Risk analysis of imported live shrimp carrying water transmitted Enterocytozoon hepatopenaei by environmental DNA method

【Objective】The purpose of this study was to analyze the risks of water-borne Enterocytozoon hepatopenaei （EHP）in imported live shrimp using environmental DNA（eDNA）method，to provide a reference basis for environmental water disease monitoring and risk assessment.【Method】In this study，60 batches of imported parent shrimp carrying water were filtered by 0.45 μm nitrocellulose membrane，eDNA of 10 filter membranes were amplified byspecific primers of Litopenaeus vannamei. All 60 batches of filter membranes were tested for EHP via real-time fluorescence quantitative PCR. At the same time，EHP positive carrying water was used for artificial infection test. After 10 d of infection，environmental water and shrimp body were taken from each group for EHP detection. The metagenome of EHP positive filter membrane was sequenced and analyzed，Krona was used to visualize the annotation results，and the results of metagenome sequencing were compared with the EHP amino acid sequences registered in NCBI by EHP.【Result】The results showed that a 347 bp specific target fragment of L.vannamei was successfully amplified from 10 filter membranes，the sequencing results showed that the homology with the original sequence was 100%. Two batches of EHP were positive in 60 batches of imported prawns. The results of artificial infection test showed that EHP positive carrying water successfully infected the post-larvae group，and mature EHP spores were isolated from the hepatopancreas of shrimps，but the shrimps in parent group body were negative in EHP detection. The species relative abundance of EHP accounted for 0.7% of eukaryotic，the results compared with the amino acid sequences registered in NCBI，213 homologous EHP amino acid sequences were obtained，the amino acid sequences of EHP obtained were similar to those of other reported evolutionary trees：Enterospora canceri（amino acid similarity 83%），Enterocytozoon bieneusi（amino acid similarity 81%）.【Conclusion】It is possible for imported animals to carry water to spread EHP. EHP detection on parent shrimp only is incomplete. eDNA method can be used to supplement the traditional shrimp disease monitoring method as an important means of water disease detection and risk assessment.