Enterocytozoon hepatopenaei Proliferate in Procambarus clarkii: A Warning for Crayfish and Shrimp Aquaculture

The pacific whiteleg shrimp ( Litopenaeus vannamei ) and the crayfish ( Procambarus clarkii ) are the most productive aquatic animals in the world. The prevalence of the microsporidium Enterocytozoon hepatopenaei (EHP), an intracellular spore-forming unicellular parasite which leading retarded growth of L. vannamei , has caused severe economic losses in most shrimp farming country. In this study, we found that the wild P. clarkii , living in shrimp ponds with EHP outbreak, excreted white faeces after temporary laboratory culture. The hepatopancreas (HP) of symptomatic crayfish exhibited a lighter color and severely atrophied. H&E-stain showed tissue lesions in both hepatopancreas and intestine, and clustered microsporidian spores were filled in the cytoplasm of the cells. PCR using EHPptp2 and two microsporidian-universal primers sets demonstrated the existence of EHP in the hepatopancreas, intestine and the white feces of P. clarkii . The EHP loads of 103-104 copies of EHPptp2 /50 ng HPgDNA were detected by qPCR. Developing stages and mature spores of EHP were observed in hepatopancreas of P. clarkii through indirect-immunofluorescence assay (IFA) and transmission electron microscopy (TEM). Considering the large-scale cultivation of L. vannamei and P. clarkii , overlap farming areas between these two species as well as the ability of crayfish to crawl on land and water, our finding indicates the potential role of P. clarkii in the transmission of EHP, and it is an early warning for crayfish and shrimp farming. ### Competing Interest Statement The authors have declared no competing interest. * Pc : Procambarus clarkii Lv : Litopenaeus vannamei EHP : Enterocytozoon hepatopenaei AHPND : acute hepatopancreatic necrosis disease WSD : White spot syndrome HP : hepatopancreas WF : white faces IT : intestine PM : peritrophic membrane EL : epithelial layer CT : connective tissue layer ML : muscle layer S : EHP mature spores MP : merogonic plasmodia Ex : exospore En : endospore Pm : plasm membrane or sporoplasm membrane P : polaroplast PF : polar filament PT : polar tube N : nucleus AD : anchoring disc EDD : electron-dense disk EHPptp2 : EHP polar tube protein 2 cox : mitochondrial cytochrome oxidase c subunit I D-loop : mitochondrial control region (Displacement loop region) gDNA : genomic DNA IFA : indirect-immunofluorescence assay TEM : transmission electron microscopy HPM : hepatopancreas microsporidiosis SPF : specific pathogen free PC : positive control NC : negative control; NTC : no template control.