Cloning and Characterization of a Serpin, DiSpn27A, Expressed in the Salivary Glands of the Larvae of <i>Drino inconspicuoides</i> (Diptera: Tachinidae)

Tachinid flies must respond to the host immune system. Drino inconspicuoides, an endoparasitoid tachinid fly, does not require the injection of immunosuppressive factors during oviposition but ensures successful larval development within the host. In this study, a serine protease inhibitor DiSpn27A, which is secreted into the host hemolymph was identified and characterized from the salivary glands of D. inconspicuoides larvae at 24 h post-parasitization. Recombinant DiSpn27A (rDiSpn27A) significantly inhibited melanization when mixed with the host Mythimna separata larvae hemolymph in vitro. Melanization was inhibited in vivo when beads coated with rDiSpn27A were implanted into host larvae or injected with rDiSpn27A into the host body. Furthermore, rDiSpn27A interacted with the host prophenoloxidase-activating enzyme derived from M. separata, leading to significant suppression of melanization. DiSpn27A may function as an immune suppressor targeting the phenoloxidase activation cascade. Moreover, the RNAi-mediated knockdown of DiSpn27A significantly reduced the parasitism success of D. inconspicuoides, indicating that the suppression of host melanization is a critical factor for the successful development of D. inconspicuoides within its hosts. These results provide novel insights into the molecular mechanisms underlying host immune regulation in tachinid flies.