Effects Of Vibrio Harveyi Injection And Salinity Changes On The Anabolism And Phenoloxidase Activity Of Hemocyanin Of Litopenaeus Vannamei

This article mainly studied the effects of hemocyanin pathogen and environmental stress on hemocyanins anabolism and phenoloxidase activity of Litopenaeus vannamei. The main contents includes:①Study on the phenoloxidase activity of hemocyanin of Litopenaeus vannamei.②Effects of Vibrio harveyi injection on the anabolism and phenoloxidase activity of hemocyanin of Litopenaeus vannamei.③Effects of salinity changes on the anabolism and phenoloxidase activity of hemocyanin of Litopenaeus vannamei.1 Study on the phenoloxidase activity of hemocyanin of Litopenaeus vannamei. The study was made to the Litopenaeus vannamei haemocytes and inhibitor-A, inhibitor-B and so on, finally screen out the inhibitor-A as the effective phenoloxidase inhibitor---10μM inhibitor-A had the best restrain effect. Then the result of native-PAGE and L-DOPA coloration showed: hemocyanin with 450kD had the phenol oxidase activity in the Litopenaeus vannamei. And the SDS-PAGE and L-DOPA associative measure suggested that the phenoloxidase activity subunit of the hemocyanin was p75-subunit.2 Effects of Vibrio harveyi injection on the anabolism and phenoloxidase activity of hemocyanin of Litopenaeus vannamei.In the present paper, effects of pathogen injection on the anabolism and phenoloxidase activity of hemocyanin of Litopenaeus vannamei were studied. The results showed that injection of Vibrio harveyi had significant effects on the hemocyanin concentration, p75- and p77-subunit and mRNA expression, and phenoloxidase activity of hemolymph, haemocyte and hemocyanin in Litopenaeus vannamei (p<0.05), while no marked difference can be observed in the control. The hemocyanin concentration and p75 and p77 subunits expression showed peak changes between 0 and 24h. The hemocyanin concentration was gradually declined between 0 and 6h and reached the minimum at 6h, but no significant impacts on p75 and p77 subunits expression were observed before 3h. Subsequently, both hemocyanin concentration and subunits expression showed obviously increasing trend and reached the maximum at 12h. Moreover, the level of subunits expression had positive correlation with the concentration of injected Vibrio harveyi, and all parameters became stable and recovered to the control level after 24h. The hemocyanin mRNA expression of injection of pathogenic bacteria group indicated a rapid increasing trend compared with the control group. The expression of hemocyanin mRNA of the 6×106 cells/mL pathogenic bacteria group showed peak changes before 6h and reached the maximum at 3h, which was much higher than the control level (p<0.05) and maintained the high level until 48h. However, the expression of hemocyanin mRNA in 6×105 cells/mL pathogenic bacteria group exhibited a slight increase tendency and showed significant difference with the control level at 6h, while in other sample time, no obvious difference could be drawed.The phenoloxidase activities of hemolymph and haemocyte as well as hemocyanin phenoloxidase activity showed peak-value changes before 12h, 12h and 24h, respectively. And all parameters peaked at 3h , 6h and 3h, and recovered to the control level at 12h ,12h and 24h. This study demonstrated that Litopenaeus vannamei started the synthesis and mRNA expression upward under the invasion of pathogen, and new synthesized hemocyanin mainly played phenoloxidase functions, which involved in immune response in coordination with other immune factors.3 Effects of salinity changes on the anabolism and phenoloxidase activity of hemocyanin of Litopenaeus vannamei.The present study examined the effect of salinity change on expression of hemocyanin gene and p77-subunit and p75-subunit and phenoloxidase activity. The results showed that:The salinity change had a significant influence on the content of hemocyanin in haemolymph and expression of p77 and p75 subunit and hemocyanin mRNA, in hepatopancreas of Litopenaeus vannamei (p<0.05), while no marked difference can be observed in the control. The content of hemocyanin in haemolymph and expression of p77 and p75 subunit in Litopenaeus vannamei exposing to the salinity declining showed peak change during 0-48h, reached the maximum at 12 h, and stabilized at 24h, 24h and 48h, respectively. The expression of hemocyanin mRNA showed the peak-value change over the control during 24h, reached to the highest level at 12h, and finally got to be stable after 12h, then declined to the level of control. A significant difference existed between the 30 and 26 group in p77-subunit expression, and then became stable over the control(P>0.05).The salinity change had notable effects on phenoloxidase activities of haemolymph, haemocyte and hemocyanin of Litopenaeus vannamei (p<0.05), while no marked difference can be observed in the control, and the two treated groups (30 and 26 group) had no significant difference. During the 48h, three parameters showed peak changes, phenoloxidase activity in haemolymph and haemocyte and hemocyanin respectively reached to the maximum and minimum at 12h, and all of them had positive or negative correlation with the salinity change. Then all parameters became stable and recovered to the control level at 24h, 24h and 48h, respectively.