Lysozyme i1-type, a crucial antimicrobial enzyme, reduces Ecytonucleospora hepatopenaei infection in Litopenaeus vannamei with potential for feed supplementation.

Ecytonucleospora hepatopenaei (EHP) is a fungal-related microsporidian parasite that infects shrimp. EHP causes a hepatopancreatic microsporidiosis (HPM), resulting in growth retardation and increasing susceptibility to several secondary infections. Yet, the innate immune responses that effectively encounter EHP infection remain largely unknown. Here, we investigate the functions of Litopenaeus vannamei lysozyme-i1 (LvLyz-i1) in the shrimp's response to the EHP infection. LvLyz-i1 expression was strongly induced during EHP infection, showing ∼ 13-fold and ∼ 6000-fold upregulation in the hepatopancreas and hemocytes, respectively. Knockdown of LvLyz-i1 increased EHP loads and altered the expression of immune-related genes associated with the JAK/STAT, Toll, and IMD pathways. Recombinant LvLyz-i1 (rLvLyz-i1) exhibited antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, and Pichia pastoris, and displayed potential isopeptidase activity. Treatment of the EHP spores with rLvLyz-i1 caused damage to the polar tube, perhaps by digesting EHP polar tube proteins and reducing the transfer of infectious cargo to host cells. Dietary supplementation with rLvLyz-i1 significantly reduced EHP copies at 7 and 9 days post-cohabitation, while increasing the gene expression of other antimicrobial peptides and stress-related proteins. These results demonstrate that LvLyz-i1 plays a critical role in shrimp innate immunity and may serve as a promising strategy to protect against EHP infection.