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Edible insects such as the mealworm (Tenebrio molitor) are emerging as an alternative animal protein resource to meet the needs of a growing global population. However, despite the successful mass production of edible insects, research into practical compound feed for edible insect is still insufficient. In this study, mealworms were fed wheat bran, layer chicken feed mash, chicken pellets and cattle pellets for 1 to 36 days, after which their body length was measured. In the groups fed with chicken feed mash, chicken pellets or cattle pellets, the mean body lengths were 2.88, 2.92 and 2.95 cm, respectively, similar to the 2.95 cm of the control T. molitor fed with wheat bran. Individual weight measurements yielded similar results: control, 7.87 g; chicken feed mash, 7.43 g; chicken pellets, 7.60 g; and cattle pellets, 7.63 g. Analysis of livestock feed intake showed that the intake was 290.4 g in control insects, 137.7 g in insects fed chicken feed mash, 220.2 g in insects fed chicken pellets and 230.3 g in insects fed cattle pellets. In the excretion analysis, the excretion of the control insects (161.60 g) was larger than that of the insects fed with chicken feed mash (93.37 g), chicken pellets (93.40 g) or cattle pellets (133.87 g). An analysis of the nutritional content of T. molitor based on nine major nutrients showed no significant differences between the control and the test groups. These results can be used to improve the price competitiveness and quality of T. molitor for commercial development. [ABSTRACT FROM AUTHOR]

Selective herbicides are used to control undesirable vegetation or weeds in fields without harming crops. Herbicide use for weed management can directly impact the densities of insect pests in agricultural communities as a result of insect mortality during and immediately after application. In insects, the glutathione S‐transferase (GST) enzyme is involved in both the detoxification process and the defense of cellular membranes against oxidative damage. In this study, two TmGSTs (TmGST‐iso1 and TmGST‐iso2) were identified and characterized to elucidate the GST family in Tenebrio molitor. Among the developmental stages of T. molitor, eggs had the highest expression levels of TmGST‐iso1. TmGST‐iso2 expression was highest in the pre‐pupal stage. TmGST‐iso1 expression was high in the guts of early and late larvae, whereas TmGST‐iso2 expression was not observed in early larvae. Adults, both male and female, had the highest levels of TmGST‐iso1 mRNA expression in the gut and reproductive organs, and the highest levels of TmGST‐iso2 expression in the reproductive organs. Quantitative polymerase chain reaction was used to investigate the impact of treatment with butachlor on the mRNA expression of TmGST‐iso1 and TmGST‐iso2 in larvae. TmGST‐iso1 expression increased in the butachlor‐treated group after 3 and 24 h, whereas TmGST‐iso2 expression peaked at 24 h after treatment. This study provides vital information about the detoxifying activities of T. molitor. [ABSTRACT FROM AUTHOR]

Innate immunity is the immediate and exclusive defense mechanism in invertebrates. It acts against foreign agents and pathogens. In insects, this defense mechanism involves a combination of humoral and cellular responses, orchestrated by specific recognition receptors and the activation of various signaling pathways. Remarkably, Toll and Toll‐related proteins play a pivotal role in insect immunity by inducing the production of antimicrobial peptides (AMPs) in response to pathogen attacks. Toll receptors, which are type I transmembrane proteins, significantly affect insect development and the innate immune response, primarily via the nuclear factor kappa‐B signaling pathway. The Malpighian tubules of insects play a crucial role in waste elimination, hemostasis maintenance, and immune defense. The Toll signaling pathway in Malpighian tubules is mainly involved in the production of AMPs. This review provides valuable insights into the mechanisms of the Toll signaling pathway and the morphological and physiological attributes of Malpighian tubules. Moreover, it discusses the classification and functionalities of AMPs synthesized in Malpighian tubules in response to pathogens, thereby contributing to our understanding of innate immunity in insects. [ABSTRACT FROM AUTHOR]

