Preliminary Study On The Etiology Of Procambarus Clarkii "Black May" Disease And Transcriptomics Analysis

Procambarus clarkii is also known as crayfish.Because of its high nutritional value and delicious meat,it is now widely distributed in major freshwater bodies in China and is an important economic aquatic species in China.However,as the aquaculture area continues to expand,the disease problem of P.clarkii has become increasingly serious.Especially in April to May every year,the P.clarkii cultured in Jiangsu Province and other regions will die in large numbers.Because of its nearly 90%mortality rate,it is called the"Black May".At present,there are few studies on the"Black May"of P.clarkii.It is generally believed that the occurrence of the"Black May"is mainly related to the white spot syndrome virus(WSSV).At present,the research on the"Black May"only stops at on the WSSV virus detection.Recent studies have shown that the occurrence of"Black May"may be related to a newly discovered small RNA virus Pc DV.In order to study the relationship between Pc DV and the"Black May"of P.clarkii,this study first established a SY-BR Green real-time fluorescent quantitative PCR detection method for Pc DV with high sensitivity and strong specificity.By optimizing the reaction conditions of the fluorescence quantitative PCR,we obtained the standard curve equation Y=-3.391x+42.341,the amplification correlation coefficient R~2=0.999,the amplification efficiency was 97.2%,and it had a good linear relationship and the melting curve was a single peak,The same Tm value,no non-specific amplification.For the sensitivity test,it was found that the minimum detectable amount of virus c DNA was 3.95×10~1copies/μL,indicating that the established method has high sensitivity.Through the test of the repeatability experiment within and between groups,it is found that the coefficient of variation of the repeatability experiment within and between groups is less than 1.5%,which has good repeatability.Later,in order to understand the load of the two viruses in P.clarkii with the onset of"Black May",we established an absolute quantitative standard curve of WSSV by q PCR,and calculated the"Black May"by using the constructed standard curve.The copy numbers of the diseased P.clarkii WSSV and Pc DV were in the gills,muscles,hepatopancreas,epidermis,intestines and heart.WSSV has a high copy number in 6 tissues,among which the WSSV viral load in the intestine is the highest,about 2.02×10~5copies/mg;followed by the gills,the WSSV viral load is about 1.76×10~5copies/mg;the viral load of the epidermal virus of the cephalothorax of the shrimp is about 1.45×10~5copies/mg;the copy number of the WSSV virus in the muscle of the diseased shrimp is about 4.20×10~4copies/mg;the copy number of the virus in the hepatopancreas and heart of the diseased P.clarkii is the lowest,respectively,about 2.92×10~4copies/mg and 7.24×10~3copies/mg.The load of Pc DV in different tissues is relatively small.Among them,the gill tissue virus copy number is the highest in P.clarkii with"Black May",which is about 9.03×10~4copies/mg,followed by the intestinal tissue.The load is about 8.78×10~4copies/mg;in muscle,the viral load is about 8.26×10~4copies/mg;in the heart tissue,the Pc DV viral load is about 7.79×10~4copies/mg;Pc DV is in the onset of P.clarkii.The virus load in the epidermal tissue and hepatopancreas tissue is relatively little,about 6.74×10~4copies/mg and 3.55×10~4copies/mg,respectively.The results showed that the copy number of WSSV virus was 2.02×10~5copies/mg,while that of Pc DV virus was9.03×10~4copies/mg.Both viruses had higher copy numbers in the diseased shrimp.Finally,we used the transcriptome to study the differences in gene expression in the gill,muscle,and hepatopancreas of P.clarkii affected by the"Black May".A total of 199094transcripts were obtained,and the numbers of differentially expressed genes in the gills,muscles,and hepatopancreas were 1703,964,and 476.We conducted KEGG and GO analysis of differentially expressed genes and found that the expression of metabolic and immune-related genes was significantly down-regulated,and the pathways that were significantly enriched in KEGG were mainly related to the mitochondrial-mediated apoptosis pathway.The changes in these pathways may eventually lead to the death of P.clarkii.Our research provides technical means and theoretical basis for more in-depth research and prevention of P.clarkii"Black May"disease.