Dorsal, a member of the nuclear factor-kappa B (NF-κB) family of transcription factors, is a critical downstream component of the Toll pathway that regulates the expression of antimicrobial peptides (AMPs) against pathogen invasion. In this study, the full-length ORF of Dorsal was identified from the RNA-seq database of the mealworm beetle Tenebrio molitor (TmDorX2). The ORF of TmDorX2 was 1,482 bp in length, encoding a polypeptide of 493 amino acid residues. TmDorX2 contains a conserved Rel homology domain (RHD) and an immunoglobulin-like, plexins, and transcription factors (IPT) domain. TmDorX2 mRNA was detected in all developmental stages, with the highest levels observed in 3-day-old adults. TmDorX2 transcripts were highly expressed in the adult Malpighian tubules (MT) and the larval fat body and MT tissues. After challenging the larvae with Staphylococcus aureus and Escherichia coli, the TmDorX2 mRNA levels were upregulated 6 and 9 h post infection in the whole body, fat body, and hemocytes. Upon Candida albicans challenge, the TmDorX2 mRNA expression were found highest at 9 h post-infection in the fat body. In addition, TmDorX2-knockdown larvae exposed to E. coli, S. aureus, or C. albicans challenge showed a significantly increased mortality rate. Furthermore, the expression of 11 AMP genes was downregulated in the gut and fat body of dsTmDorX2-injected larvae upon E. coli challenge. After C. albicans and S. aureus challenge of dsTmDorX2-injected larvae, the expression of 11 and 10 AMPs was downregulated in the gut and fat body, respectively. Intriguingly, the expression of antifungal transcripts TmTenecin-3 and TmThaumatin-like protein-1 and -2 was greatly decreased in TmDorX2-silenced larvae in response to C. albicans challenge, suggesting that TmDorX2 regulates antifungal AMPs in the gut in response to C. albicans infection. The AMP expression profiles in the fat body, hemocytes, gut, and MTs suggest that TmDorX2 might have an important role in promoting the survival of T. molitor larvae against all mentioned pathogens.
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.
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 immunerelated 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.
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 ( Tm IMD) 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 (Tm Tenecin1 , Tm Tenecin2 , Tm Tenecin4 , Tm Defensin2 , Tm Coleoptericin1 , Tm Coleoptericin2 , Tm Attacin1a , Tm Attacin1b , and Tm Attacin2 ) showed significant downregulation in TmIMD knockdown larvae challenged with E . coli . These results suggest that Tm IMD is required to confer humoral immunity against the Gram-negative bacteria, E . coli by inducing the expression of critical transcripts that encode AMPs.
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