Agricultural intensification has led to significant increases in production, but the overuse of pesticides and associated hazards pose threats to biodiversity and ecological functions. Catalase (CAT), a key antioxidant enzyme, plays a crucial role in alleviating oxidative stress by directly interacting with toxins. In this study, we identified three CAT isoforms in Tenebrio molitor (TmCAT‐iso1, TmCAT‐iso2 and TmCAT‐iso3). These CATs possess a CAT domain, tetramer interface sites and a heme‐binding pocket. We examined the expression of Tm catalases across all developmental stages and in specific tissues using quantitative real time polymerase chain reaction (qRT‐PCR) experiments. Our findings demonstrate that TmCAT‐iso1 and TmCAT‐iso3 exhibit peak expression in young and late larval stages, respectively, whereas TmCAT‐iso2 shows peak expression during the egg and pre‐pupal stages. Tissue distribution analysis revealed the high expression of TmCAT‐iso1 and TmCAT‐iso2 in larval hemocytes, whereas TmCAT‐iso3 is predominantly expressed in larval Malpighian tubules. Furthermore, injection with chlorantraniliprole significantly elevated mRNA expression levels of TmCAT‐iso1, TmCAT‐iso2, and TmCAT‐iso3 in larval groups, compared with control groups. Our study highlights the distinct developmental stages and tissues where TmCATs are expressed. We also elucidated the effects of pesticide application on the expression of each TmCAT, revealing the physiological characteristics of CATs in response to these pesticides, which are dose‐ and time‐dependent in T. molitor. [ABSTRACT FROM AUTHOR]

In insects, the production of antimicrobial peptides is considered to be the solo arm of the innate immune response. The Toll and immune deficiency pathways are two major signaling pathways that lead to the production of antimicrobial peptides as final effectors. The dynamic functions of Toll/Toll‐like receptors have been thoroughly reviewed in mammals and Drosophila. During the last decade, we have attempted to clarify the immunological roles of different Toll receptor variants and their ligands in Tenebrio molitor. Accordingly, we showed that TmToll‐8, −9, and −10 mRNA transcripts are induced following systemic injections of Gram‐negative, Gram‐positive, and fungal infections. Our data revealed harmonic expression patterns of TmToll‐8, −9, and −10 throughout the developmental stages of healthy individuals and in response to infections in the examined tissues (Malpighian tubules, gut, fat bodies, and hemolymph), illustrating similar evolutionary conversions of different Toll receptor variants in T. molitor. Taken together, our findings highlight the immunological actions of TmToll‐8, −9, and −10 in response to pathogenic insults in T. molitor. [ABSTRACT FROM AUTHOR]

Mealworms beetles, Tenebrio molitor, are the limelight next-generation food for humans due to their high nutrient contents. Since Tenebrio molitor is used as feed for pets and livestock in addition to their ability to decompose polystyrene and plastic waste, it is recognized as an insect with an industrial core value. Therefore, it is important to study the immune mechanism related to the development and infection of mealworms for mass breeding purposes. The immune deficiency (Imd) signaling is one of the main pathways with pivotal roles in the production of antimicrobial peptides (AMPs). Transforming growth factor-β activated kinase (TAK1) is one of the Imd pathway components, forms a complex with TAK1 binding protein 2 (TAB2) to ultimately help activate the transcription factor Relish and eventually induce host to produce AMPs. Relatively, little has been revealed about TAK1 in insect models, especially in the T. molitor. Therefore, this study was conducted to elucidate the function of TmTak1 in T. molitor. Our results showed that the highest and lowest mRNA expression of TmTak1 were found in egg and young larvae respectively. The tissue-specific expression patterns were reported in the gut of T. molitor larvae and the fat bodies of adults. Systemic microbial challenge illustrated TmTak1 high expression following the fungal infection in all dissected tissues except for the whole body. However, silencing TmTak1 experiments showed that the survivability of T. molitor larvae affected significantly following Escherichia coli infection. Accordingly, AMP induction after TmTak1 knock down was mainly reported in the integument and the fat bodies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Edible insects are not only good food ingredients for people, but also open the possibility of functional food and pharmaceutical raw materials. This study investigated the production of chitin‐oligosaccharide and chitosan‐oligosaccharide using edible insect, Tenebrio molitor (Tm). Chitin of Tm larvae was prepared using demineralization (2 N HCl, room temp., 3 h), deproteinization [5% NaOH (w/w) for 3.5 h at 70°C], and decolorization (3% H2O2 for 1.5 h at 80°C). Chitosan of Tm larvae was prepared by Tm chitin deacetylation at 50% NaOH (w/w) for 5 h at 105°C. The chitin of Tm yielded 50% chitosan, and the final extraction yield of chitin and chitosan from dried Tm larvae was 6.82% and 3.75%, respectively. Chitin‐oligosaccharide and chitosan‐oligosaccharide of Tm larvae were prepared via acid hydrolysis using 5.0 g of both Tm chitin and Tm chitosan. Finally, 2.12 g chitin‐oligosaccharide of Tm larvae was obtained through acid hydrolysis (con. HCl 100 mL for 4 h at 40°C). Chitosan‐oligosaccharide (2.5 g) of Tm larvae was produced via acid hydrolysis (con. HCl 50 mL for 1 h at 81°C) and purification. These results indicate that the industrial applicability of Tm chitin‐oligosaccharide and chitosan‐oligosaccharide is viable in the field of health supplements, as a large amount of these oligosaccharides can be obtained from edible insect, Tm larvae. [ABSTRACT FROM AUTHOR]

