Expression and Functional Characterisation of <scp>EhSWP</scp> 26 in <i>Ecytonucleospora hepatopenaei</i> : Insights From <scp>RNA</scp> Interference and Subcellular Localisation Analyses

ABSTRACT Ecytonucleospora hepatopenaei (EHP) is an obligate intracellular microsporidian that causes severe growth retardation in Penaeus vannamei (P. vannamei ), yet its pathogenic mechanisms remain incompletely clear. As the spore wall is essential for resisting environmental stress and mediating host invasion, characterising its protein components is critical for elucidating EHP biology. In this study, we identified and cloned a novel spore wall protein, EhSWP26. Sequence analysis revealed that EhSWP26 contains a 687 bp open reading frame encoding a 228‐amino‐acid polypeptide, lacking a signal peptide and transmembrane domain but harbouring a conserved MICSWaP (Microsporidia Spore Wall and Associated Proteins) domain characteristic of the microsporidian MICSWaPs family. Immunoelectron microscopy and indirect immunofluorescence assays demonstrated that EhSWP26 localises to the mature spore wall, where it is distributed in both the exospore and endospore layers. RNA interference further confirmed that silencing EhSWP26 significantly reduced its transcript level and led to a marked decrease in EHP spore load in shrimp hepatopancreas. Collectively, these findings indicate that EhSWP26 is a key structural and functional component associated with spore wall formation and host infection, likely playing an essential role in the EHP life cycle. This study enhances our understanding of EHP spore wall composition and function, provides a foundation for investigating its proliferation and infection mechanisms, and highlights EhSWP26 as a promising target for diagnostic development and therapeutic intervention in EHP disease of P. vannamei .