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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 ( Tm Spz1b) from RNA sequencing database of  Tenebrio molitor .  Tm Spz1b was bioinformatically analyzed, and functionally characterized for the antimicrobial function by RNA interference (RNAi). The 702 bp open reading frame of  Tm Spz1b encoded a putative protein of 233 amino acid residues. A conserved cystine-knot domain with seven cysteine residues in  Tm Spz1b was involved in three disulfide bridges and the formation of a spätzle dimer.  TmSpz1b  was mostly expressed in the hemocytes of  T. molitor  late instar larvae. The mRNA expression of  TmSpz1b  was highly induced in the hemocytes after  Escherichia coli ,  Staphylococcus aureus , and  Candida albicans  stimulation of  T. molitor  larvae.  TmSpz1b  silenced larvae were significantly more susceptible to  E. coli  infection. In addition, RNAi-based functional assay characterized  Tm Spz1b to be involved in the positive regulation of antimicrobial peptide genes in hemocytes and fat bodies. Further, the  TmDorX2  transcripts were downregulated in  TmSpz1b  silenced individuals upon  E. coli  challenge suggesting the relationship to Toll signaling pathway. These results indicate that  Tm Spz1b is involved in the  T. molitor  innate immunity, causes the sequestration of Gram-negative bacteria by the regulatory action of antimicrobial peptides, and enhances the survival of  T. molitor  larvae.

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