Innate Immune Response of TmToll-3 Following Systemic Microbial Infection in Tenebrio molitor.

Although Toll-like receptors have been widely identified and functionally characterized in mammalian models and Drosophila, the immunological function of these receptors in other insects remains unclear. Here, we explored the relevant innate immune response of Tenebrio molitor (T. molitor) Toll-3 against Gram-negative bacteria, Gram-positive bacteria, and fungal infections. Our findings indicated that TmToll-3 expression was mainly induced by Candida albicans infections in the fat bodies, gut, Malpighian tubules, and hemolymph of young T. molitor larvae. Surprisingly, Escherichia coli systemic infection caused mortality after TmToll-3 knockdown via RNA interference (RNAi) injection, which was not observed in the control group. Further analyses indicated that in the absence of TmToll-3, the final effector of the Toll signaling pathway, antimicrobial peptide (AMP) genes and relevant transcription factors were significantly downregulated after E. coli challenge. Our results indicated that the expression of almost all AMP genes was suppressed in silenced individuals, whereas the expression of relevant genes was positively regulated after fungal injection. Therefore, this study revealed the immunological involvement of TmToll-3 in T. molitor in response to systematic infections.

Immunological Roles of TmToll-2 in Response to Escherichia coli Systemic Infection in Tenebrio molitor.

The antimicrobial roles of Toll-like receptors have been mainly identified in mammalian models and Drosophila. However, its immunological function in other insects has yet to be fully clarified. Here, we determined the innate immune response involvement of TmToll-2 encountering Gram-negative, Gram-positive, and fungal infection. Our data revealed that TmToll-2 expression could be induced by Escherichia coli, Staphylococcus aureus, and Candida albicans infections in the fat bodies, gut, Malpighian tubules, and hemolymph of Tenebrio molitor young larvae. However, TmToll-2 silencing via RNAi technology revealed that sole E. coli systemic infection caused mortality in the double-strand RNA TmToll-2-injected group compared with that in the control group. Further investigation indicated that in the absence of TmToll-2, the final effector of Toll signaling pathway, antimicrobial peptide (AMP) genes and relevant transcription factors were significantly downregulated, mainly E. coli post-insult. We showed that the expression of all AMP genes was suppressed in the main immune organ of insects, namely, fat bodies, in silenced individuals, while the relevant expressions were not affected after fungal infection. Thus, our research revealed the immunological roles of TmToll-2 in different organs of T. molitor in response to pathogenic insults.

TmSpz-like Plays a Fundamental Role in Response to E. coli but Not S. aureus or C. albican Infection in Tenebrio molitor via Regulation of Antimicrobial Peptide Production.

The cystine knot protein Spätzle is a Toll receptor ligand that modulates the intracellular signaling cascade involved in the nuclear factor kappa B (NF-κB)-mediated regulation of antimicrobial peptide (AMP)-encoding genes. Spätzle-mediated activation of the Toll pathway is critical for the innate immune responses of insects against Gram-positive bacteria and fungi. In this study, the open reading frame (ORF) sequence of Spätzle-like from T. molitor (TmSpz-like) identified from the RNA sequencing dataset was cloned and sequenced. The 885-bp TmSpz-like ORF encoded a polypeptide of 294 amino acid residues. TmSpz-like comprised a cystine knot domain with six conserved cysteine residues that formed three disulfide bonds. Additionally, TmSpz-like exhibited the highest amino acid sequence similarity with T. castaneum Spätzle (TcSpz). In the phylogenetic tree, TmSpz-like and TcSpz were located within a single cluster. The expression of TmSpz-like was upregulated in the Malpighian tubules and gut tissues of T. molitor. Additionally, the expression of TmSpz-like in the whole body and gut of the larvae was upregulated at 24 h post-E. coli infection. The results of RNA interference experiments revealed that TmSpz-like is critical for the viability of E. coli-infected T. molitor larvae. Eleven AMP-encoding genes were downregulated in the E. coli-infected TmSpz-like knockdown larvae, which suggested that TmSpz-like positively regulated these genes. Additionally, the NF-κB-encoding genes (TmDorX1, TmDorX2, and TmRelish) were downregulated in the E. coli-infected TmSpz-like knockdown larvae. Thus, TmSpz-like plays a critical role in the regulation of AMP production in T. molitor in response to E. coli infection.