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 (Tm IKKε) 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. Tm IKKε contains a serine/threonine protein kinases catalytic domain. Phylogenetic analysis established the close homology of Tm IKKε to Tribolium castaneum IKKε (Tc IKKε) 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. [ABSTRACT FROM AUTHOR]

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 Tm Spz5 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 Tm tenecins, Tm attacins, Tm coleoptericins, Tm taumatin-like-proteins, and Tm cecropin-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 ds TmSpz5 -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 Tm Spz5 has a defined role in T. molitor innate immunity by regulating AMP expression in MTs in response to E. coli. [ABSTRACT FROM AUTHOR]

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 (Tm Spz1b) from RNA sequencing database of Tenebrio molitor. Tm Spz1b was bioinformatically analyzed, and functionally characterized for the antimicrobial function by RNA interference (RNAi). The 702 bp open reading frame of Tm Spz1b encoded a putative protein of 233 amino acid residues. A conserved cystine-knot domain with seven cysteine residues in Tm Spz1b 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 Tm Spz1b 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 Tm Spz1b 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. [ABSTRACT FROM AUTHOR]

The yellow mealworm beetle (Tenebrio molitor) has been exploited as an experimental model to unravel the intricacies of cellular and humoral immunity against pathogenic infections. Studies on this insect model have provided valuable insights into the phenotypic plasticity of immune defenses against parasites and pathogens. It has thus been possible to characterize the hemocoelic defenses of T. molitor that rely on the recognition of non-self-components of pathogens by pattern recognition receptors (PRRs). The subsequent signaling cascade activating pathways such as the NF-κB controlled by Toll and IMD pathways lead to the synthesis of antimicrobial peptides (AMPs), onset of hemocyte-driven phagocytosis, and activation of the prophenoloxidase cascade regulating the process of melanization. Nevertheless, the activation of autophagy-mediated defenses of T. molitor against the facultative intracellular gram-positive bacterium Listeria monocytogenes provides clear evidence of the existence of a cross-talk between autophagy and the IMD pathway. Moreover, the identification of several autophagy-related genes (Atgs) in T. molitor transcriptome and expressed sequence tag (EST) databases has contributed to the understanding of the autophagy-signaling cascade triggered by L. monocytogenes challenge. Providing further evidence of the cross-talk hypothesis, TmRelish has been shown to be required not only for regulating the synthesis of AMPs through the PGRP-LE/IMD pathway activation but also for the expression of Atgs in T. molitor larvae following L. monocytogenes challenge. Notably, L. monocytogenes can stimulate the T. molitor innate immune system by producing molecules recognized by the multifunctional PRR (Tm PGRP-LE), which stimulates intracellular activation of the IMD pathway and autophagy. Considering the conservation of autophagy components involved in combating intracellular pathogens, it will be interesting to extrapolate a dynamic cross-talk model of immune activation. This review summarizes the most significant findings on the regulation of autophagy in T. molitor during L. monocytogenes infection and on the role of the innate immunity machinery, including the NF-κB pathway, in the control of pathogenic load. [ABSTRACT FROM AUTHOR]

