Histopathological and Proteomics Analysis of Shrimp Litopenaeus vannamei Infected with Ecytonucleospora hepatopenaei

Ecytonucleospora hepatopenaei (EHP) is a microsporidian pathogen that primarily infects the hepatopancreas of Litopenaeus vannamei. Previous studies on EHP detection primarily focused on histology, in situ hybridization (ISH), and PCR, mainly concentrating on hepatopancreatic infections, with limited research on extra-hepatopancreatic tissues. This study investigates the pathogenic mechanisms of EHP infection in L. vannamei through molecular quantification, histopathology, and proteomics analysis. RT-qPCR was employed to examine the infection differences across various tissues at the molecular level, revealing that the hepatopancreas, stomach, midgut, muscle, gills, and antennal glands are susceptible tissues. Pathological analysis, combining H&E staining, Masson's trichrome staining, and immunohistochemistry, identified the EHP-targeted sites at the tissue level. Masson's staining effectively highlighted fibrosis, unveiling the histopathological characteristics of chronic EHP infection, while immunohistochemistry enhanced the specificity of EHP localization. The pathological features of EHP infection were primarily characterized by inflammation, cell degeneration and necrosis, and the accumulation of microsporidia in the cytoplasm. Proteomics analysis was used to interpret the histopathological findings, revealing significant enrichment of pathways related to inflammation, immune regulation, metabolism, and apoptosis regulation. These findings provide new insights into the infection mechanisms and tissue tropism of EHP.