Biosurfactants Induce Antimicrobial Peptide Production through the Activation of TmSpatzles in Tenebrio molitor.

Biosurfactant immunomodulatory activities in mammals, nematodes, and plants have been investigated. However, the immune activation property of biosurfactants in insects has not been reported. Therefore, here, we studied the defense response triggered by lipopeptides (fengycin and iturin A), glycolipids (rhamnolipid), and cyclic polypeptides (bacitracin) in the coleopteran insect, mealworm Tenebrio molitor. The in vitro antimicrobial activities against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria and fungi (Candida albicans) were assessed by mixing these pathogens with the hemolymph of biosurfactant-immune-activated larvae. E. coli growth was remarkably inhibited by this hemolymph. The antimicrobial peptide (AMP) induction results also revealed that all biosurfactants tested induced several AMPs, exclusively in hemocytes. The survivability analysis of T. molitor larvae challenged by E. coli (106 CFU/µL) at 24 h post biosurfactant-immune activation showed that fengycin, iturin A, and rhamnopid significantly increased survivability against E. coli. Biosurfactant-induced TmSpatzles activation was also monitored, and the results showed that TmSpz3 and TmSpz-like were upregulated in the hemocytes of iturin A-injected larvae, while TmSpz4 and TmSpz6 were upregulated in the fat bodies of the fengycin-, iturin A-, and rhamnolipid-injected larvae. Overall, these results suggest that lipopeptide and glycolipid biosurfactants induce the expression of AMPs in T. molitor via the activation of spätzle genes, thereby increasing the survivability of T. molitor against E. coli.

TmPGRP-SA regulates Antimicrobial Response to Bacteria and Fungi in the Fat Body and Gut of Tenebrio molitor.

Antimicrobial immune response is mediated by a signal-transducing sensor, peptidoglycan recognition protein-SA (PGRP-SA), that can recognize non-self molecules. Although several studies have focused on the involvement of Drosophila PGRP-SA in antimicrobial peptide (AMP) expression in response to infections, studies on its role in Tenebrio molitor are lacking. Here, we present a functional analysis of T. molitor PGRP-SA (TmPGRP-SA). In the absence of microbes, TmPGRP-SA was highly expressed in the late-larval fat body, followed by hemocytes, and gut. Interestingly, following Escherichia coli, Staphylococcus aureus, and Candida albicans infections, the mRNA level of TmPGRP-SA was significantly upregulated in both the fat body and gut. TmPGRP-SA silencing had a significant effect on the mortality rates for all the microbes tested. Moreover, TmPGRP-SA is required for regulating the expression of eight AMP genes namely TmTenecin-1, -2, and -4; TmDefensin-1 and -2; TmColeoptericin-1; and TmAttacin-1b and -2 in the fat body in response to E. coli and S. aureus infections. TmPGRP-SA is essential for the transcription of TmTenecin-2, -4; TmDefensin-2; TmColeoptericin-1, -2; and TmAttacin-1a, -1b, and -2 in the gut upon E. coli and C. albicans infections. However, TmPGRP-SA does not regulate AMP expression in the hemocytes. Additionally, TmDorsal isoform X2, a downstream Toll transcription factor, was downregulated in TmPGRP-SA-silenced larval fat body following E. coli and S. aureus challenges, and in the gut following E. coli and C. albicans challenges.

TmSpz4 Plays an Important Role in Regulating the Production of Antimicrobial Peptides in Response to Escherichia coli and Candida albicans Infections.

Spätzle family proteins activate the Toll pathway and induce antimicrobial peptide (AMP) production against microbial infections. However, the functional importance of Tmspätzle4 (TmSpz4) in the immune response of Tenebrio molitor has not been reported. Therefore, here, we have identified and functionally characterized the role of TmSpz4 against bacterial and fungal infections. We showed that TmSpz4 expression was significantly induced in hemocytes at 6 h post-injection with Escherichia coli, Staphylococcus aureus, and Candida albicans. TmSpz4 knock-down significantly reduced larval survival against E. coli and C. albicans. To understand the reason for the survivability difference, the role of TmSpz4 in AMP production was examined in TmSpz4-silenced larvae following microbe injection. The AMPs that are active against Gram-negative bacteria, including TmTenecin-2, TmTenecin-4, TmAttacin-1a, TmDefensin-2, and TmCecropin-2, were significantly downregulated in response to E. coli in TmSpz4-silenced larvae. Similarly, the expression of TmTenecin-1, TmTenecin-3, TmThaumatin-like protein-1 and -2, TmDefensin-1, TmDefensin-2, and TmCecropin-2 were downregulated in response to C. albicans in TmSpz4-silenced larvae. In addition, the transcription factor NF-κB (TmDorX1 and TmDorX2) expression was significantly suppression in TmSpz4-silenced larvae. In conclusion, these results suggest that TmSpz4 plays a key role in regulating immune responses of T. molitor against to E. coli and C. albicans.

