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The inhibitor of nuclear factor-kappa B (NF-κB) kinase (IKK) is the core regulator of the NF-κB pathway against pathogenic invasion in vertebrates or invertebrates. IKKβ, -ε and -γ have pivotal roles in the Toll and immune deficiency (IMD) pathways. In this study, a homolog of IKKε (TmIKKε) was identified from Tenebrio molitor RNA sequence database and functionally characterized for its role in regulating immune signaling pathways in insects. The TmIKKε 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. TmIKKε contains a serine/threonine protein kinases catalytic domain. Phylogenetic analysis established the close homology of TmIKKε to Tribolium castaneum IKKε (TcIKKε) and its proximity with other IKK-related kinases. The expression of TmIKKε 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. TmIKKε 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 TmIKKε 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 TmIKKε 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 TmIKKε-silenced individuals after microbial challenge. Collectively, the results implicate TmIKKε as an important factor in antimicrobial innate immune responses in T. molitor.

Immune deficiency (IMD) is a death domain-containing protein that is essential for the IMD/NF-κB humoral and epithelial immune responses to Gram-negative bacteria and viruses in insects. In the immune signaling cascade, IMD is recruited together with FADD and the caspase DREDD after the mobilization of PGRP receptors. Activated IMD regulates the expression of effector antimicrobial peptides (AMP) that protect against invading microorganisms. To date, most studies of the IMD pathway, and the IMD gene in particular, have been restricted to Drosophila; few similar studies have been conducted in other model insects. Herein, we cloned and functionally characterized an IMD homolog from the mealworm beetle Tenebrio molitor (TmIMD) and studied its role in host survival in the context of pathogenic infections. Phylogenetic analysis revealed the conserved caspase cleavage site and inhibitor of apoptosis (IAP)-binding motif (IBM). TmIMD expression was high in the hemocytes and Malpighian tubules of Tenebrio late-instar larvae and adults. At 3 and 6 hours’ post-infection with Escherichia coli, Staphylococcus aureus, or Candida albicans, TmIMD expression significantly increased compared with mock-infected controls. Knockdown of the TmIMD transcript by RNAi significantly reduced host resistance to the Gram-negative bacterium E. coli and fungus C. albicans in a survival assay. Strikingly, the expression of nine T. molitor AMPs (TmTenecin1, TmTenecin2, TmTenecin4, TmDefensin2, TmColeoptericin1, TmColeoptericin2, TmAttacin1a, TmAttacin1b, and TmAttacin2) showed significant downregulation in TmIMD knockdown larvae challenged with E. coli. These results suggest that TmIMD is required to confer humoral immunity against the Gram-negative bacteria, E. coli by inducing the expression of critical transcripts that encode AMPs.

IKK&gamma, /NEMO is the regulatory subunit of the I&kappa, B kinase (IKK) complex, which regulates the NF-&kappa, B signaling pathway. Within the IKK complex, IKK&gamma, /NEMO is the non-catalytic subunit, whereas IKK&alpha, and IKK&beta, are the structurally related catalytic subunits. In this study, TmIKK&gamma, was screened from the Tenebrio molitor RNA-Seq database and functionally characterized using RNAi screening for its role in regulating T. molitor antimicrobial peptide (AMP) genes after microbial challenges. The TmIKK&gamma, transcript is 1521 bp that putatively encodes a polypeptide of 506 amino acid residues. TmIKK&gamma, contains a NF-&kappa, B essential modulator (NEMO) and a leucine zipper domain of coiled coil region 2 (LZCC2). A phylogenetic analysis confirmed its homology to the red flour beetle, Tribolium castaneum IKK&gamma, (TcIKK&gamma, ). The expression of TmIKK&gamma, mRNA showed that it might function in diverse tissues of the insect, with a higher expression in the hemocytes and the fat body of the late-instar larvae. TmIKK&gamma, mRNA expression was induced by Escherichia coli, Staphylococcus aureus, and Candida albicans challenges in the whole larvae and in tissues such as the hemocytes, gut and fat body. The knockdown of TmIKK&gamma, mRNA significantly reduced the survival of the larvae after microbial challenges. Furthermore, we investigated the tissue-specific induction patterns of fourteen T. molitor AMP genes in TmIKK&gamma, mRNA-silenced individuals after microbial challenges. In general, the mRNA expression of TmTenecin1, -2, and -4, TmDefensin1 and -2, TmColeoptericin1 and 2, and TmAttacin1a, 1b, and 2 were found to be downregulated in the hemocytes, gut, and fat body tissues in the TmIKK&gamma, silenced individuals after microbial challenges. Under similar conditions, TmRelish (NF-&kappa, B transcription factor) mRNA was also found to be downregulated. Thus, TmIKK&gamma, is an important factor in the antimicrobial innate immune response of T. molitor.

