Effect Of Salinity, Temperature On The Non-specific Immunity Of Turbot (Scophthalmus Maximus L.) And Study On Some Immune Related Genes In Crayfish (Pacifastacus Leniusculus)

Two sections are included in the dissertation. One is effect of salinity and temperature on the non-specific immunity of turbot (Scophthalmus maximus L.); One is related to study on some immune related genes in Crayfish Pacifastacus leniusculus.1. Effect of salinity on the non-specific immunity of turbot. Studies were conducted to investigate the effects of salinity on the non-specific immune responses and disease-resistance of turbot Scophthalmus maximus L. challenged with Vibrio anguillarum. Four groups of turbot juveniles (mean weight 25±2 g) were cultivated at 8,20,32 and 40 g/L salinity, respectively, for 7 days to acclimate the experimental conditions before pathogen challenge. Three concentrations (3.75×107,3.75×108 and 3.75×109 CFU/ml) of V. anguillarum suspension were employed at each salinity to determine the 4-day LD50, in which each fish was injected with 0.1 ml of the pathogen suspension at the base of dorsal fin. For the observation of non-specific immune responses at the selected rearing salinities, each fish was injected with 0.1 ml of V. anguillarum suspension at 1.1×108 CFU/ml, then the activities of serum lysozyme, the alternative complement pathway (ACH50) and phagocytosis percentage of head kidney were recorded at 24 h,48 h and 72 h post-challenge. All treatments were conducted in triplicate. Fish reared at 20 g/L had lowest mortality, namely the highest 4-day LD50 value (8.88±0.16) that was significantly higher than that (8.04±0.04) of fish cultured at 40 g/L (P< 0.05), while no significant difference was observed compared with the other treatments (P>0.05). The activities of lysozyme, ACH50 and phagocytosis maintained the highest at the salinity of 20 g/L. In contrast, these activities at the salinity of 40 g/L were always the lowest at different sampling times (24 h,48 h and 72 h). Generally, the values of the non-specific immune parameters kept increasing within 72 h post-challenge, except that the lysozyme activity at 40 g/L significantly increased from 24 h to 48 h, and then decreased from 48 h to 72 h. The results indicate that besides growth improvement, rearing in intermediate salinity (20 g/L) is able to enhance the immunity and disease-resistance of turbot.2. Effect of temperature on the non-specific immunity of turbot. This experiment was conducted to investigate the effects of temperature on the non-specific immunity of turbot Scophthalmus maximus L. challenged with V. anguillarum. The juvenile healthy turbots (mean weight:25±2 g) were cultivated in the seawater with different temperature (10,15,20,25℃, respectively) for 7 d to acclimate the experimental conditions before pathogen challenge. The result of the 4-day LD50 showed that fish at the temperature of 15℃had lowest mortality, significantly lower than that of fish cultured at 25℃(P<0.05), while no significant difference was observed compared with the other treatments (P>0.05). The activities of lysozyme, complement of the alternative pathway (ACH50) and phagocytosis maintained the highest at the salinity of 15℃, however, they were always the lowest at different sampling time (24 h,48 h and 72 h) at the salinity of 25℃. All results indicated that turbot cultured at temperature of 15℃had relatively higher anti-infection of V. anguillarum and higher anti-disease compared with higher or lower temperature.3. RT-PCR was used to analyse the expression of a number of immune-related genes in the middle phase of crayfish Pacifastacus leniusculus embryo. Crayfish do not have larval stage as other crustacean such as penaeid shrimp, they brood their eggs until hatching and what hatches out from the eggs are miniature crayfish known as juveniles. In order to address the question whether immune genes are initially expressed during the embryo development in the egg stage, the expression of some immune-related genes:prophenoloxidase (proPO), peroxinectin, hemocyanin, anti-lipopolysaccharide factor (ALF), plcrustin, astakine 1,2 and transglutaminase (TGase) were determined in the middle phase of crayfish embryo development. Furthermore, immune challenge was used to determine the immune response of eggs by immersing them in a solution of the highly pathogenic bacterium Aeromonas hydrophila. Semi-quantitative RT-PCR analysis showed that all tested genes are present except proPO in this phase of crayfish embryo development and none of the genes tested changed their expression following immersion in A. hydrophila. The proPO transcript has been reported from hemocytes in crustaceans and it plays crucial roles in crustacean immune response. This may indicate that the development of immune-competent hemocytes in this stage of crayfish embryo is not completed and the egg shell as such plays an important role as a shield in protecting the embryo from bacteria and maybe also other pathogens.4. Expression of Kazal-type proteinase inhibitors in different types of hemocytes in ccrayfish and sequence alignment. Crustacean hemocytes were found to produce a large number of transcripts coding for Kazal-type proteinase inhibitors (KPIs). A detailed study performed with the crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon revealed the presence of at least 26 and 20 different Kazal domains from the hemocyte KPIs, respectively. Comparisons with KPIs from other taxa indicate that the sequences of these domains evolve rapidly. A few conserved positions, e.g. six invariant cysteines were present in all domain sequences whereas the position of PI amino acid, a determinant for substrate specificity, varied highly. A study with a single crayfish animal suggested that even at the individual level considerable sequence variability among hemocyte KPIs produced exist. Expression analysis of four crayfish KPI transcripts in hematopoietic tissue cells and different hemocyte types suggest that some of these KPIs are likely to be involved in hematopoiesis or hemocyte release as they were produced in particular hemocyte types or maturation stages only.5. Expression of recombiant properoxinectin from crayfish Pacifastacus leniusculus in Bac-to-Bac Baculovirus Expression System. The full-length cDNA of preoperoxinectin was amplified by PCR with 6×His Tag on 5', cloned the gene into pFastBacTM1, sequencing. Transform purified plasmid DNA into DH10BacTM E. coli for transposition into the bacmid. Use blue/white selection to identify colonies containing the recombinant bacmid, analyzing recombinant bacmid DNA by PCR, transfecting insect cells to produce recombinant baculovirus, harvest the virus from the cell culture medium to amplify and express the protein, the recombinant properoxinectin expressed in the insect cell. It's analysed with SDS-PAGE and identified by WesternBlot, and purified by Ni-NTA.