Tenebrio molitor larvae were reared in the dark at 27°C ± 1°C and 60% ± 5% relative humidity in an environmental chamber established in the laboratory. The reared larvae were fed 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, sterilized by autoclaving at 121°C for 15 min). Healthy 10th to 12th instar larvae (1.2–1.5 cm length) were used for experiments.

Three microorganisms—the gram-negative bacterium E. coli strain K12, gram-positive bacterium S. aureus strain RN4220, and the fungus C. albicans—were used for immune challenge experiments. E. coli and S. aureus were cultivated in Luria-Bertani (LB) broth (MB cell, South Korea). C. albicans suspension was prepared by culturing the fungi in Sabouraud dextrose broth (MB cell) at 37°C overnight. The microorganisms were harvested and washed twice in 1× phosphate-buffered saline (PBS) (8.0 g NaCl, 0.2 g KCl, 1.42 g Na₂HPO₄, 0.24 g of KH₂PO₄ in 1 L of distilled water, pH 7.0) and centrifuged at 1,700 × g for 10 min. The washed microorganisms were resuspended in 1× PBS and the optical density at 600 nm (OD₆₀₀) of the suspension was measured using a spectrophotometer (Eppendorf, Germany). The concentration of microbial cells was adjusted to 1 × 10⁶ cells/μL of E. coli and S. aureus and 5 × 10⁴ cells/μL of C. albicans for the immune challenge studies. The relevant optimization of microbial concentration has been adjusted based on the previous studies (Chae et al., 2012; Jo et al., 2017; Park et al., 2019).

The TmIKKε sequence was retrieved from T. molitor RNA sequencing (RNA-seq) (unpublished) and expressed sequence tag (EST) databases. Local-tblastn analysis was performed using T. castaneum IKKε amino acid sequence (EEZ99267.2) as a query. The full-length cDNA and deduced amino acid sequences of TmIKKε were determined using the blastx and blastp algorithm, respectively, on the National Center for Biotechnology Information (NCBI) website. Complementary DNA (cDNA) translation and predictions of the deduced protein were analyzed using BioPHP mini tools software (http://www.biophp.org). FGENESH eukaryotic gene prediction was used to predict the TmIKKε open reading frame (ORF) region. The domain architecture of the protein sequences was retrieved using the InterProScan domain analysis program. Representative IKKε protein sequences from other insects were obtained from GenBank and were used for multiple sequence alignments, and percentage identity analysis using Clustal X2.1 (Larkin et al., 2007). A phylogenetic tree was constructed based on the amino acid sequence alignments using the Maximum likelihood method (bootstrap trial set to 1000) with IKKε/TBK1 proteins from representative insects (Supplementary Table 1). The phylogram was analyzed using Tree Explorer view with the Molecular Evolutionary Genetics Analysis (MEGA) version 7.0 program (Kumar et al., 2016) (https://megasoftware.net).

The TmIKKε mRNA expression was investigated in different developmental stages of T. molitor, eggs (EG), young instar larvae (YL; 10th–12th instar larvae), late-instar larvae (LL; 19th–20th instar larvae), prepupae (PP), 1- to 7-day-old pupae (P1–P7), and 1- to 5-day-old adults (A1–A5). TmIKKε mRNA expression was also measured in the different tissues that included integument (IT), hemocytes (HC), GT, fat body (FB), and Malpighian tubules (MT) dissected under a stereoscopic microscope (SMZ645, Nikon, Japan). The tissues were dissected from both LL and adults. Ovary (OV) and testis (TE) were additionally dissected from 5-day-old adults.

To investigate induction patterns of TmIKKε mRNA against microbial challenge, 1 × 10⁶ cells/larva of E. coli and S. aureus, or 5 × 10⁴ cells/larva of C. albicans were injected into 12th–15th instar larvae using microinjector with microcapillary. Samples (whole body, HC, GT, and FB) were collected at 3, 6, 9, 12, and 24 h following injection of microorganisms.

The total RNA was isolated from the developmental stages, tissues, and time-course samples using a Clear-S™ Total RNA extraction kit (Invirustech Co., Gwangju, South Korea) according to the manufacturer’s instructions. The total RNA (2 μg) was used as the template to synthesize cDNA using the Oligo(dT)_12–18 primer on MyGenie96 Thermal Block (Bioneer, South Korea) and AccuPower^® RT PreMix (Bioneer) according to the manufacturer’s instructions. The cDNA was stored at −20°C until required.