These contributions expand our understanding of the novel components of innate immunity signaling cascades, elucidation of physiological fitness traits and pathogen evasion mechanisms intricately attributed to invertebrate survival. With the lack of adaptive immunity, invertebrates rely on the plasticity conferred by innate immunity to eliminate pathogens, and perhaps more importantly discriminating them from other microorganisms. Keywords: innate immunity; Tenebrio molitor; immune priming; physiological plasticity; host-pathogen interaction EN innate immunity Tenebrio molitor immune priming physiological plasticity host-pathogen interaction 1 3 3 06/16/22 20220614 NES 220614 Innate immunity mechanisms have provided an effective means of surveillance and protection against parasites and pathogens in invertebrates. [Extracted from the article]

Antimicrobial peptides (AMPs) are considered to be candidate effectors for eliciting humoral immune responses against infectious pathogens in the host. Cecropins are α‐helical peptides of 30–40 amino acids, which are known to permeabilize bacterial membranes, and play authoritative roles in the innate immune system of insects. In the present study, we identified the full‐length open reading frame (ORF) encoding the Tenebrio molitor cecropin‐2 (TmCec2) gene using the Tribolium castaneum cecropin‐2 (TcCec2) gene to query a T. molitor Rnaseq database. Phylogenetic analysis revealed close identity of TmCec2 with TcCec2. TmCec2 was ubiquitously expressed in the insect during all developmental stages, with the highest expression observed in the adult. Tissue‐specific TmCec2 expression was highest in larval hemocytes and in the adult integument and hemocytes. Microbial challenge experiments revealed that TmCec2 was highly induced in response to gram‐positive and gram‐negative bacteria, and fungi. These data provide credible evidence for a putative role of TmCec2 in insect innate immunity against a plethora of pathogens. [ABSTRACT FROM AUTHOR]

Defensins are a major family of antimicrobial peptides that serve as the innate immune defense of both vertebrates and invertebrates. Due to their antimicrobial, chemotactic, and regulatory activities, Defensins have been exploited for their therapeutic potential. Insect Defensins are cysteine‐rich and contain an N‐terminal loop, α‐helix, and antiparallel β‐sheet, forming a "cysteine‐stabilized alpha beta (CSαβ)" or "loop–helix‐sheet" structure. In this study, we identified the full‐length open reading frame (ORF) sequences of Defensin (TmDef) and Defensin‐like (TmDef‐like) genes from the mealworm beetle Tenebrio molitor using in silico methods. TmDef and TmDef‐like genes encode the peptides of 72 and 71 amino acid residues, respectively. TmDefensin is comprised of a Defensin domain and the TmDefensin‐like is comprised of a signal peptide of 21 amino acid residues. Phylogenetic analysis revealed close similarities of TmDefensin with the Defensin of Acalolepta luxuriosa of the longhorn beetle family. The expression of TmDef mRNA was found to be greater than that of TmDef‐like mRNA and was mostly expressed in the pupal and adult stages. Tissue distribution showed high expression of TmDef‐like mRNA in larval hemocytes, gut, integument, and fat body, while in adults, the expression was high in gut and hemocytes. Following bacterial and fungal stimulation in vivo, TmDef was upregulated at 24 h post‐infection in whole body, fat body, and hemocytes of the larvae. Even TmDef‐like mRNA was upregulated in the gut and hemocytes at 12 and 9 h post‐infection respectively. These results suggest that TmDef and TmDef‐like genes play important roles in protecting T. molitor from microbial contact. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) are effector candidates that elicit humoral immunity in invertebrates. AMPs facilitate bacterial clearance by either physically disrupting the microbial membranes or the intracellular targets. In the Coleopteran pest, Tenebrio molitor, transcriptional regulation of AMPs has been studied in the context of innate immune signaling cascades and antimicrobial immunity. Here, we report the transcriptional response of three AMP genes, Coleoptericin A, B, and C (TmCole A, B and C) in T. molitor in response to bacterial (Escherichia coli, Staphylococcus aureus), and fungal (Candida albicans) challenges. These genes were expressed essentially in the gut and hemocytes followed by the integument tissue of the T. molitor larva. Further, these genes were highly expressed in the late‐larval, pupal, and early adult stages. Furthermore, while all of these transcripts were highly upregulated in the fat body and Malpighian tubules after bacterial challenge, TmCole B and TmCole C were induced in the gut after E. coli challenge. Fungal stimulation was not required for the upregulation of the transcription of Coleoptericin genes in T. molitor. [ABSTRACT FROM AUTHOR]

