Immune Response And Detoxification Mechanism Of Swimming Crab Portunus Trituberculatus Exposed To Ambient Ammonia

Ammonia is one of the important environmental toxic factors in pond. In this study, some important immune-related genes were cloned and characterized from the haemocytes of swimming crab Portunus trituberculatus. Then the effects of ammonia on immunity indicators, immune-related gene expression and some ammonia detoxification parameters were determined to detect the immunity toxicity mechanism and ammonia detoxification mechanism. Three bodies are:(1) Cloning characterization and expression analysis of immune-related genes in swimming crab P. trituberculatus, including crustin, anti-lipopolysaccharide factor (ALF), lysozyme, prophenoloxidase (proPO) andα2-macroglobulin (α2-M); (2) Effects of ammonia on immunity evaluate parameters of swimming crab P. trituberculatus; (3) Effects of ammonia on ammonia detoxification parameters of swimming crab P. trituberculatus. The results are as follows:1 Cloning, characterization and expression analysis of immune-related genes in swimming crab P. trituberculatusIn this study, the cDNAs of a crustin and an ALF were cloned from the hemocytes of swimming crab P. trituberculatus using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of P. trituberculatus crustin (denoted as PtCrustin) was of 584 bp with an open reading frame (ORF) of 333 bp encoding a 21 amino acids signal peptide and a mature polypeptide of 89 amino acids with the predicted molecular mass of 10.08 kDa and pI of 7.93. PtCrustin contains a WAP domain at the C-terminal composed by eight conserved cysteine (Cys) residues forming a four-disulphide core (4-DSC). PtCrustin displayed 52%-81% high similarity to other crustins. Moreover, the full-length cDNA of P. trituberculatus ALF (denoted as P/ALF) was of 1050 bp, including an ORF of 372 bp encoding a 26 amino acids signal peptide and a mature polypeptide of 97 amino acids with the predicted molecular mass of 11.35 kDa and pI of 10.19. PtALF contained two conserved cysteine residues and showed 58%-87% high similarity to other crustacean ALFs. Using fluorescent real-time quantitative PCR, PtCrustin mRNA was expressed at the highest level in hemocytes and higher level in stomach while extremely low in hepatopancreas, muscle, gill and heart. In contrast, the PtALF mRNA expression was observed in all tissues. The relative expression levels of PtCrustin and PtALF in haemocytes of P. trituberculatus were increased obviously (P<0.05) and showed a clear time-dependent response after challenge with Vibrio alginolyticus. These results indicate that PtCrustin and PtALF are two acute-phase proteins involved in the immune responses of P. trituberculatus.The full-length cDNA of a c-type lysozyme was cloned from the haemocytes of swimming crab P. trituberculatus using RACE approaches. The full-length cDNA of lysozyme was of 984 bp, including an ORF of 672 bp encoding an 18 amino acids signal peptide and a mature polypeptide of 205 amino acids with the predicted molecular weight of 23.76 kDa and pI of 7.57. The swimming crab c-type lysozyme displayed 75%-77% high similarity to other shrimp lysozyme. The conserved catalytic sites Glu51 and Asp69, and the eight conserved cysteine residues identified in lysozyme suggesting it belonged to c-type lysozyme. Also, the swimming crab c-type lysozyme may belong to calcium-binding c-type lysozymes because of containing the three Asp residues (Asp98, Asp103 and Asp104), which are conserved in calcium-binding c-type lysozymes. Quantitative real-time RT-PCR analysis revealed that the mRNA transcripts of lysozyme were expressed at the highest level in haemocytes, with higher level in gill, moderately in hepatopancreas and muscle while low in stomach and heart. The mRNA expression of lysozyme in haemocytes was down-regulated significantly after challenge with V. alginolyticus at 3 h (P<0.05), and then kept at the low expression level until 24 h. As time progressed, the mRNA expression of c-type lysozyme was recovered to the control level after 24 h injection. The results indicate swimming crab c-type lysozyme may be an inducible acute-phase protein and bacterial challenge could decline its mRNA expression. The partial cDNA sequences of proPO andα2-M were cloned from the haemocytes of swimming crab P. trituberculatus using degenerate primer and PCR metnods. The partial cDNA of proPO was of 551 bp encoding 183 amino acids containing a conserved histidine residue and two possible glycosylation sites Asn-Ser-Asn (NSN) and Asn-Thr-Leu (NTL). The proPO amino acids showed 73%-94% high similarity to other crustacean proPO. In addition, the partial cDNA ofα2-M was of 2685 bp with 3'UTR 1165 bp and