The relative expression level of TmIKKε mRNA was investigated by performing quantitative real-time polymerase chain reaction (qRT-PCR) using an AccuPower^® 2× Greenstar™ qPCR Master Mix (Bioneer, Daejeon, Korea) and synthesized cDNAs, and TmIKKε gene-specific primers were designed using the Primer 3 plus program (https://primer3plus.com/cgi-bin/dev/primer3plus.cgi) (Supplementary Table 2). The PCR conditions included an initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s, and annealing and extension at 60°C for 30 s. The qRT-PCR assays were performed on an AriaMx Real-Time PCR System (Agilent Technologies, United States). The results were analyzed using AriaMx Real-Time PCR software. The 2^–ΔΔCt method (Livak and Schmittgen, 2001) was employed to analyze the TmIKKε mRNA expression levels. The mRNA expression levels were normalized to those of T. molitor ribosomal protein L27a (TmL27a), which acted as an internal control. The results represent mean ± standard error (SE) of three biological replicates (3 pools of 20 T. molitor larvae).

For the RNA interference (RNAi) experiments of TmIKKε, a double-strand RNA (dsRNA) fragment of TmIKKε gene was synthesized. Briefly, dsDNA fragment of TmIKKε was amplified using PCR with gene-specific primers conjugated with T7 promoter sequences (Supplementary Table 2). The primers were designed using Snapdragon software (https://www.flyrnai.org) to prevent any cross-silencing effects. The primary PCR for the TmIKKε gene was carried out using an AccuPower Pfu PCR PreMix (Bioneer) with cDNA and specific primers for the TmIKKε gene (Supplementary Table 2). The second PCR was conducted with primers tailed with T7 promoter sequences and 100× dilution of the second PCR products.

Polymerase chain reaction was conducted using an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 53°C for 1 min, and extension at 72°C for 30 s on a MyGenie96 Thermal Block (Bioneer). The PCR products purified using the AccuPrep^® PCR Purification Kit (Bioneer) were used to synthesize the dsRNA using the EZ™ T7 High Yield in vitro transcription kit (Enzynomics, South Korea). The dsRNA for enhanced green fluorescent protein (dsEGFP) synthesized from pEGFP-C1 plasmid DNA as described above acted as a negative control. The dsRNA products were purified using the phenol:chloroform:isoamyl alcohol (PCI) method, precipitated with 5 M ammonium acetate, and washed with 70 and 90% ethanol by centrifugation at 10,000 × g for 15 min at 4°C. The dried pellet was resuspended in DNase and RNase-free water. The synthesized dsRNA was stored at −20°C until required. For the knockdown of TmIKKε mRNA, 1 μg of dsRNA for EGFP and TmIKKε were injected into T. molitor 10th to 12th instar larvae.

To measure mortality, microorganisms (1 × 10⁶ cells/μl of E. coli or S. aureus, and 5 × 10⁴ cells/μl of C. albicans) were injected into TmIKKε gene-silenced T. molitor larvae. Dead larvae were counted each day for up to 10 days following the injection of the microorganisms. Ten insect larvae were used for each group in the mortality assay. Each assay was performed in triplicate. Kaplan–Meier method was used to plot cumulative survival curves of larvae after inoculation, the log-rank chi-squared test was used to assess differences in survival between treatments (Goel et al., 2010).

To further characterize the function of the TmIKKε gene in the humoral innate immune response, the TmIKKε silenced individuals were challenged with microorganisms and the expression levels of 14 AMP genes as well as three NF-κB genes were investigated. After treatment of TmIKKε dsRNA into T. molitor 10th to 12th instar larvae, E. coli (1 × 10⁶ cells/larva), S. aureus (1 × 10⁶ cells/larva), or C. albicans (5 × 10⁴ cells/larva) were injected into T. molitor larvae. Twenty-four hours post-injection, over 20 larvae (as a group) were dissected and the samples (HC, GT, and FB) were collected. 1× PBS was used as an injection control. The expression levels of the following 14 AMP genes were measured by qRT-PCR with 14 AMP gene-specific primers (Supplementary Table 2): TmTenecin 1, 2, 3, and 4 (TmTene1, 2, 3, and 4), TmDefensin and TmDefensin-like (TmDef and TmDef-like), TmColeoptericin A and B (TmColeA and B), TmAttacin 1a, 1b, and 2 (TmAtt1a, 1b, and 2), TmCecropin 2 (TmCec2), and TmThaumatin-like protein 1 and 2 (TmTLP1 and 2). In addition, the expression profiles of NF-κB genes such as TmDorsal isoform X1 and X2 (TmDorX1 and X2), and TmRelish, were investigated by qRT-PCR. A relative quantitative PCR was performed as mentioned above using the AMP and NF-κB gene specific primers.

