gills. The mRNA expression levels of XU were significantly up-regulated in gills and muscles, while significantly down-regulated in hepatopancreas; The mRNA expression levels of CPSIII) and ARG were significantly up-regulated in gills and muscles and reached the maximum at 12h, while significantly down-regulated during earlier stage of ammonia exposure and significantly up-regulated after 48h and 24h of ammonia exposure in hepatopancreas; The expression pattern of URI was similar to that of XO; Trace amounts of uric acid were detected in hepatopancreas, muscles, gills and hemolymph, but significant changes were only found at a few sampling points, uric acid content in muscles was higher than that in hemolymph followed by gills and hepatopancreas. Urea content was significantly changed during the experiment time compared with the control, especially in 5mg/L NH4Cl group, urea content in hemolymph was higher than that in gills followed by hepatopancreas and muscles; The mRNA expression of UT in gills was significantly increased. It is concluded that the crabs would converted excess ammonia into urea and trace amounts of uric acid to release the ammonia toxicity when exposed to ammonia. The uric acid may generate via purine nucleotides catabolism and mainly stored in muscles. The urea may generate via ornithine-urea cycle, but it can’t exclude the possibility of producing urea by hydrolysis of arginase, meanwhile, urea may also be produced by decomposition of uric acid via URI. Urea generated from tissues may be release to hemolymph and excreted via UT in gill cells when pathways of ammonia excretion are decreased after long term ammonia exposure.