TmIKK epsilon Is Required to Confer Protection Against Gram-Negative Bacteria, E. coli by the Regulation of Antimicrobial Peptide Production in the Tenebrio molitor Fat Body

The inhibitor of nuclear factor-kappa B (NF-kappa B) kinase (IKK) is the core regulator of the NF-kappa B pathway against pathogenic invasion in vertebrates or invertebrates. IKK beta, -epsilon and -gamma have pivotal roles in the Toll and immune deficiency (IMD) pathways. In this study, a homolog of IKK epsilon (TmIKK epsilon) was identified from Tenebrio molitor RNA sequence database and functionally characterized for its role in regulating immune signaling pathways in insects. The TmIKK epsilon gene is characterized by two exons and one intron comprising an open reading frame (ORF) of 2,196 bp that putatively encodes a polypeptide of 731 amino acid residues. TmIKK epsilon contains a serine/threonine protein kinases catalytic domain. Phylogenetic analysis established the close homology of TmIKK epsilon to Tribolium castaneum IKK epsilon (TcIKK epsilon) and its proximity with other IKK-related kinases. The expression of TmIKK epsilon mRNA was elevated in the gut, integument, and hemocytes of the last-instar larva and the fat body, Malpighian tubules, and testis of 5-day-old adults. TmIKK epsilon expression was significantly induced by Escherichia coli, Staphylococcus aureus, and Candida albicans challenge in whole larvae and tissues, such as hemocytes, gut, and fat body. The knockdown of the TmIKK epsilon messenger RNA (mRNA) expression significantly reduced the survival of the larvae against microbial challenges. Further, we investigated the induction patterns of 14 T. molitor antimicrobial peptides (AMPs) genes in TmIKK epsilon gene-silencing model after microbial challenges. While in hemocytes, the transcriptional regulation of most AMPs was negatively regulated in the gut and fat body tissue of T. molitor, AMPs, such as TmTenecin 1, TmTenecin 4, TmDefensin, TmColeoptericin A, TmColeoptericin B, TmAttacin 1a, and TmAttacin 2, were positively regulated in TmIKK epsilon-silenced individuals after microbial challenge. Collectively, the results implicate TmIKK epsilon as an important factor in antimicrobial innate immune responses in T. molitor.