All experiments were carried out in triplicate and the data were subjected to one-way ANOVA. Tukey’s multiple range tests were used to evaluate the difference between groups (p < 0.05).

The organization of the TmIKKε gene was deciphered from the T. molitor nucleotide database using the TcIKKε amino acid sequence as a query in a tBLASTn analysis. TmIKKε includes two exons interrupted by a single intron (Figure 1A and Supplementary Figure 1). Exon 1 and exon 2 of TmIKKε were 423 bp and 1,773 bp, respectively. The ORF sequence of 2,196 bp starts with the “ATG” initiation codon and ends with the “TGA” stop codon. The ORF sequence of TmIKKε encoded a protein of 731 amino acid residues (Supplementary Figure 2). Domain analysis predicted a serine/threonine-protein kinase catalytic domain (residues 12–258), a ubiquitin-like domain (residues 310–387), and a TANK-binding kinase 1 coiled-coil domain 1 (residues 412–653) in TmIKKε protein. A 5′-untranslated region (UTR) sequence of 581 bp and 3′-UTR of 930 bp was also predicted for TmIKKε. The 3′-UTR sequence contains a consensus polyadenylation signal (AATAAA) located in 173–178 bp following termination codon (TGA). The TmIKKε cDNA sequence and deduced protein sequence have been submitted to GenBank (GenBank ID: MZ708789).

The alignment of the predicted amino acid sequence (restricted to the serine/threonine protein kinase catalytic domain) of TmIKKε with IKKε and serine/threonine protein kinase TBK1 from other known insect IKKs revealed a high degree of conservation (Figure 1B). A phylogenetic tree was constructed using the Clustal X2 alignment file and MEGA 7.0 program to assess the evolutionary position of TmIKKε among the orthologs (Figure 1C). Homo sapiens IKKε (HsIKKε) was used as the outgroup in the phylogenetic analysis. Two clear clade divisions were observed. TmIKKε was placed with TcIKKε and TBK1 from other beetles (AglTBK1, LdTBK1, and AtTBK1). The same clade also placed the IKKε isoforms from the order Diptera in a separate cluster. The high bootstrap values supported the tree topology in this clade. While the mosquito IKKε/TBK1 formed one sub-cluster, the Drosophila and blowfly IKKε/TBK1 formed the other sub-cluster. In the second clade, the orthologs of other insect IKKε proteins were placed.

In order to examine the TmIKKε mRNA expression during development and in different tissues of T. molitor, we performed qRT-PCR using SYBR Green dye-binding assay. Developmental expression patterns indicate that TmIKKε mRNA was highly expressed in the pupal stage-2, -3, and -4 followed by variable expression in the adult stages (Figure 2A). Further, the expression of TmIKKε mRNA was measured in tissues of T. molitor late-instar larvae and 5-day old adults using qRT-PCR. In the late-instar larval tissues, the TmIKKε mRNA was expressed in a tissue-dependent manner (Figure 2B). TmIKKε mRNA was found to be higher (especially in FB, MT, and TE) in the 5-day old adult T. molitor (Figure 2C).

To understand the biological function of TmIKKε in innate immunity of T. molitor, the mRNA expression levels were monitored at different time-points (3, 6, 9, 12, and 24 h) in whole-larvae, HC, GT, and FB after exposure to PBS (as injection controls), and microorganisms (E. coli, S. aureus, and C. albicans) challenge (Figure 3). The expression level of TmIKKε at various time points was analyzed relative to PBS control. In the whole body, TmIKKε mRNA was induced early at 3 h and declined at 6, 9, and 12 h post-injection of microorganisms towards the level of PBS-injected control. At 24 h, the expression of TmIKKε mRNA was found to be lower compared to the expression at 3 h (Figure 3A). In hemocytes, TmIKKε mRNA was induced at 3 and 12 h post-injection by E. coli. S. aureus induced TmIKKε mRNA early at 3 and 9 h post-challenge (Figure 3B). But in the gut tissue, expression of TmIKKε mRNA was drastically high at 6 h-post-injection of S. aureus, E. coli, and C. albicans (Figure 3C). Expression of TmIKKε mRNA in the FB was higher at 9 h post-injections with E. coli and is decreased at 12 hpi relative even to 3 hpi (Figure 3D).

