Identification And Epidemiological Investigation Of Microsporidium In Eriocheir Sinensis

Eriocheir sinensis has become a hot spot of cultivation and consumption over the recent years.The development is highly rapid and has become the most distinctive and potential aquaculture new species,and also has highly edible and economic value.Jiangsu is a large province of E.sinensis culture,and its regional superiority are apparant.However,in recent years,hepatopancreas necrosis has caused enormous losses to the E.sinensis breeding industry,which has affected the healthy development of the industry.The studies have indicated that the Hepatospora Eriocheir is closely related to the outbreak of the epidemic.In this paper,we carried out a series of researches around morphological identification of H.eriocheir,pathological characteristics of infection and epidemic situation in key region.Specifically including:1.Morphological identification of the microsporidian H.eriocheirIdentifications of the morphology and structure of the microsporidian from E.sinensis are fundamental requirements for further research.Hypertrophic and hyperchromatic eosinophilic granular bodies were observed after Masson staining.The results showed that the hepatopancreas infected with microsporidian were filled with large amounts of spores and formed inclusion bodies compared with normal cells after observing the undyed tissue rubbings.It was proved that the mature spores were circular through the negative staining technique,with a diameter of 0.8 ±0.1 μm.The characteristic disc anchor structure of the sporn front-end is obvious,which was consistent with the previous results.2.Microscopical and ultramicroscopic pathological analysis of H.eriocheirThis part was carried out from the light and electron microscopy levels respectively.After microsporidian infection,the hepatopancreas of E.sinensis were treated with paraffin section and H.E staining.Local inflammation was found under the light microscopy,including hypertrophy of the nucleus,aggregation of blood cells,exfoliation of epithelial cells,etc.After microsporidian infection,the samples treated with ultrathin sections were observed under the electron microscopy:the nuclei of the hepatopancreas of E.sinensis had a large number of mitochondria aggregation,indicating that the microsporidian worm depended on the mitochondria of the host hepatopancreas cells.The filamentous microsporidian appears and fills the cell;The microsporidian is characterized by a thick layer of cell wall indicating its maturity.The In Situ Hybridization(ISH)detection method was further established.The results showed that the digoxin labeled oligonucleotide probe designed and synthesized by ourselves could be better combined with the microsporidian in the cells of E.sinensis,and after being stained by dimethylbenzidine(DAB),the positive region was dark brown(some regions were stained too deep to show black),and the normal nucleus was blue with hematoxylin,but the healthy E.sinensis was no specific signal.3.Epidemiological survey of H.eriocheir in Jiangsu province.In light of this important disease,the follow-up investigation and continuous monitoring of typical incidence areas were carried out to analyze the prevalence of microsporidian in the culture area of E.sinensis in our province.The results showed that E.sinensis cultured in 2016 had obvious symptoms of albinism or necrosis of the hepatopancreas.The hepatopancreatic tissue of the dying E.sinensis had been infected with the pathogen of microsporidian by PCR detection technique.The results of sequence alignment showed that the pathogen causing hepatopancreas necrosis was 99.77%similar to the H.eriocheir.In 2017,the incidence of the disease has a significantly decline compare with previous years,and the positive rate of microsporidian in each monitoring area was low.