Due to the exposure of insects to various sources of pathogens during different stages of their life cycle and their vulnerability towards subsequent infections, moreover lack of morality issues, economical breeding and short‐term‐life cycle, insect's immunity has been considered as a high‐potential candidate to study underlying mechanisms of defense responses against all sort of invaders, as well as pandemic diseases. Currently, the world is enduring monumental pressure to meet global food production demands. One viable option would be to mass rear edible insects, such as coleopteran meal worm Tenebrio molitor, which is thought to be a substantial protein source. In addition, using antimicrobial peptides as an alternative for antibiotic source against multidrug‐resistant pathogens, makes insects a valuable option to solve this health issue. As a consequence, sufficient knowledge of insect immunity will lead us to reach advanced diagnostic and treatment technologies. To accomplish these goals, crucial importance of identification and functional characterization of the main signaling pathways, such as Toll and immune deficiency (IMD), prompted us to review the mechanisms of the signaling pathways involved in immune response in well‐known insect models. [ABSTRACT FROM AUTHOR]

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 (Tm PGRP-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 ds EGFP -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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: The purpose of this study was to investigate the production of chitin and chitosan from both the exuvium and whole body of mealworm (Tenebrio molitor) larvae. Chitin from the exuvium and whole body of T. molitor larvae was chemically extracted with acid and alkali solutions to achieve demineralization (DM) and deproteinization (DP), respectively. The average DM (%) and DP (%) on a dry weight (DW) basis was 32.56 and 73.16% from larval exuvium, and 41.68 and 91.53% from whole body, respectively. To obtain chitosan, chitin particles from the exuvium and whole body of T. molitor larva were heated at various temperatures in different concentrations of NaOH. Average chitin yields were 18.01% and 4.92% of DW from the exuvium and whole body, respectively. The relative average yield of chitosan from whole body was 3.65% of DW. On average, over 90% of chitosan derived from whole body was deacetylated. The viscosity of chitosan from whole body was ranged from 48.0 cP to 54.0 cP. The chitin content of dry and wet byproducts from whole body were 17.32% and 16.94% respectively, compared to dry weight. The chitosan contents of byproducts on a DW basis were 14.48% in dry and 13.07% in wet byproduct. These results indicate that the exuvium and whole body of T. molitor larva may serve as a source of chitin and chitosan for use in domestic animal feed. [ABSTRACT FROM AUTHOR]

Target of rapamycin ( TOR) is an evolutionarily conserved serine-threonine kinase that affects various cellular functions including growth, development, ageing, immunity and autophagy. Inhibition in TOR activity stimulates autophagy, a self-eating process that ensures survival by maintaining metabolic homeostasis and energy retention at both cellular and organismal levels. To understand the interplay between TOR and autophagy during development and immunity, we screened and identified a TOR homologue from the coleopteran insect, T enebrio molitor ( T m TOR). The full-length cDNA of TmTOR contained an open reading frame (ORF) of 7,197 bp encoding a protein of 2,398 amino acids, and a 5′- and 3′- untranslated region (UTR) of 156 and 457 bp, respectively. Deduced amino acid sequence of T m TOR shows characteristic Huntington, EF3A, ATM, TOR ( HEAT) repeat, focal adhesion kinase targeting ( FAT), rapamycin-binding, phosphatidylinositol 3-/4-kinase and FRAP, ATM and TRRAP C-terminal ( FATC) domains known to be conserved among TOR orthologs. T m TOR showed high sequence identity (92%) with T ribolium castaneum TOR ( T c TOR) and the two were placed in the same cluster of the phylogenetic tree. T m TOR shows high m RNA expression level in the fat body and integument of T . molitor larvae. Significantly, a reduced expression of T m TOR m RNA during pupation correlated to an increase in the area of autolysosomes. Intriguingly, T . molitor larvae showed an increase in Tm TOR transcripts upon a challenge with various bacterial pathogens. This suggests a negative role of Tm TOR in the regulation of autophagy during development. [ABSTRACT FROM AUTHOR]

