Melanization and Toll pathway activation are essential innate immune mechanisms in insects, which result in the generation of reactive compounds and antimicrobial peptides, respectively, to kill microbes. These two processes are mediated by phenoloxidase (PO) and Spätzle (Spz) through an extracellular network of serine proteases. While the melanization and Toll pathways have been investigated mostly in adult and larval insects, their regulation in insect eggs remains unclear. Here, we present evidence from transcriptome and proteome analyses that extra-embryonic tissues of early-stage M. sexta eggs are immune competent, with expression of immune effector genes including prophenoloxidase (proPO) and antimicrobial peptides (AMPs). We identified gene products of the melanization and Toll pathways in M. sexta eggs. Through in vitro reconstitution experiments, we demonstrated that constitutive and infection-induced serine protease cascade modules that stimulate immune responses exist in extra-embryonic tissues of M. sexta eggs. The constitutive module (HP14b-SP144-GP6) may promote rapid early immune signaling by forming a cascade activating the cytokine Spz and regulating melanization by activating proPO. The inducible module (HP14a-HP21-HP5) may trigger enhanced activation of Spz and proPO at a later phase of infection. Crosstalk between the two modules may occur in transition from the constitutive to the induced response in eggs inoculated with bacteria. Examination of data from two other well-studied insect species, Tribolium castaneum and D. melanogaster supports a role for a serosa-dependent constitutive protease cascade in protecting early embryos against invading pathogens. Although certain proteases involved in regulating melanization and Toll pathway activation have been identified in genetic studies on D. melanogaster, the exact order of proteolytic activation events remains controversial. Here, we reconstituted the serine protease framework in Drosophila by biochemical methods. This system comprises ten proteases, i.e. ModSP, cSP48, Grass, Psh, Hayan-PA, Hayan-PB, Sp7, MP1, SPE and Ser7, which form cascade pathways that recognize microbial molecular patterns and pathogen virulence factors, and generate PO1, PO2, and Spz from their precursors. Furthermore, the serpin Necrotic negatively regulates the immune response progression by inhibiting ModSP and Grass. The biochemical approach, when combined with genetic analysis, is useful for addressing problems that long stand in this important research field.
Tisheng Shan (Tue,) studied this question.