Study On Enterocytozoon Hepatopenaei Infection At Various Stages In The Breeding Stage Of Litopenaeus Vannamei

Litopenaeus vannamei is the main species of shrimp farming in China,and excellent seedlings are the basis for the healthy development of the aquaculture industry.Enterocytozoon hepatopenaei(EHP)is an important pathogen that has harmed the shrimp industry in recent years,and is a microsporidium that mainly parasitizes the hepatopancreas of shrimp.After infection with EHP,Shrimps show severe growth retardation,which eventually leads to a significant decline in production,and seriously affects the income on culture and future production plans.Besides,EHP is prone to co-infected with other pathogens,leading breeding failure.EHP can be transmitted horizontally by water and ingestion,but its vertical transmission is inconclusive.The nursery period is a critical stage for the growth and development of shrimp.Is this period susceptible to EHP infection?Is there a sensitive period for EHP infection in each developmental stage of the nursery period?It is a major problem that must be clarified in the shrimp breeding process.In this study,the immunofluorescence labeling method of EHP at the proliferation stage was established,which provided a technical basis for the subsequent histological detection of EHP in the early stage of proliferation in shrimp tissues.The infection of EHP in hepatopancreas and ovaries of EHP-carrying female broodstocks as well as eggs with or without EHP incubation and subsequential developed larvae at various stages were analyzed by fluorescence microscopy,scanning electron microscopy and real-time quantitative PCR.It is hoped that this study will explore the infection of EHP in shrimp at various stages during the nursery period of P.vannamei and the infection route of transmission from parent shrimp to larvae.The main findings are as follows:1.Establishment of a method for labeling the proliferative phase ofEnterocytozoon hepatopenaeiEh SWP1-specific polyclonal antibodies were prepared by immunizing rabbits by expressing and purifying the recombinant protein of Enterocytozoon hepatopenaei spore wall protein 1(Eh SWP1).Eh SWP1 was proved to recognize merogonial plasmodium,the early proliferative stage of EHP,by indirect immunofluorescence.The indirect immunofluorescence microscopy,Fluorescent Brightener and Propidium Iodide co-staining were combined to establish a trichromatic fluorescence microscopy method that could label the merogonial plasmodium,the early proliferative stage of EHP,and spores respectively,so as to facilitate the subsequent histological detection of the early proliferation of EHP.2.Analysis of EHP infection in female broodstockTwenty-five Litopenaeus vannamei females broodstocks were collected,and the hepatopancreas were proved to be infected with EHP by trichromatic fluorescence microscopy and transmission electron microscopy.The real-time quantitative PCR against EHP-ptp2 showed that the EHP load in the hepatopancreas of female broodstocks ranged from 2×10~1to 6×10~2copies/50 ng Hp DNA.No infection was detected in the ovaries of most broodstocks,while only a few were detected but at very low loads(EHP-ptp2<2×10~1copies/50 ng Hp DNA).The grinding fluid and tissue sections of female broodstock ovaries were observed by Fluorescent Brightener staining or trichromatic fluorescence microscopy,respectively,and no EHP was observed.3.Analysis of EHP infection in eggs and larvae of Litopenaeus vannameiThe eggs and larvae of 5 female broodstocks carrying EHP were detected by EHP-ptp2-q PCR.Low EHP loads(EHP-ptp2<5×10~1copies/50 ng DNA)were detected in the eggs and larvae of all groups,but mature spores were not observed by fluorescence microscopy,and no spores were observed to be adhered to the surface of shrimp eggs by scanning electron microscopy.In view of natural conditions,the amount of egg and EHP spore contact is small,which is not conducive to early detection.In this study,crudely purified EHP spores were used to incubate with shrimp eggs to carry out the following investigation.Fluorescence microscopy and scanning electron microscopy showed that the spores could closely adhere to the surface of shrimp eggs.The shrimp eggs can hatch and develop smoothly after EHP bathing.The6 groups of immersed eggs and samples of each larval stage cultured to larvae were collected,and q PCR were performed to detect EHP infection in eggs,nauplius,zoea I,II,III,mysis I,II,III and postlarvae.The results showed that in the nauplius period,the minority group had slightly higher detection amount but with lower copy number than the control group(EHP-ptp2<5×10~1copies/50 ng DNA).The detection results of postlarvae and larvae showed that the copy number of EHP-ptp2 in zoea was less than5×10~1copies/50 ng DNA,and EHP infection could not be detected in most groups of mysis and postlarvae.Mature spores of EHP were not observed in larvae by fluorescence microscopy.In conclusion,this study investigated the EHP infection in the eggs and larvae of female broodstocks clearly carrying EHP by fluorescence microscopy and quantitative PCR,and investigated the possibility of EHP transmission through the egg surface by immersion bath experiment.Given that the quantitative detection results showed that the copy number of EHP-ptp2 in larvae was<5×10~1copies/50 ng DNA,and EHP could not be observed by fluorescence microscopy and transmission electron microscopy,it was not possible to draw a clear conclusion that EHP has vertical transmission.The trichromatic fluorescence microscopy method stablished in this study can be used to further clarify the infection status from histological aspects.