TmAtg6 Plays an Important Role in Anti-Microbial Defense Against Listeria monocytogenes in the Mealworm, Tenebrio molitor.

Autophagy-related gene-6 (Beclin-1 in mammals) plays a pivotal role in autophagy and is involved in autophagosome formation and autolysosome maturation. In this study, we identified and characterized the autophagy-related gene-6 from Tenebrio molitor (TmAtg6) and analyzed its functional role in the survival of the insect against infection. The expression of TmAtg6 was studied using qRT-PCR for the assessment of the transcript levels at various developmental stages in the different tissues. The results showed that TmAtg6 was highly expressed at the 6-day-old pupal stage. Tissue-specific expression studies revealed that TmAtg6 was highly expressed in the hemocytes of late larvae. The induction patterns of TmAtg6 in different tissues of T. molitor larvae were analyzed by injecting Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, or Candida albicans. The intracellular Gram-positive bacteria, L. monocytogenes, solely induced the expression of TmAtg6 in hemocytes at 9 h-post-injection, whilst in the fat body and gut, bimodal expression times were observed. RNAi-mediated knockdown of the TmAtg6 transcripts, followed by a challenge with microbes, showed a significant reduction in larval survival rate against L. monocytogenes. Taken together, our results suggest that TmAtg6 plays an essential role in anti-microbial defense against intracellular bacteria.

Critical Roles of Spätzle5 in Antimicrobial Peptide Production Against Escherichia coli in Tenebrio molitor Malpighian Tubules.

The dimeric cytokine ligand Spätzle (Spz) is responsible for Toll pathway activation and antimicrobial peptide (AMP) production upon pathogen challenge in Tenebrio molitor. Here, we indicated that TmSpz5 has a functional role in response to bacterial infections. We showed that the highest expression of TmSpz5 is induced by Candida albicans. However, TmSpz5 knockdown reduced larval survival against Escherichia coli and Staphylococcus aureus. To evaluate the molecular mechanism underlying the observed survival differences, the role of TmSpz5 in AMP production was examined by RNA interference and microbial injection. T. molitor AMPs that are active against Gram-negative and -positive bacteria, including Tmtenecins, Tmattacins, Tmcoleoptericins, Tmtaumatin-like-proteins, and Tmcecropin-2, were significantly downregulated by TmSpz-5 RNAi in the Malpighian tubules (MTs) following a challenge with E. coli and S. aureus. However, upon infection with C. albicans the mRNA levels of most AMPs in the dsTmSpz5-injected group were similar to those in the control groups. Likewise, the expression of the transcription factors NF-κB, TmDorX2, and TmRelish were noticeably suppressed in the MTs of TmSpz5-silenced larvae. Moreover, E. coli-infected TmSpz5 knockdown larvae showed decreased antimicrobial activity in the MTs and hindgut compared with the control group. These results demonstrate that TmSpz5 has a defined role in T. molitor innate immunity by regulating AMP expression in MTs in response to E. coli.

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

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 (TmSpz1b) from RNA sequencing database of Tenebrio molitor. TmSpz1b was bioinformatically analyzed, and functionally characterized for the antimicrobial function by RNA interference (RNAi). The 702 bp open reading frame of TmSpz1b encoded a putative protein of 233 amino acid residues. A conserved cystine-knot domain with seven cysteine residues in TmSpz1b 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 TmSpz1b 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 TmSpz1b 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 PGRP-LE Plays a Critical Role in Gut Antimicrobial Peptide Production in Response to Escherichia coli.

