Introduction

The yellow mealworm, Tenebrio molitor (T. molitor) is a well-known insect not only because of its high nutritional values but also as a crop pest1,2. T. molitor exposure to different pathogenic factors in their natural inhabitants has made them a great immune study model3,4,5. Considering T. molitor industrial importance, their relatively larger size, and their convenient rearing condition, it is important to discuss disease resistance defense mechanisms related to the development and health of mealworms6,7. Like other insects, yellow mealworms have unique physical and physiological mechanisms to prevent foreign invaders or reduce infection3. Among all the shields, T. molitor uses its innate immune arms to produces antimicrobial peptides (AMPs) through a specific mechanism8,9,10,11,12. Insects’ innate immunity perceive Lipopolysaccharide (LPS) and Peptidoglycan (PGN) from various microbial sources via their pattern recognition receptors (PRRs)13,14. Functional immune response is divided into cellular and humoral immunity15. Cellular immunity includes blood cell-mediated immune responses including phagocytosis, nodulation, and encapsulation16, whereas humoral immunity involves Toll, immune deficiency (Imd), and the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways triggering immune related gene expression including but not limited to AMPs production5,17,18,19,20,21. While in Drosophila both Toll and Imd pathways are important, Imd pathway regulates most of the AMPs expression18. In Drosophila, peptidoglycan recognition proteins (PGRPs) which recognize bacterial cell wall compartments can be divided into PGRP-SA, PGRP-LC, and PGRP-LE22. Lys-type PGN, a cell wall component of Gram-positive bacteria, is recognized by PGRP-SA23, activates the Toll pathway, and induce AMPs production. On the other hand, DAP-type PGN, a cell wall component of Gram-negative bacteria, is recognized by PGRP-LC and LE24 and forms a complex, which activates the Imd pathway and induces an immune response through a complex pathway leading to AMP production.

The Imd pathway is activated by forming a complex containing Fas-associated death domain protein (FADD) and death-related ced-3/Nedd2-like protein (DREDD). DREDD is a caspase that cleaves the Imd protein and creates a new binding site for Inhibitor of apoptosis-2 (IAP-2). Then, the E2-ubiquitin complex binds to the IAP-2 binding site and forms a Transforming growth factor-β (TGF-β) activated kinase (TAK1)/TAK1 binding protein2 (TAB2) complex. The TAK1/TAB2 complex then induces phosphorylation of the inhibitor of Nuclear factor-κB kinase (IKK) complex to activate Relish. Subsequently, activated Relish mediates transcription of the AMP genes5. Studies on Drosophila TAK1 mutations revealed that TAK1 plays role in Gram-negative bacteria infection by activating IKK complex25.

The Imd pathway is often compared to mammalian tumor necrosis factor (TNF) signaling26. Essentially, activation of the Imd pathway and TNF-α target genes require activation of the transcription factors Relish and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) respectively26. In mammalian NF-κB signaling, activation of transcription factors occurs through a pathway in which the Inhibitor of NF-κB (IκB) kinase complex phosphorylates IκB and the phosphorylated IκB is degraded to activate NF-κB27. It has been demonstrated that TAK1 is required for activation of NF-κB following TNF or Interleukin-1 (IL-1) stimulation in mammals28,29,30.

TAK1 is a kinase that can be activated by TGF-β and was first discovered in 199531. It has also been found to be an important mediator of inflammatory responses activated by LPS, TNF-α, and IL-130,32,33, and it plays an important role in immune responses in mammals and insects.

Until recently, most studies on the Imd pathway were confined to Drosophila34 and few similar studies have been conducted in other insect models. Here, we investigated immunological roles of TmTak1, an understudied compartment in Imd pathway involves in humoral immunity, in T. molitor.

