Transcriptome Analysis Of Enterocytozoon Hepatopenaei Infected Litopenaeus Vannamei And Germination-related Studies Of EHP Spores

Enterocytozoon hepatopenaei(EHP)belongs to the genus Enterocytozoon of the phylum Microsporidia,an obligate intracellular parasitic microsporidian,EHP can cause slow growth in shrimp and the disease is known as hepatopancreatic microsporidiosis(HPM),which has caused significant economic losses in shrimp aquaculture worldwide.After infection with EHP,Litopenaeus vannamei will show symptoms of slow growth,including immune,metabolic and hormonal regulation pathways will be changed to some extent as a result.EHP,like other microsporidia,has a typical microsporidian life history,germination as an important process of EHP from the Infective Phase to the Proliferative Phase is a key biological process for EHP to complete the infestation.There are relatively few studies on hormone-related changes after EHP infection in shrimp and on the molecular biology of EHP before and after germination.In order to gain a more comprehensive understanding of the EHP infection process and the physiological changes in shrimp after infection,we designed and conducted relevant experiments and obtained the following main research results.We used the Illumina Nova Seq 6000 sequencing platform to perform transcriptome sequencing analysis of hepatopancreas tissues from healthy shrimp and EHP-infected shrimp.There were 157 Differentially Expressed Genes(DEGs)at the hormone regulation level,of which 32 were upregulated and 125 were downregulated.GO and KEGG analysis of hormone-regulation-related DEGs revealed that among them,six genes were involved in molt growth,one in immunity and one in osmotic pressure regulation in shrimp.The pathways with the most significant differences were mostly related to molting and growth regulation,including EGFR tyrosine kinase inhibitor resistance,thyroid hormone signaling pathway,and steroid hormone biosynthesis.This part of the experiment obtained experimental data before and after EHP infection during shrimp growth,revealing the effects of EHP infection on the hormonal level of shrimp and providing data to support a comprehensive understanding of the effects of EHP on shrimp growth.Morphological observations of purified EHP spores,fixated EHP spores by fixative and EHP spores with completed polar tube ejection were made under different staining conditions by light microscopy(Nikon DS-Ri2).By staining germinated and ungerminated spores with dyes such as Phloxin B,Malachite green,Crystal violet,Fuchsin basic,Trypan blue,5X India ink and observing spore morphology,the results showed that Phloxin B,Malachite green,and Crystal violet were more effective for coloring and morphological observation of EHP spores.Moreover,these dyes appeared to stimulate the extrusion of polar tubes,likely due to their acidic nature.This part of the experiment provides some methodological reference for morphological observation and germination studies of EHP.Transcriptome sequencing analysis was performed on EHP non-germinated spore(NGS)and germinated spore(GS)after germinating spores were obtained by in vitro stimulation.This analysis identified 137 DEGs,of which 84 were up-regulated and 53 were down-regulated.Of the up-regulated genes,those associated with translation,cell growth and death,and folding,sorting and degradation pathways were enriched,while down-regulated genes were enriched in replication and repair and translation pathways.Although most of the genes were functionally unidentified and annotated,there were still important genes related to germination such as Invasin,asn A,and ksh that were changed significantly at the m RNA level before and after germination.This part of the experiment can help to further investigate the mechanism of EHP infection.In summary,this study used bioinformatics technology to study and analyze the differential changes of m RNA at the hormone level after EHP infection in Litopenaeus vannamei,and EHP infection would significantly affect the expression levels of ecdysone and other related hormones in Litopenaeus vannamei.Phloxin B,Malachite green and Crystal violet were found to have good staining effects and certain germination stimulation on EHP spores under different staining conditions by optical microscopy.Analysis of transcriptome data from non-germinated and germinated spores revealed that although the functions of some genes are still unknown,important data on the transcriptome of EHP before and after germination were provided.These studies provide some theoretical support for a comprehensive understanding of the infection mechanism of EHP.