To further substantiate the function of TmIKKε in the host immunity against pathogens, we silenced TmIKKε mRNA by synthesizing dsRNA and injected it into the T. molitor larvae. The silencing of TmIKKε mRNA was compared relative to the injection of dsEGFP (as negative control) to a separate set of larvae. The RNAi efficiency of TmIKKε was found to be approximately 75% relative to the dsEGFP control in the whole body of the larvae (Figure 4A). In addition, the tissue-specific knockdown efficiency by injecting TmIKKε dsRNA was investigated, which showed the results that the expression of TmIKKε was significantly down-regulated by dsTmIKKε-treatment in all tissues including FB (86%), GT (68%), HC (89%), and integuments (84%) (Supplementary Figure 3).

The survival of the larvae was recorded for 10 days after injection of E. coli, S. aureus, and C. albicans to TmIKKε-silenced larvae. After E. coli challenge to TmIKKε-silenced larvae, the mortality observed was 100% at 6-day post-challenge but was not significant compared to dsEGFP-treated larvae (log-rank chi-squared test; p = 1.28E-09) (Figure 4B). The mortality of TmIKKε-silenced larvae was 70% at 10-day post-challenge with S. aureus (log-rank chi-squared test; p = 0.1129) (Figure 4C). After infection with the fungus, C. albicans, the mortality was close to 90% in TmIKKε-silenced larvae (log-rank chi-squared test; p = 0.0378) (Figure 4D). The results indicate that depletion of TmIKKε caused increased larval mortality against E. coli, not S. aureus and C. albicans.

In order to investigate the requirement of TmIKKε gene in the regulation of AMP production in immune organs (hemocytes, gut, and fat body) of T. molitor larvae, we injected gram-negative bacteria E. coli, gram-positive bacteria S. aureus or fungus C. albicans into TmIKKε knockdown T. molitor larvae. The transcriptional expression levels of fourteen T. molitor AMP genes were measured in TmIKKε knockdown individuals in comparison with dsEGFP-treated group. In the hemocytes of T. molitor larvae, the expression of TmTene2 was significantly upregulated in TmIKKε knockdown individuals post-inoculation with E. coli, S. aureus, and C. albicans (Figure 5B). TmTene1 (Figure 5A) and TmTene3 (Figure 5C) expression were also upregulated post-inoculation of E. coli and C. albicans, respectively. Further, the expression of TmAtta1a was upregulated post-inoculation of E. coli (Figure 5J) and TmAtta1b upregulated post-inoculation of E. coli and S. aureus (Figure 5K) in TmIKKε knockdown individuals. The upregulation of AMPs in response to TmIKKε gene knockdown suggests negative regulation during the pathogenic challenge.

Alternatively, in the gut, the expression of eight AMP genes including TmTene1 (Figure 6A), TmTene4 (Figure 6D), TmDef (Figure 6E), TmColeA (Figure 6G), TmColeB (Figure 6H), TmAtta1a (Figure 6J), TmAtta1b (Figure 6K), and TmAtta2 (Figure 6L) out of the fourteen AMP genes were significantly decreased in TmIKKε knockdown individuals. On the other hand, the expression of only two AMP genes including TmTene2 (Figure 6B) and TmDef-like (Figure 6F) was significantly upregulated by TmIKKε RNAi. Overall, in the gut of T. molitor larvae, TmIKKε RNAi leads to decreased transcriptional regulation of most AMPs and might be putatively involved in the survival of the larvae against pathogenic stress. We also noticed the downregulation of TmIKKε transcripts in the gut tissue following systemic injection of dsTmIKKε. Moreover, in the silenced individuals, eight AMPs were downregulated suggesting putative role in gut immunity.

In T. molitor FB, the expression of twelve AMP genes including TmTene1 (Figure 7A), -2 (Figure 7B) and -4 (Figure 7D), TmDef (Figure 7E) and -2 (Figure 7F), TmColeA (Figure 7G) and -2 (Figure 7H), TmAtta1a (Figure 7J), -1b (Figure 7K), and -2 (Figure 7L), TmTLP1 (Figure 7M), and -2 (Figure 7N), out of fourteen AMP genes were significantly decreased by TmIKKε RNAi. Downregulation of AMPs in TmIKKε knockdown individuals after challenge with microorganisms ascertains the role of TmIKKε in the innate immunity of the insect.

Furthermore, to understand the effect of TmIKKε RNAi on expression of Tenebrio NF-κB genes, TmIKKε-silenced T. molitor larvae were challenged with microorganisms and the expression patterns of Tenebrio NF-κB genes such as TmRelish, TmDorX1, and TmDorX2 were investigated at 24 h by qPCR analysis. The results showed that the mRNA level of three NF-κB genes was dramatically decreased by TmIKKε RNAi in the FB of T. molitor larva. In addition, the expression of TmDorX1 and TmDorX2 transcripts were significantly decreased by TmIKKε RNAi in the GT (Figure 8).