incompete ORF 1520 bp encoding 505 deduced amino acids. Theα2-M deduced amino acids sequence contains a GCGEQNM thioester region and a receptor-binding domain which are present in other invertebrate and vertebrateα2-Ms. Besides, theα2-M of P. trituberculatus contains a possible glycosylation sites Asn-Glu-Ser (NES). Sequence comparison showed thatα2-M deduced amino acid sequence of P. trituberculatus has an overall similarity of 65%-71% to other crustaceanα2-Ms.2 Effects of ammonia on immunity evaluate parameters of swimming crab P. trituberculatusThe effects of ammonia (0,1,5 and 20 mg/L) on dopamine (DA) concentration, the proPO system, immune parameters and immune-related gene expression were determined in swimming crab P. trituberculatus. The results showed that DA concentration in haemolymph of the experimental groups increased until 6 h, then recovered to similar with the control level after 12 h. Total haemocyte count (THC), differential haemocyte count (DHC) and phagocytic activity decreased significantly after ammonia exposure, and display significant negative correlation with ammonia concentration (P<0.05). proPO activity were decreased significantly and then tend to be stable after 12 h, then the 1 mg/L group were recovered to the control level, the other groups were negative correlation with the ammonia concentration (P<0.05).α2-M activity of all ammonia exposure groups decreased significantly, then recovered to the control level rapidly and tended to be stable after 12 h. The antibacterial and bacteriolytic activity decreased significantly to the minimum at 6 h or 12 h respectively, then recovered to the control level except bacteriolytic activity exposed to 5 and 20 mg/L ammonia (P<0.05). After ammonia exposure, the proPO mRNA levels of 5 and 20 mg/L were increased obviously and became stable after 6 h ammonia exposure.α2-M mRNA expression was induced by ammonia exposure significantly and up to the maximum at 6 h (P<0.05), recovered to the control level after 24 h. With crabs exposed to 20 mg/L ammonia-N, the gene expression levels of crustin and lysozyme decreased significantly (P<0.05), then recovered to the control level after 12 h. ALF expression also decreased significantly when exposed to 5 and 20 mg/L ammonia-N, then remained stable and significantly lower than the control group after 6 h (P<0.05). The results indicate that ammonia could decrease immunity of crab and affect the immune-related gene expression levels.3 Effects of ammonia on ammonia detoxification parameters of swimming crab P. trituberculatusSwimming crab P. trituberculatus exposed to 0,1,5 and 20 mg/L ammonia were examined for arginase activity, urea content, glutamine synthetase (GS) activity and glutamine content in the hepatopancreas, gill, haemolymph and muscle. The results showed ammonia could affect these ammonia detoxification parameters significantly (P<0.05). The arginase activity in hepatopancreas, gill and haemolymph increased directly with ambient ammonia, reached to the maximum at 6 h or 12 h respectively, and arginase activity increased by 233.1%,111.1% and 151.5% in those tissue respectively for the crabs exposed to 20 mg/L ammonia; After becoming stable at 12 h or 24 h, arginase activity of 5 and 20 mg/L groups showed positive correlation with the ammonia (P<0.05). Urea content in all those tissues was increased significantly except the 1 mg/L group of haemolymph and muscle, up to the maximum at 6 h,12 h or 24 h respectively (P<0.05), and urea content increased by 132.2%,230.4%,48.7% and 118.2% in the four tissues for 20 mg/L ammonia group. GS activity of 5,20 mg/L in hepatopancreas and all gill groups increased obviously, became stable at 12 h or 24 h respectively and display significant positive correlation with the ammonia concentration (P<0.05); No significant difference of GS activity was observed in 1 mg/L group in haemolymph, however the GS activity of the other two groups were increased until 48 h (P<0.05);GS activity in muscle groups were increased directly, tended to stable at 24 h, but only 1 mg/L ammonia group were significant higher than the control (P<0.05). After ammonia exposure, glutamine content in all those tissues was increased significantly, up to the maximum at 6 h,12 h or 24 h respectively (P<0.05), and glutamine content increased by 195.7% in hepatopancreas, by 332.3% in gill, by 93.5% in haemolymph and by 69.6% in muscle for 20 mg/L ammonia group. Glutamine content in hepatopancreas and gill displays significant positive correlation with the ammonia concentration (P<0.05). Those results suggest that ammonia could induce the urea and glutamine synthesis and the mechanism to detoxification of ammonia may be to transform ammonia to urea and glutamine.