Result

In silico analysis of TmTAK1

As a result of homology mapping of the TmTak1 cDNA sequence based on the T. molitor DNA-seq database, 8 exons intervening with 7 introns were identified (Supplementary Fig. 1). Among the 8 exons of TmTak1, exon 2 was the longest with 330 sequences, followed by exon 4 with 249 nucleotide sequences and exon 8 with 220 nucleotide sequences. Among the 7 introns, intron 6 was longer than the other introns, so the size of the introns was not uniform. In addition, the coding sequence consisted of nucleotides of 1527 bp and coded 508 amino acid residues. The full-length coding sequence of TmTak1 was submitted to GenBank, accession number OR373077 has been granted. As a result of protein domain analysis of TmTAK1, it was confirmed that the protein tyrosine kinase domain from amino acid residues no. 24 to 256 and four tyrosine kinase catalytic domains were located at no. 93 to 106, no. 134 to 152, no. 179 to 189, and no. 247 to 269 residues (Supplementary Fig. 2). TAK1 orthologues and Multiple Sequence Alignment (MSA) of insect was analyzed using ClustalX v2.0. Through the MSA results, it was confirmed that protein tyrosine kinase and tyrosine kinase catalytic domains in the TmTAK1 amino acid sequence is well conserved in other insects (Supplementary Fig. 3). The phylogenetics of TmTAK1 were analyzed using TAK1 homologues from other insect species (Supplementary Fig. 4). TmTAK1 clustered with TcMKKK7 which has 85% sequence identity with TmTAK1 and was isolated under the same cluster as other coleopteran species.

Developmental and tissue-specific expression patterns of TmTak1

During the development of T. molitor, the expression pattern of TmTak1 was investigated by qPCR. As a result, the expression level of TmTak1 was highest in young larvae (YL) and lowest in 7-day old pupae (P7) (Fig. 1A). As a result of examining the tissue-specific expression pattern of TmTak1 in T. molitor, high expression level of TmTak1 was confirmed in the gut (GT) of the larval stage and low expression level was confirmed in the integument (INT) (Fig. 1B). Next, in the adult stage, the expression level of TmTak1 was the highest in the fat bodies (FBs), and low expression levels were confirmed in the other tissues (Fig. 1C).

Figure 1
figure 1

Relative expression levels of TmTak1 mRNA in developmental stages and tissues of T. molitor. (A) Expression levels of TmTak1 in T. molitor at the egg, the young larvae (YL), late larvae (LL), pre-pupae (PP), 1–7-day-old pupae (P1–7), and 1–5-day-old adult (A1–5) stages. Distribution of TmTak1 transcripts in larva and adult tissues. Fat bodies (FB), gut (GT), hemocytes (HC), integument (INT), and Malpighian tubules (MT) of (B) late instar larvae and (C) adults, in addition to ovaries (OV) and testes (TT) of adults, were dissected and collected from 20 late larvae and 5-day-old adults for analysis. T. molitor 60S ribosomal protein L27a (TmL27a)-encoding gene was used as an internal control.

Full size image

TmTak1 mRNA expression level after microbial challenge

T. molitor larvae were infected with PBS, E. coli, S. aureus, and C. albicans, and after 3 h, 6 h, 9 h, 12 h, and 24 h, WB and five tissues (GT, FBs, MTs, INT, and HCs) were dissected (Fig. 2). TmL27a was used as a house keeping gene, and the relative expression level of TmTak1 was investigated using qPCR using PBS as a control. As a result of examining the expression level, high expression levels specifically for C. albicans were confirmed in the gut, fat bodies, Malpighian tubules, integument, and hemocytes compared to E. coli in whole body. In particular, the expression level of TmTak1 was relatively higher in MTs than other tissues against C. albicans infection (Fig. 2E).

Figure 2
figure 2

TmTak1 mRNA expression profiles after microbial challenge. Expression of TmTak1 mRNA in the whole body (A), hemocytes (B), gut (C), fat bodies (D), Malpighian tubules (E), integument (F) of larvae infected with Escherichia coli, Staphylococcus aureus, and Candida albicans. The expression was analyzed by qPCR using L27a (T. molitor) as the internal control. For each time point, the expression level in the phosphate-buffered saline (PBS)-injected control (mock control) was set to 1; this is represented by a dotted line. vertical bars represent mean ± standard error from three biological replicates.