Insect apolipophorin- III is an exchangeable protein that is abundantly found in the hemolymph, and serves an important role in lipid transport, development, and innate immunity. In this study, we examined the role of apolipophorin- III ( TmapoLp- III) during the adult eclosion stages of Tenebrio molitor by RNA interference ( RNAi) analysis. After silencing of the m RNA transcripts at both larval and pupal stages, adult phenotypic defects were noticed. Defects included the incomplete shedding of pupal skin, shorter extension of the elytra, and improper folding of the hind wings. Most of the adults were malformed and died possibly due to dehydration. We also showed the involvement of TmapoLp- III in conferring resistance to T. molitor larvae against Listeria monocytogenes infection. Mortality was found to be lower in non-silenced intoxicated larvae while the TmapoLp- III silenced larvae showed a significant susceptibility after 7 days post-injection with a dose of 106 cfu/larvae. [ABSTRACT FROM AUTHOR]

Tenebrio molitor has been seriously investigated as a model insect in elucidating Toll signaling pathway and related regulators of innate immunity. However, little is known with regards to the genomic information in T. molitor. In an attempt towards exploiting the rich transcriptomics data that would offer further insights into the study on insect immunity through potential screening of immune-related genes in the model insect, we constructed a cDNA library (library titer = 5.0 × 105pfu/ml) of T. molitor larvae and analyzed expressed sequence tag ( EST) sequences from 1056 clones. The base calling and quality check of obtained chromatogram files were performed by using the Phred program (trim_alt 0.05 ( P-score > 20). After removal of vector and 100 bp and less sequences, 1023 sequences were generated having an average insert size of 792 bp, including 160 clusters, 164 contigs and 387 singletons through clustering and assembling process using the TGI Clustering Tools ( TGICL) package. The unique EST sequences were searched against the NCBI nr database by local BLAST (blastx, E < e−5) with 940 sequences showing significant hits. Subsequently, KOG (clusters of orthologous groups for eukaryotic complete genomes) analysis was conducted (blastx, E < e−10) towards prediction of transcriptomal functions, leading to the categorization of 638 genes. The majority of genes belonged to Z category (cytoskeleton-related genes), R category (general function prediction), and C category (energy production and conversion related genes). These basic EST datasets and their bioinformatics analysis will be helpful in investigating the biological mechanism and molecular pathway related genes involved in innate immunity mechanisms of T. molitor. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 IKKβ 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 3- and 6-day-old pupae, while remarkable expression was also observed in other stages. 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. [ABSTRACT FROM AUTHOR]

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 Tm14-3-3ζ) was identified from the expressed sequence tags (ESTs) and RNA sequencing (RNA-Seq) database of the coleopteran pest, Tenebrio molitor. Tm14-3-3ζ 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 Tm14-3-3ζ, a peptide-based Tm14-3-3ζ polyclonal antibody was generated in rabbit and the specificity was confirmed using Western blot analysis. Immunostaining and confocal microscopic analyses indicate that Tm14-3-3ζ is mainly expressed in the membranes of midgut epithelial cells, the nuclei of fat body and the cytosol of hemocytes. Gene silencing of Tm14-3-3ζ increases mortality of the larvae at 7 days post-infection with E. coli and C. albicans. Our findings demonstrate that 14-3-3ζ in T. molitor is essential in the host defense mechanisms against bacteria and fungi. [ABSTRACT FROM AUTHOR]