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.

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.

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.

Aedes albopictus Autophagy-Related Gene 8 (AaAtg8) Is Required to Confer Anti-Bacterial Gut Immunity.

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.

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.

IKKγ/NEMO Is Required to Confer Antimicrobial Innate Immune Responses in the Yellow Mealworm, Tenebrio Molitor.

IKKγ/NEMO is the regulatory subunit of the IκB kinase (IKK) complex, which regulates the NF-κB signaling pathway. Within the IKK complex, IKKγ/NEMO is the non-catalytic subunit, whereas IKKα and IKKß are the structurally related catalytic subunits. In this study, TmIKKγ 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 TmIKKγ transcript is 1521 bp that putatively encodes a polypeptide of 506 amino acid residues. TmIKKγ contains a NF-κB 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 IKKγ (TcIKKγ). The expression of TmIKKγ 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. TmIKKγ 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 TmIKKγ 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 TmIKKγ 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 TmIKKγ-silenced individuals after microbial challenges. Under similar conditions, TmRelish (NF-κB transcription factor) mRNA was also found to be downregulated. Thus, TmIKKγ is an important factor in the antimicrobial innate immune response of T. molitor.

Transcriptome analysis of air-breathing land slug, Incilaria fruhstorferi reveals functional insights into growth, immunity, and reproduction.

BACKGROUND: Incilaria (= Meghimatium) fruhstorferi is an air-breathing land slug found in restricted habitats of Japan, Taiwan and selected provinces of South Korea (Jeju, Chuncheon, Busan, and Deokjeokdo). The species is on a decline due to depletion of forest cover, predation by natural enemies, and collection. To facilitate the conservation of the species, it is important to decide on a number of traits related to growth, immunity and reproduction addressing fitness advantage of the species. RESULTS: The visceral mass transcriptome of I. fruhstorferi was enabled using the Illumina HiSeq 4000 sequencing platform. According to BUSCO (Benchmarking Universal Single-Copy Orthologs) method, the transcriptome was considered complete with 91.8% of ortholog genes present (Single: 70.7%; Duplicated: 21.1%). A total of 96.79% of the raw read sequences were processed as clean reads. TransDecoder identified 197,271 contigs that contained candidate-coding regions. Of a total of 50,230 unigenes, 34,470 (68.62% of the total unigenes) annotated to homologous proteins in the Protostome database (PANM-DB). The GO term and KEGG pathway analysis indicated genes involved in metabolism, phosphatidylinositol signalling system, aminobenzoate degradation, and T-cell receptor signalling pathway. Many genes associated with molluscan innate immunity were categorized under pathogen recognition receptor, TLR signalling pathway, MyD88 dependent pathway, endogenous ligands, immune effectors, antimicrobial peptides, apoptosis, and adaptation-related. The reproduction-associated unigenes showed homology to protein fem-1, spermatogenesis-associated protein, sperm associated antigen, and testis expressed sequences, among others. In addition, we identified key growth-related genes categorized under somatotrophic axis, muscle growth, chitinases and collagens. A total of 4822 Simple Sequence Repeats (SSRs) were also identified from the unigene sequences of I. fruhstorferi. CONCLUSIONS: This is the first available genomic information for non-model land slug, I. fruhstorferi focusing on genes related to growth, immunity, and reproduction, with additional focus on microsatellites and repeating elements. The transcriptome provides access to greater number of traits of unknown relevance in the species that could be exploited for in-depth analyses of evolutionary plasticity and making informed choices during conservation planning. This would be appropriate for understanding the dynamics of the species on a priority basis considering the ecological, health, and social benefits.