Full size image

TmTak1 knockdown increased the mortality of E. coli-injected T. molitor larvae

The mortality was investigated after RNAi using dsTmTak1 in T. molitor (Fig. 3A). The concentration of bacteria used in the experiment was 1 × 107 CFU/μl for E. coli and S. aureus, and 5 × 105 CFU/μl l for C. albicans, and n = 10 were repeated three times. It was confirmed up to the 10th day after infection with the microorganisms (Fig. 3B–D). There was no significant difference between S. aureus and C. albicans, but after E. coli infection, the survival rate of the group injected with dsTmTak1, the experimental group, was reduced to about 10% on the first day and 35% on the second day, compared to the control group injected with dsEGFP (Fig. 3B).

Figure 3
figure 3

Effect of TmTak1 RNAi on T. molitor Larval Survival. (A) TmTAK1 knockdown efficiency measured using RT-qPCR at day 5 post-injection. The survivability of larva was measured after TmTak1 knockdown and infection with Escherichia coli (B), Staphylococcus aureus (C), or Candida albicans (D) (n = 30). Data are presented as average of three biologically independent replicate experiments. Asterisks indicate significant differences between TmTak1-knocked down and dsEGFP-treated groups (p < 0.05). Survival rates were analyzed based on the Kaplan–Meier plots (log-rank chi-square test; *p < 0.05).

Full size image

Effects of TmTak1 RNAi on AMP expression

After knockdown of TmTak1 using dsTmTak1, the amount of AMP expression was examined 24 h after systemic infection in each. GT, INT, MTs, and HCs, were the major tissues in which AMP expression was reduced after gene silencing of TmTak1.

In the GT, mRNA expression of ten AMPs including TmTene1, 2, 4, TmDef-like, TmColeA, B, C, TmAtta1a, 1b, and 2 were significantly decreased by TmTak1 knockdown after E. coli (Fig. 4). TmTene2 and TmAtta1b were the AMPs most affected by TmTak1 gene silencing in the GT (Fig. 4B, L). In the INT, although the number of decreased AMP expression is smaller than that of the three tissues mentioned above, four AMPs showed most significantly reduced mRNA expression are including TmTene2, TmColeB, C, and TmAtta1a were the AMPs most affected by TmTak1 gene silencing (Fig. 5). In the MTs, mRNA expression of ten AMPs including TmTene2, 4, TmDef, TmColeA, B, C, TmAtta1a, 1b, 2, and TmTLP1 were significantly decreased by TmTak1 knockdown after E. coli (Fig. 6). Six AMPs showed most significantly reduced mRNA expression in the MTs are including TmTene2, 4, TmDef, TmColeB, TmAtta1b and 2 were the AMPs most affected by TmTak1 gene silencing (Fig. 6B, D, E, I, L, M). In the HCs, mRNA expression of twelve AMPs including TmTene1, 2, 4, TmDef, TmDef-like, TmColeA, B, C, TmAtta1a, 1b, 2, and TmTLP1 were significantly decreased by TmTak1 knockdown after E. coli (Fig. 7). TmTene1, TmDef and TmAtta2 were the AMPs most affected by TmTak1 gene silencing in the HCs (Fig. 7E, M).

Figure 4
figure 4

AMPs mRNA expression levels in the gut against E. coli, S. aureus, and C. albicans after TmTak1 knockdown. To evaluate the functional properties of TmTak1 in regulating AMP genes expression in response to pathogens, we used dsTmTak1, the amount of AMP expression was examined 24 h after systemic infection. AMP genes, including TmTene1 (A), TmTene2 (B), TmTene3 (C), TmTene4 (D), TmDef (E), TmDef-like (F), TmCec2 (G), TmColeA (H), TmColeB (I), TmColeC (J), TmAtt1a (K), TmAtt1b (L), TmAtt2 (M), TmTLP1 (N), and TmTLP2 (O).

