The Epidemiology Of Enterocytozoon Hepatopenaei And Mechanism Of Retarded Growth In Shrimp

In recent years,lots of retarded growth in shrimp has been reported in Southeast Asia.Enterocytozoon hepatopenaei was detected in these slow-growing shrimps.It is believed that EHP is one of the most important pathogens which caused the slow-growing in shrimp in aquaculture industry.EHP has seriously affected the yield of shrimp aquaculture industry and caused serious economic losses.In this study,through the perspective of several different organizations,epidemiological survey,analysising the realatinship between different tissue’s EHP loading and growth parameters of shrimp,hemolymph ATP and protein content determination,in situ hybridization and other technical means,aimed at to search the epidemic situation of EHP in shrimp,and the mechanism of retarded growth in shrimp.Growth related parameters of four populations of Litopenaeus vannamei from Huanghua of Heibei Province(HH),Pingdu of Shandong Province(PD),Wujiang of Jiangsu Province(WJ),and Rizhao of Shandong Province(RZ)were measured.Enterocytozoon hepatopenaei(EHP)in hepatopancreas of all populations,and in multiple tisses of population RZ were quantitated with Taq Man based quantitative PCR.Results showed that population RZ had the optimal growth related parameters and lowest EHP load amoug 4 populations.The histograms of cases-logarithmic EHP of the 4 populations had different mode.The case distributions of logarithmic EHP of population HH and PD showed double peaks,while that of population WJ and RZ showed single peak.The different modes of distribution may indicate different EHP spread in different populations.The population with a single peak mode or the higher logarithmic EHP subpopulation isolated from the population with multiple peak mode had a significant negative correlation in shrimp body length or body weight to logarithmic EHP.EHP significantly increased the variable coefficient of ponderal index.In order to further research the distribution of EHP in infected shrimp,a total of 48 Litopenaeus vannamei that collected from Rizhao of Shandong Province.Hepatopancreas,gut,muscle,hemolymph and gill was taken out.EHP was detected by Taqman q PCR,The results showed that the positive rates of EHP were 94%,79%,40%,67% and 38% respectively,The logarithm of EHP copies was 1.26 ± 0.72,1.06 ± 0.65,0.19 ± 0.16,0.96 ± 0.50 and 0.79 ± 0.47,respectively.The regression analysis between the EHP load and Pi of Litopenaeus vannamei showed that the logarithm of EHP copies in the hepatopancreas and hemolymph showed a positive correlation with the body mass index of shrimp.The EHP load in different tissues of population RZ ranked from great to small as follows: hepatopancreas,midgut,haemolymph,gills and muscle.The logarithmic EHP in hepatopancreas,midgut,and gills had very significant correlation level above 99.9% significance(P<0.001),while the correlation level of logarithmic EHP of other two tissues had very significant(P<0.01)or significant(P<0.05)levels,except midgut-hemolymph and hepatopancreas-hemolymph.There was a negative correlation between the logarithm of EHP load and Pi of the shrimp in the gut and gill tissues.Results of the protein content in haemolymph and muscle tissue,and the relationship between EHP loading and shrimp growth parameters showed that protein in muscle had a negative correlation with EHP load in hemolymph and muscle,up to 95% level.There was a significant negative correlation between the concentration of ATP in haemolymph and the EHP load in the hepatopancreas and the logarithm of the sum of EHP load in the five tissues.Though the analysis using Sigma Plot 12.0,the relationship between the ATP content in the haemolymph and the logarithm of the EHP load in all of the 5 tissues was that ATP concentration =-(0.060 ± 0.019)Log(EHP loading)+(2.326 ± 0.238).It is proved that EHP does affect the growth mechanism of shrimp by intercepting host ATP,and formed a certain extent that EHP affected the accumulation of muscle protein in shrimp.In order to verify that EHP distribution in shrimp,a PCR primers were designed according to the EHP SSU r DNA sequence(KF362129)published in Gen Bank.In-situ hybridization of DIG labeled EHP in hepatopancreas,muscle,gills,and midgut showed that hepatopancreas is the major target tissue of EHP infection.Minor and weak hybrization signals were observed in other tissues indicated a few cells in those tissue were also suseptible to EHP.In this study,we obtained the accurate data of EHP load in different populations and different tissues of Litopenaeus vannamei by Taqman q PCR.It was observed that there was a significant negative correlation between EHP load and the growth parameters of shrimp when EHP infecting different individuals at the nearest time point.It will significantly increase the coefficient variation of Ponderal index when EHP infected the group.Besides,EHP can be detected in multiple tissues,which has the largest distribution in hepatopancreas and midgut.In situ hybridization showed that EHP existed in other organizations,so EHP can not only infecting hepatopancreas.Also,it proved that EHP could affect the growth of shrimp by intercepting ATP of host,and affected the accumulation of protein in muscle in shrimp.This study expounded the infection and distribution of EHP in shrimp populations and individual,it provided the basic data for the diagnosis and prevention of this disease.