Studies On Screening And Applying Disease Resistance Effectors Of Shrimps After White Spot Syndrome Virus(WSSV) Infection

The rapid development of the mariculture industry in China is now facingthe problems such as diseases and degradation of mariculture environment, amongwhich the shrimp-culture industry is particularly impacted by the disease of white spotsyndrome virus (WSSV). Focusing on this topic, we studied systematically thevariation of immune parameters of Chinese shrimp Fenneropenaeus chinensis andKuruma prawn Marsupenaeus japonicus after infection of WSSV by oral route, withattention of comparing the immunocompetence of the two species to WSSV infection.The immune parameters were also evaluated in Kuruma prawn M. japonicus under thestress of ammonia-N and WSSV infection. Based on these works, we discriminatedthe immune parameters closely related to WSSV infection from all the parametersused, and found nitric oxide synthase (NOS) a reliable parameter for indicatingdisease resistance ability to WSSV infection. Based on these findings, we developed nitro blue tetrazolium (NBT) method andcellular morphological approaches to identify the inducible NOS (iNOS) in thehaemocytes of Kuruma prawn M. japonicus and Chinese shrimp F. chinensis. Theconditions for NBT method were modified to make them suitable for the analysis ofiNOS in shrimp blood cells. Results showed that the optimal concentrations ofL-arginine, lipopolysaccharide (LPS) and Ca2+ for NBT method are 2.5mmol l-1,100μg ml-1 and 2.5mmol l-1, respectively. Meanwhile, two methods for iNOS activityassay based on the analysis of nitrite and L-citrulline concentrations were also appliedto study iNOS activity in the haemocytes of shrimp. The results showed that anincubation time of 8 hours is most suitable for the analysis of iNOS enzyme activity.The four methods could identify iNOS in shrimp haemocytes and analyze the activityeffectively, thus provide a good way for the further study of role of iNOS in innate 2对虾抗白斑综合症病毒的免疫学研究immunology of shrimps. Furthermore, the iNOS was identified in the haemocytes of Chinese shrimp F.chinensis and Kuruma prawn M. japonicus, and the methods developed wereemployed to evaluate their immunocompetence to WSSV infection in shrimps. Thevariations of iNOS activity in both penaeid species post-challenge of WSSV wereassayed with the methods of L-citrulline analysis and nitrite analysis. The resultshowed that iNOS activity in WSSV infected Chinese shrimp F. chinensis andKuruma prawn M. japonicus increased at 12 and 48 hours post-inoculum, butdecreased sharply after 36 and 72 hours post-inoculum respectively. LPS inducibleNOS activity also decreased significantly with the time elapsed in WSSV infectedshrimps. Results of WSSV detection with the Dig-DNA Probe dot blot hybridizationtest showed that WSSV of the two penaeid species couldn't be detected until iNOSstarted decreasing. It can be concluded that WSSV infection can stimulate the iNOSsynthesis in shrimps. However, the enzyme activity decreased sharply after theproliferation of virus. Therefore, iNOS can be considered as a valuable index forindicating the health status of Chinese shrimp F. chinensis and Kuruma prawn M.japonicus. In comparison with Chinese shrimp F. chinensis, the iNOS in thehaemocytes of Kuruma prawn M. japonicus are more inclined to be induced by LPSafter WSSV infection at early stage of infection, which might account for therelatively longer surviving time of M. japonicus after WSSV infection. Based on the findings in the comparative study of immune parameters betweenChinese shrimp Fenneropenaeus chinensis and Kuruma prawn Marsupenaeusjaponicus, this thesis firstly carry on the researches of iNOS activity in shrimps, andfind it a reliable parameter for indicating the disease resistance ability in shrimps. Theresults here could offer a sound scientific basis for the better understanding of themechanism of shrimp disease resistance ability to WSSV infection, as well as thedevelopment of countermeasures for...