Full size image
Figure 5
figure 5

AMPs mRNA expression levels in the integument against E. coli, S. aureus, and C. albicans after TmTak1 knockdown. To evaluate the functional properties of TmTak1 in regulating AMP genes expression in response to pathogens, we used dsTmTak1, the amount of AMP expression was examined 24 h after systemic infection. AMP genes, including TmTene1 (A), TmTene2 (B), TmTene3 (C), TmTene4 (D), TmDef (E), TmDef-like (F), TmCec2 (G), TmColeA (H), TmColeB (I), TmColeC (J), TmAtt1a (K), TmAtt1b (L), TmAtt2 (M), TmTLP1 (N), and TmTLP2 (O).

Full size image
Figure 6
figure 6

AMPs mRNA expression levels in the Malpighian tubules against E. coli, S. aureus, and C. albicans after TmTak1 knockdown. To evaluate the functional properties of TmTak1 in regulating AMP genes expression in response to pathogens, we used dsTmTak1, the amount of AMP expression was examined 24 h after systemic infection. AMP genes, including TmTene1 (A), TmTene2 (B), TmTene3 (C), TmTene4 (D), TmDef (E), TmDef-like (F), TmCec2 (G), TmColeA (H), TmColeB (I), TmColeC (J), TmAtt1a (K), TmAtt1b (L), TmAtt2 (M), TmTLP1 (N), and TmTLP2 (O).

Full size image
Figure 7
figure 7

AMPs mRNA expression levels in the hemocytes against E. coli, S. aureus, and C. albicans after TmTak1 knockdown. To evaluate the functional properties of TmTak1 in regulating AMP genes expression in response to pathogens, we used dsTmTak1, the amount of AMP expression was examined 24 h after systemic infection. AMP genes, including TmTene1 (A), TmTene2 (B), TmTene3 (C), TmTene4 (D), TmDef (E), TmDef-like (F), TmCec2 (G), TmColeA (H), TmColeB (I), TmColeC (J), TmAtt1a (K), TmAtt1b (L), TmAtt2 (M), TmTLP1 (N), and TmTLP2 (O).

Full size image

Effects of TmTak1 RNAi on the expression of Tenebrio NF-κB genes

The expression of T. molitor NF-κB genes implicated in the IMD (TmRelish), JNK (TmKayak), and Toll (TmDorX1 and TmDorX2) pathways were studied in TmTak1-silenced individuals upon microbial challenge (Fig. 8). The study was conducted to address the antimicrobial activity of TmTak1. The mRNA expression of TmRel and TmKay transcripts were significantly decreased in the MTs, GT, and HCs of TmTak1-silenced T. molitor in a descending manner. Conversely, relevant expression of TmRel upregulated in the TmTak1 silenced INT and FBs, suggesting involvement of TmTak1 in the canonical pathway of AMP production which effects TmRel mRNA expression MTs, GT, and HCs. Furthermore, the relevant downregulation of TmRel and TmKay mRNA expression was more significant after E. coli challenge compared to S. aureus and C. albicans challenge. Regarding the TmDorX1 and TmDorX2 identical result was reported, while the most significant depletion of transcription factor expression was related to TmDorX2 following E. coli challenge in the GT of silenced larvae. Accordingly, these results justified the significant mortality of the T. molitor larvae following E. coli challenge.

Figure 8
figure 8

Effects of TmTak1 RNAi on NF-κB genes. To investigated the effect of dsTmTak1 on the expression of NF-κB genes, including TmRelish (A), TmKayak (B), TmDorX1 (C), and TmDorX2 (D). Gut (GT), Malpighian tubules (MT), hemocytes (HC), and integument (INT) were dissected and collected from 20 early larvae for analysis. T. molitor 60S ribosomal protein L27a (TmL27a)-encoding gene was used as an internal control.

Full size image

Discussion

The innate immune system is the first line of defense against invading pathogens and is comprised of a variety of cellular and humoral components35. In terms of immunological effects, TAK1 has been shown to play a critical role in the regulation of the innate immune response in insects20,25,36,37,38. In this study, we found that TAK1 might be as regulator Imd and JNK signaling pathway which response to pathogenic infection such as E. coli.

