Diagnosis Of Co-infection Pathogen And Study On Rab7 Immunological Function In Procrayfish Clarkii

Procambarus clarkii,commonly known as crayfish,is one of the most economically valuable crustaceans in freshwater aquaculture in China.However,with the increase of breeding density and the deterioration of the breeding environment,the frequent occurrence of diseases has seriously restricted the development of its breeding industry.Thus,it is urgent to carry out basic research on identification and diagnosis of pathogenic agents and the molecular mechanism of natural immune prevention of P.clarkii to provide theoretical guidance for the prevention and control of the disease.In this study,the pathogenic isolation,identification and biological function of immune-related genes were studied.The results are as follows:1.Isolation,identification and pathological studies of pathogens from P.clarkii.In May 2019,the naturally diseased crayfish from the local farm in Xinxiang,Henan Province,showed the following clinical symptoms:lethargy,loss of appetite,black gills,the weaken shell,the hepatopancreas with the swollen and yellow,and the massive death.A dominant bacterial strain（LY-1）was firstly isolated from the gill of diseased crayfish.The isolated was identified as A.veronii through molecular biology experiments.Further studies showed that the strain carried four virulence genes（Hlya,Aera,act and alt）,and the 50%lethal dose to crayfish was about 3.02×10~7CFU/m L,indicating that LY-1was highly virulent.The isolated LY-1 was highly sensitive to 7 kinds of antibiotics according to the drug sensitivity test.Besides,white spot syndrome virus（WSSV）was also detected from the gill of diseased crayfish by PCR test.Finally,artificial infection experiment was carried out,and the results showed that A.veronii and WSSV co-infection group indicated the similar symptoms and pathological changes with natural infection,and the mortality rate（100%）was higher than that in single pathogen infection group.To sum up,the natural diseased P.clarkii was co-infected with A.veronii and WSSV.The results may provide a reference for prevention and control of bacterial and viral co-infection in P.clarkii.2.The immune responses of P.clarkii after infected with A.veronii.To study the immune responses of P.clarkii upon A.veronii infection,the changes of total haemocytes count（THC）,enzyme activity and m RNA expressions of immune-related genes in P.clarkii after infected with A.veronii were studied.The results showed that the THC decreased sharply after infection.Meanwhile,the activities of Acid phosphatase（ACP）,Alkaline phosphatase（AKP）,phenoloxidase（PO）and Lysozyme（LSZ）in serum were higher than those in control group.However,the activities of antioxidant enzymes,including Superoxide dismutase（T-SOD）and Glutathione Peroxidase（GSH-PX）,were lower than those of the control group.After A.veronii infection,eight immune-related genes in the intestine,hepatopancreas and gills of crayfish showed significant transcriptional expression changes.In addition,the m RNA expression of Rab7 changed significantly after A.veronii challenge,which indicated that it was involved in the immune response of the P.clarkii.The result of this study provides a theoretical basis for understanding and studying the natural immune response of P.clarkii.3.The gene cloning and molecular characterization of Rab7 in P.clarkii.To further explore the structural characteristics,tissue distribution and immune response of Rab7 in P.clarkii,the Pc Rab7 gene was cloned and related studies were carried out.The results showed that the full length of Pc Rab7 gene with 741 bp contained a 468 bp ORF,encoding 155 amino acids.The molecular weight of Pc Rab7 protein was predicted to be 17.61 k Da.Pc Rab7 was widely expressed in various tissues including hepatopancreas,intestines and haemocytes,and the highest expression level was found in lymphocyte and intestines.The expressions of Pc Rab7 in the gills,intestines and hepatopancreas were up-regulated in P.clarkii after stimulated by A.veronii or WSSV.Subsequently,based on the constructed Rab7 prokaryotic expression vector the polyclonal antibody was prepared,with the antibody titer of1:90,000,and could specifically recognize the prokaryotic and endogenous Rab7 protein.These results could provide a basis for further study the immunological function of Pc Rab7.4.The immune functional analysis of Rab7 in P.clarkiiIn this study,Rab7 interfering gene and eukaryotic expression vector were designed to ascertain the immune function of Rab7 in P.clarkii.The effect of Rab7 on the phagocytic function of haemocytes and the mortality of P.clarkii after the infection were further studied.After gene interference,the A.veronii-infected crayfish died faster,with the survival rate of 0%,while the WSSV-infected crayfish died slower with the increased survival rate.The ability of haemocytes to phagocytize A.veronii was significantly reduced after Rab7 was interferred.After Rab7 was overexpressed,in P.clarkii,after A.veronii infection,the abiligy to phagocytize A.veronii increased significantly,and the survival rate of P.clarkii was 50%.After WSSV infection,the course of the disease was accelerated,with the survival rate of0%,and the haemocytes phagocytosis increased significantly（49.8%）.The results demonstrated that Rab7plays an important role in the phagocytosis of haemocytes and participates in the process of anti-pathogen infection.