Prophenoloxidase-activating system plays a crucial role in innate immune responses to Enterocytozoon hepatopenaei infection in shrimp Litopenaeus vannamei

The microsporidian Enterocytozoon hepatopenaei (EHP) is an emerging pathogen that causes high economic losses in shrimp industry. The knowledge on shrimp's immune response to EHP infection to properly handle this outbreak is poorly understood. The prophenoloxidase (proPO)-activating system is an important invertebrate innate immune systems that produces melanin and toxic reactive intermediates against invading pathogens. In this study, we investigated the role of the proPO-activating system during EHP infection in the Pacific white shrimp Litopenaeus vannamei. The expression of proPO-activating system-related genes was highly responded to the EHP infection and the hemolymph PO activity was significantly increased and tightly regulated during the infection. The melanization products, generated by the proPO activation, exhibit in vitro cytotoxicity effect on the EHP spores and inhibit spore germination. Suppression of the proPO-activating system by RNA interference significantly decreased hemolymph PO activity and resulted in increased EHP copy number and reduced expression of several genes in the JAK/STAT and Toll signaling pathways as well as antimicrobial peptides. Furthermore, suppression of the proPO system also reduced hemocyte adhesion and encapsulation of the EHP spores. These results demonstrated that the proPO system plays a vital role in reducing EHP infectivity and cross-talks with other humoral and cellular responses to coordinately defend the EHP infection.