TAK1 is a serine/threonine kinase that is involved in a wide range of signaling pathways in various organisms, including mammalian and insects39,40,41. In mammalian, TAK1 is complexed with adaptor proteins TAB1, TAB2 and TAB3. TAB1 interact with TAK1 and triggers autophosphorylation of TAK1, which is essential for TAK1 kinase activity. TAB2 and TAB3 are involved in complex structure and share 48% of both amino acid sequence. TAB1 binds to the kinase domain in N-terminus of TAK1, and TAB2 and TAB3 bind to the C-terminal region of TAK142. Complex between the kinase domain of TAK1 and the C-terminal TAB1 stimulates phosphorylation-dependent TAK1 activation43. Herein, we found TmTAK1 includes the protein tyrosine kinase domain from amino acid residues no. 24 to 256 and four tyrosine kinase catalytic domains were located at no. 93 to 106, no. 134 to 152, no. 179 to 189, and no. 247 to 269 residues (Supplementary Fig. 2). Further, the sequence alignment results revealed the presence of homologs of TAK1 in many insect species, this study assigned Odonata, Isoptera, Hemiptera, Hymenoptera, Coleoptera, Lepidoptera, and Diptera (Supplementary Fig. 3) and phylogenetic characteristics of TmTAK1 disclose adjacent association within the Coleoptera (Supplementary Fig. 4).

In this study, mRNA of TmTak1 was expressed in all developmental stage and the highest level of expression was detected in Egg and YL stages (Fig. 1A). In Xenopus embryos, xTAK1 and xTAB1 is relative with ventral mesoderm development. The ventralization caused by constitutively active the bone morphogenetic protein receptor IA (BMPR-IA) and Smad1/5 is reversed by the kinase-negative form of xTAK1. Ectopic expression of xTak1 led cell death in early embryos44. It must be borne in mind that previous studies in Drosophila and silkworm demonstrated that during the developmental stages and metamorphosis further to pathogenic infections 20-hydroxyecdysone (20E), one of the two most important developmental hormones, improves the insect innate immune response, via upregulation of the PGRP-LCs45,46. Accordingly, our findings also support the solid interactions of developmental and immune system compartments.

TmTak1 is required for activation of both Imd and JNK NF-κB after three type of pathogen such as Gram negative-, Gram positive-bacteria, and Fungi infection (Fig. 2). In Drosophila, PGN stimulation triggered TAK1 activation in both NF-κB and JNK mitogen-activated protein kinase (MAPK) signaling25,37,47,48. In Drosophila infected with Gram-negative bacteria, negative regulation of JNK signaling was demonstrated to be associated with proteasomal degradation of TAK136. In crustacean, TAK1 activates the Imd and JNK pathways in response to Gram negative infections49. The endogenous TAK1 is phosphorylated in LPS-stimulated RAW 264.7 cells50. In mammalian, TAK1 is critical mediator of the antifungal immune response and a key component of the signaling cascade downstream of the c-type lectin receptor51.

The pathogen-derived metabolite, phenazine-1-carboxamide, has recently been discovered to be recognized by nuclear hormone receptors and to induce anti-pathogen defense in C. elegans52. Because a live microorganism was employed in this study, it is possible that the bacterial metabolite activates a non-canonical immunological pathway. Moreover, it has be reported repeatedly that various bacterial metabolite compositions which differ in distinct bacterial species including but not limited to E. coli might sequentially and cooperatively interfere with humoral and cellular defense response53,54,55. While these secondary metabolites might highly effect immune related signaling pathways via induction of host immunosuppression little is known about the relevant interaction. Hence, more research is needed to clarify the effect of bacterial metabolites on the immune system of T. molitor.

