Toll and IMD pathways play an important role in producing antimicrobial peptides (AMPs) through NF-κB in insects. The functions of IκB kinase (IKK) complex regulating the NF-κB signaling cascade have not yet been investigated in Tenebrio model. Here, we identified TmIKK-β (or TmIrd5) which contains 2,112 bp encoding 703 amino acid residues. Domain analysis shows that TmIKK-β contains one Serine/Threonine protein kinases catalytic domain. Developmental expression patterns indicate that TmIKK- β gene was highly expressed in early pupal (P1) and adult (A5) stages. Tissue specific profiles show that TmIKK-β was highly expressed in the integuments in last instar larvae, and fat body and hemocytes in 5 day-old adults. TmIKK-β1 transcripts were strongly induced at 3 and 12 h-post injection of E. coli, and 3 h-post injection of S. aureus or C. albicans in hemocytes. In gut, TmIKK-β transcripts were slightly induced by E. coli (at 6, 9 and 24 h) and C. albicans (at 24 h), while it was not induced by S. aureus challenge. Moreover, it was highly induced at 6 h-post injection of E. coli and then it was gradually decreased in the fat body. To understand the immunological role of TmIKK-β, gene specific RNAi and mortality assay was performed. Depletion of TmIKK-β mRNA leads to increase microbial susceptibility of larvae against E. coli, S. aureus and C. albicans. In addition, induction patterns of fourteen AMP genes in response to microbial challenge was tissue specifically investigated in TmIKK-β–silenced T. molitor larvae. The results suggest that expression of ten AMP genes out of fourteen genes were drastically decreased by TmIKK-β RNAi in fat body, suggesting that TmIKK-β plays an important role in antimicrobial innate immune responses.

Autophagy is an important self-eating process to eliminate damaged or unused organelles. We identified nine autophagy-related genes (Atg) including AaAtg-1, -3, -4b, -4d, -5, -6, -8, -12 and -13 from the Asian tiger mosquito, Aedes albopictus. Developmental expression patterns indicate that mRNA levels of AaAtg-1, -3, -4b, -4d, -5, -6, -12 and -13 were highly expressed in egg, whereas expression of AaAtg8 was high in 1stand3rdinstarlarvalstages. TissuespecificexpressionofthesegenesindicatesthatAaAtg1 was highly expressed in thorax and midgut in blood-fed adult female mosquitoes (BF), and head and thorax in sugar fed adult female mosquitoes (SF). Transcript level of AaAtg3 was high in thorax in BF, but head, thorax and Malpighian tubules in SF. AaAtg4b, -4d mRNA levels were significantly high in Malpighian tubules in BF, and head in SF, respectively. AaAtg-5 and -6 transcripts were highly expressed in head in BF, and expression of AaAtg-8 was high in Malpighian tubules in BF. Levels of AaAtg-12 and -13 mRNAs were significantly high in head and midgut in BF. Induction patterns of AaAtg genes against pathogens showed that AaAtg-1, -3, -4b, -8, -12 and -13 were strongly induced at 6 h-post injection of S. aureus, and mRNA levels of AaAtg-1, -3 and -13 were significantly induced by E. coli challenge after 3 h-post injection in SF abdominal carcass. In SF midgut, AaAtg-1, -3, -4b, -4d, -5, -6, -12 and -13 transcripts were drastically induced at 9 h-injection of E. coli and S. aureus, while expression of AaAtg-8 was highly induced by S. aureus and C. albicans at 9 h-post injection. Each AaAtg gene was slightly induced by E. coli, S. aureus or C. albicans at different time points in abdominal carcass in BF. Interestingly, AaAtg-8 was not induced by microbial challenge. While eight other Atg genes except AaAtg-8 were highly influenced by S. aureus at 6 and 9 h-post injection, E. coli at 3 h-post-treatment, and 3, 6, and 9 h-post inoculation. In the future, we will characterize the functional roles of autophagy during mosquito-microbes interaction.