Toll and IMD pathways regulate antimicrobial innate immune responses in insect model systems. The transcriptional activation of antimicrobial peptides (AMPs) confers humoral immunity in the host against invaded pathogens. The IKK kinase complex (IKKα, IKKβ, and the regulatory subunit IKKγ/NEMO) centrally regulates the NF-κB response to various stimuli. It triggers an appropriate antimicrobial immune response in the host. In this study, a Tm IKKβ (or Tm Ird5) homolog was screened from the RNA-seq database of the coleopteran beetle, Tenebrio molitor. A single exon characterizes the Tm IKKβ gene, and the open reading frame (ORF) comprises of 2112 bp that putatively encodes a polypeptide of 703 amino acid residues. Tm IKKβ contains a serine/threonine kinase domain and is phylogenetically close to Tribolium castaneum IKKβ homolog (Tc IKKβ). TmIKKβ transcripts were highly expressed in the early pupal (P1) and adult (A5) stages. Among the tissues, TmIKKβ showed higher expression in the integument of the last instar larvae and the fat body and hemocytes of 5-day-old adults. TmIKKβ mRNA was upregulated post- E. coli challenge to the host. Moreover, RNAi-based TmIKKβ mRNA silencing increased host larvae' susceptibility against E. coli, S. aureus and C. albicans. TmIKKβ RNAi in the fat body led to a downregulation in mRNA expression of ten out of fourteen AMP genes, including TmTenecin1, -2, and -4 ; TmDefensin, and -like ; TmColeoptericinA, and -B ; and TmAttacin1a, - 1b, and -2 , suggesting the requirement of the gene in antimicrobial innate immune responses. Further, a decrease in the mRNA expression of NF-κB factors such as TmRelish, TmDorsal1 , and TmDorsal2 in the fat body of T. molitor larvae was observed post-microorganisms challenge. Thus, Tm IKKβ regulates antimicrobial innate immune responses in T. molitor. • We have newly identified IκB kinase β gene from the Tenebrio molitor , named TmIKKβ and domain analysis indicated that it contains one Serine/Threonine protein kinase catalytic domain, suggesting that TmIKKβ may regulate cell signaling by phosphorylation. • The temporal and spatial expression patterns of TmIKKβ transcripts and its induction patterns in responses to microbial challenges indicated that the TmIKKβ may have important roles in development as well as immune responses against microbial challenges. • We have investigated the effects of TmIKKβ RNAi on larval survivability, and expression patterns of antimicrobial peptide (AMP) genes and transcription factors for Toll and Imd pathways to understand the critical function of TmIKKβ in antimicrobial immune responses. [ABSTRACT FROM AUTHOR]

A bstract Scavenger receptors (SRs) constitute a family of membrane-bound receptors that bind to multiple ligands. The SR family of proteins is involved in removing cellular debris, oxidized low-density lipoproteins, and pathogens. Specifically, class C scavenger receptors (SR-C) have also been reported to be involved in phagocytosis of gram-positive and -negative bacteria in Drosophila and viruses in shrimp. However, reports are unavailable regarding the role of SR-C in antifungal immune mechanisms in insects. In this study, a full-length Tenebrio molitor SR-C ( Tm SR-C) sequence was obtained by 5′- and 3′-Rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The TmSR-C full-length cDNA comprised 1671 bp with 5′- and 3′-untranslated regions of 23- and 107-bp, respectively. Tm SR-C encodes a putative protein of 556 amino acid residues that is constitutively expressed in all tissues of late instar larvae and 2-day-old adults, with the highest transcript levels observed in hemocytes of larvae and adults. TmSR-C mRNA showed a 2.5-fold and 3-fold increase at 24 and 6 h after infection with Candida albicans and β-glucan, respectively. Immunoassay with Tm SR-C polyclonal antibody showed induction of the putative protein in the cytosols of hemocytes at 3 h after inoculation of C. albicans . RNA interference (RNAi)-based gene silencing and phagocytosis assays were used to understand the role of Tm SR-C in antifungal immunity. Silencing of TmSR-C transcripts reduced the survivability of late instar larvae at 2 days post-inoculation of C. albicans , Escherichia coli , or Staphylococcus aureus . Furthermore, in TmSR-C -silenced larvae, there was a decline in the rate of microorganism phagocytosis. Taken together, results of this study suggest that Tm SR-C plays a pivotal role in phagocytosing not only fungi but also gram-negative and -positive bacteria in T. molitor . [ABSTRACT FROM AUTHOR]

Macroautophagy (hereinafter called autophagy) is a highly regulated process used by eukaryotic cells to digest portions of the cytoplasm that remodels and recycles nutrients and disposes of unwanted cytoplasmic constituents. Currently 36 autophagy-related genes ( ATG ) and their homologs have been characterized in yeast and higher eukaryotes, including insects. In the present study, we identified and functionally characterized the immune function of an ATG8 homolog in a coleopteran insect, Tenebrio molitor ( TmATG8 ). The cDNA of TmATG8 comprises of an ORF of 363 bp that encodes a protein of 120 amino acid residues. TmATG8 transcripts are detected in all the developmental stages analyzed. TmAtg8 protein contains a highly conserved C-terminal glycine residue (Gly116) and shows high amino acid sequence identity (98%) to its Tribolium castaneum homolog, TcAtg8. Loss of function of TmATG8 by RNAi led to a significant increase in the mortality rates of T. molitor larvae against Listeria monocytogenes . Unlike ds EGFP -treated control larvae, TmATG8- silenced larvae failed to turn-on autophagy in hemocytes after injection with L. monocytogenes . These data suggest that TmATG8 play a role in mediating autophagy-based clearance of Listeria in T. molitor . [ABSTRACT FROM AUTHOR]