In T. molitor, TAK1 has been shown to be involved in the regulation of immune-related processes. For example, TAK1 activation has been shown to induce the expression of AMPs in T. molitor larvae. After infection with bacterial and fungal pathogens, the highest expression levels of TmTak1 were observed specifically in response to C. albicans infection in multiple tissues including the gut, fat bodies, Malpighian tubules, integument, and hemocytes (Fig. 2). In mosquito, two entomopathogenic fungi Beauveria bassiana and Isaria javanica trigger Imd pathway component and induce AMPs in the gut and fat bodies56. In mice, fungal infection induce JNK1 which negatively controls innate immune response by suppressing CD23 expression57.

Like Drosophila, AMP production is regulated by Dorsal and Relish, which are transcription factors of Toll and Imd signaling pathways58,59,60. Moreover, dTAK1 is a factor involved in the JNK pathway36. JNK pathway is one of the main conserved signaling branch of the MAPK signaling pathway61,62. Kayak is one of the components of AP-1 transcription factor complex63. The sequences of TmKayak were retrieved from the T. molitor RNAseq and expressed sequence tag (EST) databases using local-tblastn analysis (unpublished). After gene silencing of TmTak1, the mRNA expression pattern of AMPs was significantly decreased by pathogen infection, proving that TmTAK1 plays a critical role in AMP production. In order to confirm which transcription factors caused the absence of AMP expression due to gene silencing of TmTak1, the expression levels of Toll, Imd, and JNK former factors were examined. Despite systemic infection with E. coli and S. aureus, TmRel and TmKay were significantly decreased after TmTak1 silencing in the MTs proposing critical roles of TmRel and TmKay in AMP induction. According to our finding, Gram-negative bacteria infection in shrimp revealed that TAK1 activated AMP production via c-jun and Relish49.

Knockdown of TmTak1 using RNA interference resulted in increased mortality in T. molitor larvae following E. coli infection (Fig. 3B). TAK1 activates downstream kinase the inhibitor of NF-κB (IκB) kinase (IKK) complex64. Several studies have been conducted on immunological roles of IKK complex components65,66,67. For instance it has been shown that similar to TmTak1, knock-down of TmIKKε causes higher mortality rate post E. coli systemic infection67. Moreover, TmIKKβ and TmIKKγ/NEMO are critical immune factor for T. molitor survival following E. coli, S. aureus and C. albicans infections65,66.

Our observations bring out the importance of reaching a better perspective over TmTAK1 regulatory acts on each IKK components independently and IKKs complex. In addition, the direct action of TmTAK1 on components of the JNK pathway remains to be elucidated. This study suggests that TmTAK1 plays a role in the innate immune response of T. molitor to Gram negative infection and also triggers Imd and JNK pathway.

Materials and method

Insect rearing

Insects were reared in a plastic container measuring 29 cm in width, 21 cm in length, and 9.5 cm in height, and wheat bran was used as food. The mealworms used in the experiment were separately fed to an artificial diet (170 g wheat flour, 20 g roasted soy flour, 10 g protein, 100 g wheat bran, 0.5 g sorbic acid, 0.5 mL propionic acid, and 0.5 g chloramphenicol in 200 mL of distilled water) and sterilized water was supplied once a day to maintain the temperature of 26 °C ± 1 °C and humidity of 60% ± 5% in an incubator. The larvae used in all experiments except for the developmental experiment were 10–12 years old (1.34–1.88  cm), and the adult was 5 days old after eclosion.