Invading pathogens are recognized by peptidoglycan recognition proteins (PGRPs) that induce translocation of NF-κB transcription proteins and expression of robust antimicrobial peptides (AMPs). Tenebrio molitor PGRP-LE (TmPGRP-LE) has been previously identified as a key sensor of Listeria monocytogenes infection. Here, we present that TmPGRP-LE is highly expressed in the gut of T. molitor larvae and 5-day-old adults in the absence of microbial infection. In response to Escherichia coli and Candida albicans infections, TmPGRP-LE mRNA levels are significantly upregulated in both the fat body and gut. Silencing of TmPGRP-LE by RNAi rendered T. molitor significantly more susceptible to challenge by E. coli infection and, to a lesser extent, Staphylococcus aureus and C. albicans infections. Reduction of TmPGRP-LE levels in the larval gut resulted in downregulation of eight AMP genes following exposure to E. coli, S. aureus, and C. albicans. However, the transcriptional levels of AMPs more rapidly reached a higher level in the dsEGFP-treated larval gut after challenge with E. coli, which may suggest that AMPs induction were more sensitive to E. coli than S. aureus and C. albicans. In addition, TmPGRP-LE RNAi following E. coli and C. albicans challenges had notable effects on TmRelish, TmDorsal X1 isoform (TmDorX1), and TmDorX2 expression level in the fat body and gut. Taken together, TmPGRP-LE acts as an important gut microbial sensor that induces AMPs via Imd activation in response to E. coli, whereas involvement of TmPGRP-LE in AMPs synthesize is barely perceptible in the hemocytes and fat body.

Two Roles for the Tenebrio molitor Relish in the Regulation of Antimicrobial Peptides and Autophagy-Related Genes in Response to Listeria monocytogenes.

Relish is a key NF-κB transcription factor of the immune-deficiency (Imd) pathway that combats infection by regulating antimicrobial peptides (AMPs). Understanding of the fundamental role of Tenebrio molitor Relish (TmRelish) in controlling of Listeria monocytogenes virulence through the regulation of both AMPs and autophagy-related (ATG) genes is unclear. Here, we show that TmRelish transcripts were highly abundant in the larval fat body and hemocytes compared to the gut upon L. monocytogenes infection. Furthermore, significant mortality was observed in TmRelish-silenced larvae after intracellular insult. To investigate the cause of this lethality, we measured the induction of AMPs and ATG genes in the TmRelish dsRNA-treated T. molitor larvae. The expression of TmTenecin-1, TmTenecin-4, TmColeptericin-1, TmAttacin-2, and TmCecropin-2 were suppressed in the fat body and hemocytes of dsTmRelish-injected larvae during L. monocytogenes infection. In addition, TmRelish knockdown led to a noticeable downregulation of TmATG1 (a serine-threonine protein kinase) in the fat body and hemocytes of young larvae 6 h post-infection (pi). The notable increase of autophagy genes in the early stage of infection (6 h pi), suggesting autophagy response is crucial for Listeria clearance. Taken together, these results suggest that TmRelish plays pivotal roles in not only regulation of AMP genes but also induction of autophagy genes in response to L. monocytogenes challenge in fat body and hemocytes of T. molitor larvae. Furthermore, negative regulation of several AMPs by TmRelish in the fat body, hemocytes, and gut leaves open the possibility of a crosstalk between Toll and Imd pathway.

TmSpz6 Is Essential for Regulating the Immune Response to Escherichia Coli and Staphylococcus Aureus Infection in Tenebrio Molitor.

Spätzle is an extracellular protein that activates the Toll receptor during embryogenesis and immune responses in Drosophila. However, the functions of the spätzle proteins in the innate immune response against bacteria or fungi in T. molitor are not well understood. Therefore, in this study, the open reading frame (ORF) of TmSpz6 was identified and its function in the response to bacterial and fungal infections in T. molitor was investigated using RNAi. The highest expression of TmSpz6 was in prepupae and in 3- and 6- day-old pupae. While in other stages, remarkable expression was also observed. The tissue-specific expression analysis showed that TmSpz6 expression was highest in the hemocytes of larvae. TmSpz6 expression was highly induced when challenged with Escherichia coli, Staphylococcus aureus, or Candida albicans at 6 h post-injection; however, TmSpz6-silenced larvae were significantly more susceptible to only E. coli and S. aureus infection. The antimicrobial peptides (AMPs) gene expression analysis results show that TmSpz6 mainly positively regulated the expression of TmTencin-2 and -3 in response to E. coli and S. aureus infection. Collectively, these results suggest that TmSpz6 plays an important role in regulating AMP expression and increases the survival of T. molitor against E. coli and S. aureus.

TmRelish is required for regulating the antimicrobial responses to Escherichia coli and Staphylococcus aureus in Tenebrio molitor.

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.

Author Correction: TmRelish is required for regulating the antimicrobial responses to Escherichia coli and Staphylococcus aureus in Tenebrio molitor.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

TmDorX2 positively regulates antimicrobial peptides in Tenebrio molitor gut, fat body, and hemocytes in response to bacterial and fungal infection.

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.