Myeloid differentiation factor 88 (MyD88), an intracellular adaptor protein involved in Toll/Toll-like receptor (TLR) signal processing, triggers activation of nuclear factor-kappaB (NF-κB) transcription factors. In the present study, we analyzed the gene structure and biological function of MyD88 in a coleopteran insect, Tenebrio molitor (TmMyD88). The TmMyD88 gene was 1380 bp in length and consisted of five exons and four introns. The 5′-flanking sequence revealed several putative transcription factor binding sites, such as STAT-4, AP-1, cJun, cfos, NF-1 and many heat shock factor binding elements. The cDNA contained a typical death domain, a conservative Toll-like interleukin-1 receptor (TIR) domain, and a C-terminal extension (CTE). The TmMyD88 TIR domain showed three significantly conserved motifs for interacting with the TIR domain of TLRs. TmMyD88 was grouped within the invertebrate cluster of the phylogenetic tree and shared 75% sequence identity with the TIR domain of Tribolium castaneum MyD88. Homology modeling of the TmMyD88 TIR domain revealed five parallel β-strands surrounded by five α-helices that adopted loop conformations to function as an adaptor. TmMyD88 expression was upregulated 7.3- and 4.79-fold after 12 and 6 h, respectively, of challenge with Staphylococcus aureus and fungal β-1,3 glucan. Silencing of the TmMyD88 transcript by RNA interference led to reduced resistance of the host to infection by S. aureus . These results indicate that TmMyD88 is required for survival against Staphylococcus infection. [ABSTRACT FROM AUTHOR]

Abstract: Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5′-flanking region. BLAST and phylogenetic analyses reveal that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic alpha helices, including a short helix 3′. The ‘helix–short helix–helix’ motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly upregulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor. [Copyright &y& Elsevier]

Highlights: [•] A novel ricin-type (R-type) lectin was identified from T. molitor. [•] The cDNA sequence consists of 495bp encoding 164 amino acid residues. [•] Domain analysis shows that there is a ricin-type beta-trefoil structure. [•] The transcript was upregulated by peptidoglycans, beta 1, 3 glucan, and Listeria. [Copyright &y& Elsevier]

Vertebrates rely on the most sophisticated adaptive immunity to defend themselves against various pathogens. This includes immunologic memory cells, which mount a stronger and more effective immune response against an antigen after its first encounter. Unlike vertebrates, invertebrates' defense completely depends on the innate immunity mechanisms including humoral and cell-mediated immunity. Furthermore, the invertebrate equivalent of the memory cells was discovered only recently. Since the discovery of transgenerational immune priming (TGIP) in crustaceans, numerous findings have proven the IP in invertebrate classes such as insects. TGIP can be induced through maternal priming pathways such as transcriptional regulation of antimicrobial peptides, and also paternal IP including the induction of proPO system activity. We appraise the diversity and specificity of IP agents to provide sustained immunologic memory in insects, particularly T. molitor in the review. An understanding of IP (more so TGIP) response in T. molitor will deepen our knowledge of invertebrate immunity, and boost the mass-rearing industry by reducing pathogen infection rates. • Unlike mammals, solo arm of defense mechanism in invertebrates is innate immunity, particularly, antimicrobial peptide production. • The ability of being immunized against a preexisting elicitor, was a matter of uncertainty until a wile ago. • This phenomenon known as "immune priming", can be transferred to progenies by both parents. • The diversity and specificity of immune priming in insects more precisely, Tenebrio molitor has been investigated in numbers of studies. • Understanding of immune priming in insects can provide us with novel strategies and applications at the field during T. molitor mass rearing. [ABSTRACT FROM AUTHOR]