It has been well known that IKK-β, -ε and –γ play a pivotal role in IMD pathway. In this study, TmIKK-ε was identified and their functions in countering pathogenic infections were investigated. We identified TmIKK-ε gene which including 2,196 bp nucleotides (encoding 731 amino acid residues). Domain analysis of TmIKK-ε indicates that there is one Serine/Threonine protein kinases catalytic domain. TmIKK-ε gene was highly expressed in 2 day-old pupal stage and the expression was gradually decreased until 1 day-old adults. Then the expression was slightly increased until 4 day-old adult stage. Tissue specific expression of TmIKK-ε mRNA was high in the gut, integuments and hemocytes in last instar larvae, and fat body, Malpighian tubules and testis in 5-daysold adult. In hemocytes, TmIKK-ε was drastically induced by E. coli injection after 3 h and by S. aureus at 3 and 12 h-post injection. In gut, expression level of TmIKK-ε was high at 6 h-post injection of microbial injection. Expression of TmIKK-ε in fat body was drastically induced by E. coli at 3 and 24 h-post injection while it was not significantly induced by S. aureus and C. albicans. To understand the immunological role of TmIKK-ε, gene specific RNAi and mortality assay were performed. TmIKK-ε RNAi caused increased larval mortality against E. coli, not S. aureus and C. albicans. Finally, to investigate the induction patterns of Tenebrio fourteen AMP genes in response TmIKK-ε RNAi, three microorganisms were treated into TmIKK-ε-silenced T. molitor larvae. Nine out of fourteen AMP genes were not induced by microbial challenge in TmIKK-β dsRNA-injected group. Taken together, our results indicate that TmIKK-ε may regulates nine antimicrobial peptide genes in response to microbial challenge in T. molitor fat body.

Host defense against pathogen invasion highly relies on immune defense machinery that is controlled by the nuclear factor-κB (NF-κB) of transcription factors. The Toll pathway are well known as an insect innate immune mechanism to protect host itself from invaded pathogens. Basically, in the edible insect, Tenebrio molitor, the Toll pathway is primarily activated by polymeric Lys-type peptidoglycans (PGNs), and components of fungal cell walls, β-1,3-glucan. Based on the current studies, the tremendous study has been focused on recognition and subsequent activation of spätzle in haemolymph, hence, there is a grave gap for intracellular event. Herein, in order to understand intracellular event of Toll signaling pathway, the Dorsal gene were identified. Moreover, domain analyses of TmDorsal2 gene indicate that there are two major domains such as Rel homology domain (RHD), ig-like, plexins, and transcription factors (IPT) domains. Based on the achieved results, TmDorsal2 mRNA was highly expressed in 1-day old pupa. Furthermore, TmDorsal2 was highly expressed in Malpighian tubules and fat body in last instar larvae (LL), and likewise mainly expressed in Malpighian tubules during adult 5-day old period, also the lowest expression of TmDorsal2 was observed in gonads. Moreover, TmDorsal2 mRNA levels after infection with E. coli appreciably went up at 6 and 9h time points. To investigate the effects of TmDorsal2 RNAi on larval susceptibility against various pathogens namely E. coli, S. aureus or C.albicans, dsRNA of TmDorsal2 has been synthesized the larvae dissected after 24h. As a result, TmAttacin1a, 1b and 2, TmDefencine1 and 2, TmTenecin1, 2, 3 and 4, TmCecropin2, TmColeoptericin1 and 2, Thaumatin-like protein 1 and 2 markedly reduced in the gut after injecting all mentioned microbes. In contrast, TmTenecin 2, Thaumatin-like protein 1 and 2 strikingly increased after microbe injection in the fat body. Interestingly, the most AMPs gene expression in whole body experimental case were upregulated. On the horizon, we will investigate effects of TmDorsal1 RNAi on larval susceptibility against various pathogens. Taken together, our studies may aid to understand insect innate immunity.