Spätzle 1b Is Required to Confer Antibacterial Defense Against Gram-Negative Bacteria by Regulation of Antimicrobial Peptides.

Innate immunity is the ultimate line of defense against invading pathogens in insects. Unlike in the mammalian model, in the insect model, invading pathogens are recognized by extracellular receptors, which activate the Toll signaling pathway through an extracellular serine protease cascade. In the Toll-NF-κB pathway, the extracellular spätzle protein acts as a downstream ligand for Toll receptors in insects. In this study, we identified a novel Spätzle isoform (Spz1b) from RNA sequencing database of . Spz1b was bioinformatically analyzed, and functionally characterized for the antimicrobial function by RNA interference (RNAi). The 702 bp open reading frame of Spz1b encoded a putative protein of 233 amino acid residues. A conserved cystine-knot domain with seven cysteine residues in Spz1b was involved in three disulfide bridges and the formation of a spätzle dimer. was mostly expressed in the hemocytes of late instar larvae. The mRNA expression of was highly induced in the hemocytes after , , and stimulation of larvae. silenced larvae were significantly more susceptible to infection. In addition, RNAi-based functional assay characterized Spz1b to be involved in the positive regulation of antimicrobial peptide genes in hemocytes and fat bodies. Further, the transcripts were downregulated in silenced individuals upon challenge suggesting the relationship to Toll signaling pathway. These results indicate that Spz1b is involved in the innate immunity, causes the sequestration of Gram-negative bacteria by the regulatory action of antimicrobial peptides, and enhances the survival of larvae.