Identification and in silico characterization

The sequences of TmTak1 were retrieved from the T. molitor RNAseq (unpublished) and expressed sequence tag (EST) databases using local-tblastn analysis. For the retrieval, the amino acid sequences of Tribolium castanuem TAK1 (TcTAK1) (XP_968547. 1) were used as a query. For TmTak1 gene architecture, local-tblastn analysis was performed with TmTak1 nucleotide sequence as query against the T. molitor DNASeq database (unpublished) as the subject. Conserved domains were identified using InterProScan (https://www.ebi.ac.uk/interpro/search/sequence-search) and blastp (https://blast.ncbi.nlm.nih.gov/Blast.cgi) programs. cDNA translation and predictions of the deduced protein were analyzed using BioPHP minitools software (http://www.biophp.org/minitools/dna_to_protein/demo.php). FGENESH eukaryotic gene prediction was used to predict TmTak1 open reading frame (ORF) region (http://www.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=gfind). The complete cDNA sequence was formatted using UltraEdit64-bit text editor (http://www.ultraedit.com). For the reverse complement of the nucleotide sequence, ExPASy Translate tool was used (https://web.expasy.org/translate/). Multiple sequence alignment was performed with representative TAK1 protein sequences from other insects retrieved from Genbank (https://www.ncbi.nlm.nih.gov/) using Clustal X268. The percentage identity and phylogenetic analysis were conducted using Clustal X2 and MEGA 769. The Maximum likelihood approach was used to investigate phylogenetic relationships using the Jones–Taylor–Thorton model, with a bootstrap consensus of 1000 replications. The following protein sequences were used in the multiple sequence alignment and phylogenetic analysis: IeMKKK7-like-isoX2, Ischnura elegans MKKK7-like-isoX2 (XP_046405324. 1); IeMKKK7-like-isoX1, Ischnura elegans MKKK7-like-isoX1 (XP_046405323. 1); CsMKKK7-isoX2, Cryptotermes secundus MKKK7-isoX2 (XP_023703195. 1); ZnMKKK7-isoX1, Zootermopsis nevadensis (XP_021932762. 1); DvMKKK7-like, Daktulosphaira virifoliae MKKK7-like (XP_050524364. 1); SfMKKK7-like, Sipha flava MKKK7-like (XP_025413188. 1); AcMKKK7-like, Aphis craccivora MKKK7-like (KAF0770127. 1); FvMKKK7-like, Frieseomelitta varia MKKK7-like (XP_043516360. 1); VcMKKK7-like, Venturia canescens MKKK7-like (XP_043281270. 1); VcMKKK7-like, Venturia canescens MKKK7-like (XP_043281270. 1); BkMKKK7-like, Belonocnema kinseyi MKKK7-like (XP_033208032. 1); ArMKKK7-like, Athalia rosae MKKK7-like (XP_012253187. 2); TmTAK1, Tenebrio molitor TAK1 (OR_373077); TcMKKK7, Tribolium castaneum MKKK7 (XP_968547. 1); PpMKK7-like, Photinus pyralis MKKK7-like (XP_031330485. 1); ApMKKK7, Agrilus planipennis MKKK7 (XP_018333073. 1); HkMKK7-like, Hyposmocoma kahamanoa MKKK7-like (XP_026321167. 1); BmMKKK7, Bombyx mori MKKK7 (XP_021207294. 1); HzMKKK7, Helicoverpa zea MKKK7 (XP_047031840. 1); HaMKKK7, Helicoverpa armigera MKKK7 (XP_021198433. 2); DmTAK1-like1, Drosophila melanogaster TAK1-like1 (NP_732554. 1); DmTAK1, Drosophila melanogaster TAK1 (NP_524080. 1); DmTAK1-like2, Drosophila melanogaster TAK1-like2 (NP_651090. 2); PvMKKK7-like, Penaeus vannamei MKKK7-like (XP_027228781. 1).

Preparation of microorganisms

To investigate the immune function of TmTAK1 against infection, we used three microorganisms including Escherichia coli K12, Staphylococcus aureus RN4220 and Candida albicans AUMC 13,529. E. coil and S. aureus were cultured in Luria–Bertani (LB) broth and C. albicans in Sabouraud Dextrose (SD) broth at 37 °C for 16 h. Then, the microorganisms were suspended by centrifugation at 3500 rpm for 10 min, washed with phosphate buffered saline (PBS, pH 7.0), and the concentration was measured at OD600. Finally, 1 × 106 cells/μl of E. coli and S. aureus and 5 × 104 cells/μl of C. albicans were used in all infection experiment, and only in the survivability assay, they were used in increments of 10 times more units.