Autophagy is an important process by which pathogens and damaged or unused organelles are eliminated. The role of autophagy in development and the immune response to pathogens is well established. Autophagy-related protein 8 (Atg8) is involved in the formation of the autophagosome and, with the help of the serine protease Atg4, mediates the delivery of both vesicles and the autophagosome to the vacuole. Here, we cloned the Aedes albopictus autophagy-related protein 8 (AaAtg8) gene and characterized its role in the innate immunity of the mosquito against microbial infections. AaAtg8 is comprised of an open reading frame (ORF) region of 357 bp encoding a polypeptide of 118 amino acid residues. A domain analysis of AaAtg8 revealed an Atg8 ubiquitin-like domain, Atg7/Atg4 interaction sites, and peptide binding sites. The AaAtg8 mRNA expression was high in the Malpighian tubules and heads of both sugar-fed and blood-fed adult female mosquitoes. The expression level of AaAtg8 mRNA increased in the midgut and abdominal carcass following being challenged with Listeria monocytogenes. To investigate the role of AaAtg8 in the innate immune responses of Ae. albopictus, AaAtg8 gene-silenced adult mosquitoes were challenged by injection or by being fed microorganisms in blood. High mortality rates were observed in mosquitoes in which AaAtg8 was silenced after challenges of microorganisms to the host by blood feeding. This suggests that Atg8-autophagy plays a critical role in the gut immunity in Ae. albopictus.

Relish, a transcription factor, is a critical downstream component of the immune deficiency (Imd) pathway and regulates host defense against bacterial infection by mediating antimicrobial peptide (AMP) synthesis. Understanding the immunological function of the mealworm beetle, Tenebrio molitor Relish (TmRelish) will be instructive in understanding insect immunity. In the present study, full-length ORF of TmRelish was retrieved from T. molitor-expressed sequence tags and RNA-seq database. The predicted TmRelish amino acid sequence contained an N-terminal Rel-homology domain; an Ig-like, plexin, and transcription factor domain; ankyrin repeat motifs; a nuclear localization signal; and a C-terminal death domain and shared the highly conserved structure of the Relish proteins of other insect species. TmRelish mRNA was detected in all developmental stages of the insect; however, the highest levels were detected in the larval gut tissue and adult hemocytes. TmRelish mRNA level was upregulated in the fat body, hemocyte, and gut tissue 9 h after infection of T. molitor larvae by the gram-negative bacteria, Escherichia coli. Furthermore, TmRelish knockdown led to significantly higher mortality of the E. coli-infected larvae, and significantly lower mortality of larvae infected with Staphylococcus aureus or Candida albicans. To elucidate the possible cause of mortality, we measured AMP transcription in the fat body, hemocytes, gut, and Malpighian tubules (MTs) of T. molitor larvae. TmRelish knockdown suppressed the expression of nine AMP genes in the larval fat body and gut tissue during E. coli infection, suggesting that TmRelish positively regulates AMP expression in both immune-related tissues, in response to E. coli challenge. Furthermore, negative regulation of some AMPs by TmRelish in the MTs, gut and hemocytes in response to C. albicans infection suggests a crosstalk between the Toll and Imd pathways.

Although it is known that the Drosophila Toll-7 receptor plays a critical role in antiviral autophagy, its function in other insects has not yet been reported. Here, we have identified a Toll-like receptor 7 gene, TmToll-7, in the coleopteran insect T. molitor and examined its potential role in antibacterial and antifungal immunity. We showed that TmToll-7 expression was significantly induced in larvae 6 h after infection with Escherichia coli and Staphylococcus aureus and 9 h after infection with Candida albicans. However, even though TmToll-7 was induced by all three pathogens, we found that TmToll-7 knockdown significantly reduced larval survival to E. coli, but not to S. aureus, and C. albicans infections. To understand the reasons for this difference, we examined the effects of TmToll-7 knockdown on antimicrobial peptide (AMP) gene expression and found a significant reduction of E. coli-induced expression of AMP genes such as TmTenecin-1, TmDefensin-1, TmDefensin-2, TmColeoptericin-1, and TmAttacin-2. Furthermore, TmToll-7 knockdown larvae infected with E. coli showed significantly higher bacterial growth in the hemolymph compared to control larvae treated with Vermilion dsRNA. Taken together, our results suggest that TmToll-7 plays an important role in regulating the immune response of T. molitor to E. coli.

The 14-3-3 family of proteins performs key regulatory functions in phosphorylation-dependent signaling pathways including cell survival and proliferation, apoptosis, regulation of chromatin structure and autophagy. In this study, the zeta isoform of 14-3-3 proteins (designated as Tm14-3-3&zeta, ) was identified from the expressed sequence tags (ESTs) and RNA sequencing (RNA-Seq) database of the coleopteran pest, Tenebrio molitor. Tm14-3-3&zeta, messenger RNA (mRNA) is expressed at higher levels in the immune organs of the larval and adult stages of the insect and exhibit almost five-fold induction within 3 h post-infection of the larvae with Escherichia coli and Candida albicans. To investigate the biological function of Tm14-3-3&zeta, a peptide-based Tm14-3-3&zeta, polyclonal antibody was generated in rabbit and the specificity was confirmed using Western blot analysis. Immunostaining and confocal microscopic analyses indicate that Tm14-3-3&zeta, is mainly expressed in the membranes of midgut epithelial cells, the nuclei of fat body and the cytosol of hemocytes. Gene silencing of Tm14-3-3&zeta, increases mortality of the larvae at 7 days post-infection with E. coli and C. albicans. Our findings demonstrate that 14-3-3&zeta, in T. molitor is essential in the host defense mechanisms against bacteria and fungi.