Total RNA extraction, cDNA synthesis and expression of TmTak1 mRNA

RNA extraction, cDNA synthesis, and quantitative real-time PCR (qPCR) were commonly performed to investigate the mRNA expression level of TmTak1 in developmental stages (n = 20) of T. molitor and each tissue (n = 20) and against microorganism. Total RNA extraction was performed according to the manual of RNA extraction kit (Invirustech, Gwangju, South Korea). The extracted RNA was quantified to 1 μg and synthesized into cDNA using AccuPower® RT PreMix (Bioneer, Daejeon, South Korea) and Oligo (dT)12–18 primer on a MyGenie 96 thermal block (Bioneer). Then, to investigated the mRNA expression level of TmTak1, AccuPower® 2X GreenStar™ qPCR Master Mix (Bioneer), synthesized cDNA and specific primers (TmTAK1_qPCR_Fw, TmTAK1_qPCR_Rv, Supplementary Table 1) were used for qPCR was performed under conditions of initial denaturation of 95 °C for 5 min, followed by 40 cycle at 95 °C for 15 s, and 60 °C for 30 s. T. molitor ribosomal protein (TmL27a) was used as a reference gene and the results were analyzed using the 2−ΔΔCq method70,71.

TmTak1 gene silencing

To investigate the survival rate of T. molitor larvae, RNA interference (RNAi) was first performed using double-stranded RNA (dsRNA) of TmTak1. The dsTmTak1 primer was designed using SnapDragon software (https://www.flyrnai.org/snapdragon) and synthesized by Bionics (Seoul, South Korea). PCR was performed using AccuPower® Pfu PCR PreMix & Master Mix (Bioneer), initial-denaturation of 94 °C for 5 min, followed by 39 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 1 min, and final extension at 72 °C for 5 min was performed under conditions. The PCR product was purified according to the manual of the Clear-STM PCR/Gel DNA fragment purification kit (Invirustech), and dsRNA was prepared according to the manual of the EZTM T7 high yield in vitro transcription kit (Enzynomics, Daejeon, South Korea). The dsRNA product was purified using 5 M ammonium acetate and 99% ethanol and was finally quantified to 1 μg/μl and stored at − 80 °C. As a negative control, dsEGFP was used, and injection was performed by quantifying at 1 μg/μl. After the injected samples were sampled, RNA was extracted and used for RT-qPCR, reverse-transcription step at 50 °C for 10 min, initial denaturation step at 95 °C for 5 min, followed by 39 cycles at 95 °C for 15 s, and 60 °C for 30 s.

Effects of TmTak1 RNAi on the expression mRNA pattern of AMP and NF-κB genes

To evaluate the functional properties of TmTak1 in regulating AMP gene expression in response to pathogens, we used dsTmTak1 to RNAi and then injected E. coli, S. aureus, and C. albicans into larvae. dsEGFP and PBS served as negative and injection controls, respectively. Twenty-four hours after injection, whole body (WB) and five tissues which are Malpighian tubules (MTs), gut (GT), Integument (INT), fat bodies (FBs), Hemocytes (HCs) were dissected, total RNA was extracted from each tissue (n = 20), and cDNA was synthesized as described above. Next, by performing qPCR with specific primers, 15 AMP genes: TmTenecin-1, -2, -3, -4 (TmTene1, 2, 3, 4), TmAttacin-1a, -1b, -2 (TmAtta1a, 1b, 2), TmDefensin (TmDef), TmDefensin-like (TmDef-like), TmColeoptericin-A, -B, -C (TmColeA, B, C), TmCecropin-2 (TmCec2) and TmThaumatin like protein-1, -2 (TmTLP1, 2). To investigate the effect of dsTmTak1 on the expression of NF-κB genes, including TmRelish (TmRel), TmKayak (TmKay) and TmDorsal isoform -X1, -X2 (TmDorX1, X2) bacteria were injected, dissected, and qPCR was performed after RNAi as in the method for examining AMP expression.

Statistical analysis

All experiments were carried out in triplicate. These data were subjected to t test or a one-way analysis of variance (ANOVA). Tukey’s multiple range tests were used to evaluate the difference between groups (p < 0.05).