T. molitor larvae were reared under dark conditions at 26 ± 1°C and 60 ± 5% relative humidity in an environmental chamber established in the laboratory. Larvae were fed an artificial diet consisting of 1.1 g sorbic acid, 1.1 ml propionic acid, 20 g bean powder, 10 g brewer’s yeast powder, and 200 g wheat bran in 4,400 ml distilled water. The feed was autoclaved at 121°C for 15 min and fed to healthy 10th–12th instar larvae for all experiments.

The Gram-negative bacterium Escherichia coli (strain K12), Gram-positive bacterium Staphylococcus aureus (strain RN4220), and fungus Candida albicans (strain AUMC 13529) were used as pathogenic invaders. E. coli and S. aureus were cultured in Luria–Bertani (LB) broth, and C. albicans was cultured in Sabouraud’s dextrose broth overnight at 37°C. The microorganisms were harvested and washed twice in phosphate-buffered saline (PBS; pH 7.0) and then centrifuged at 3,500 × g for 15 min. Subsequently, the samples were suspended in PBS, and concentrations were measured at 600 nm (OD₆₀₀) by spectrophotometry (Eppendorf, Hamburg, Germany). E. coli and S. aureus were diluted to 1 × 10⁶ cells/µl, and C. albicans was diluted to 5 × 10⁴ cells/µl for immune challenge studies.

The TmSpz5 gene sequence (accession number: MW916536) was obtained from the T. molitor RNAseq analysis (unpublished) and NCBI Expressed Sequence Tag (EST) database. The Tribolium castaneum Spz5 amino acid sequence (accession number: XP_008193940.1) was used as the query for identification by local-tblastn searches. The full-length open reading frame (ORF) and deduced amino acid sequences of TmSpz5 were analyzed using BLASTp (NCBI; https://blast.ncbi.nlm.nih.gov/Blast.cgi). The domain architectures of the protein sequences were retrieved using InterProScan (https://www.ebi.ac.uk/interpro/search/sequence-search). Signal peptides were predicted using the SignalP 5.0 server (http://www.cbs.dtu.dk/services/SignalP/).

A multiple-sequence alignment of the TmSpz5 amino acid sequence with representative Spätzle amino acid sequences from other insects (retrieved from GenBank) was generated using ClustalX 2.1 (46). Estimation of the percent identity and phylogenetic analyses were performed using ClustalX 2.1 (pim as the output file) and MEGA version 7.0 (47), respectively. Evolutionary relationships were inferred using the neighbor-joining method (48), and the bootstrap consensus tree was inferred from 1,000 replicates. Several protein sequences were used to generate the phylogenetic tree, including those of TcSpz5, Tribolium castaneum spätzle 5 isoform X1 (XP_008193940.1); TcSpz5, Tribolium castaneum spätzle 5 isoform X2 (XP_015836109.1); AtSpz5like, Aethina tumida spätzle 5-like (XP_019879590.1); SoSpz5like, Sitophilus oryzae spätzle 5-like (XP_030767938.1); MsSpz5, M. sexta spätzli 5 (XP_037299529.1); BmSpz5, B. mori spätzli 5 (XP_004924790.1); AaSpz5like, Anopheles albimanus spätzle 5-like (XP_035790066.1); DmeSpz5, D. melanogaster spätzle5 (NP_647753.1); DmaSpz5, Drosophila mauritiana spätzle 5 (XP_033160799.1); ArSpz5, Athalia rosae spätzli 5 (XP_012261687.1); PgSpz5, Pseudomyrmex gracilis spätzle 5 isoform X3 (XP_020284715.1); and PvSpz4, Penaeus vannamei spätzle 4 (ANJ04742.1).

The protocols for the developmental stage- and tissue-specific analyses have been reported previously (44, 45, 49). Briefly, total RNA was isolated from different developmental stages (eggs, young larvae (instars 10–12), late larvae (instars 14-15), pre-pupae, 1- to 7-day-old pupae, and 1- to 5-day-old adults) and tissues [integument, gut, fat bodies, Malpighian tubule (MT), hemocytes of last instar larvae and 5-day-old adults, and ovary and testis of 5-day-old adults] of T. molitor.

To analyze the induction of TmSpz5, suspensions containing 1 × 10⁶ cells/µl of E. coli and S. aureus and 5 × 10⁴ cells/μl of C. albicans were injected into T. molitor larvae at instars 10–12 (n = 20). PBS-injected T. molitor larvae were used as the control group. Samples were collected at 3, 6, 9, 12, and 24 h post-microbial challenge.

Total RNA was isolated using the Clear-S Total RNA Extraction Kit (Invirustech Co., Gwangju, South Korea) according to the manufacturer’s instructions. Then, 2 μg of total RNA was used as the template to synthesize cDNA using the Oligo (dT)12–18 primers under the following reaction conditions: 72°C for 5 min, 42°C for 1 h, and 94°C for 5 min. The MyGenie96 Thermal Block (Bioneer, Daejeon, Korea) and AccuPower^® RT PreMix (Bioneer) were used according to the manufacturer’s instructions. cDNA was stored at -20°C until further use.

Relative quantitative PCR (qRT-PCR) was performed using AccuPower^® 2X GreenStar qPCR Master Mix (Bioneer) with synthesized cDNAs and specific primers (TmSpz5_qPCR_Fw and TmSpz5_qPCR_Rv), as depicted in Table 1 , with an initial denaturation of 95°C for 20 s, followed by 40 cycles at 95°C for 5 s and 60°C for 20 s. T. molitor ribosomal protein L27a (TmL27a) was used as an internal control, and the results were analyzed using the 2^-ΔΔCt method (50). The results are presented as means ± standard error (SE) of three biological replicates.

To synthesize the double-stranded RNA (dsRNA) of the TmSpz5 gene, primers containing the T7 promoter sequence at their 5′ ends were designed using SnapDragon-Long dsRNA Design ( Table 1 ). PCR was performed using AccuPower^® Pfu PCR PreMix with the TmSpz5_Fw and TmSpz5_Rv primers ( Table 1 ) and according to the developmental expression pattern of TmSpz5, cDNA synthesized from pre-pupae (whole bodies) as a template under the following cycling conditions: an initial denaturation step at 94°C for 2 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 30 s, with a final extension step at 72°C for 5 min. PCR products were purified using the AccuPrep PCR Purification Kit (Bioneer), and dsRNA was synthesized using the AmpliScribe T7-Flash Transcription Kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer’s instructions. After synthesis, the dsRNA was purified by precipitation with 5 M ammonium acetate and 80% ethanol, followed by quantification using an Epoch spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). The dsRNA for enhanced green fluorescent protein (dsEGFP) was synthesized for use as a control and was stored at -20°C until use.

To study the importance of TmSpz5 in the T. molitor immune response, dsTmSpz5 (1 µg/µl) was first injected into early-instar larvae (instars 10–12; n = 30) using disposable needles mounted onto a micro-applicator (Picospritzer III Micro Dispense System; Parker Hannifin, Hollis, NH, USA). An equal amount of dsEGFP was injected in the larvae at the same stage as the negative control. The efficiency of TmSpz5 knockdown was evaluated by qRT-PCR, and over 86% knockdown was achieved at 4 days postinjection. The TmSpz5-silenced and dsEGFP-injected larval groups were challenged with E. coli (10⁶ cells/µl), S. aureus (10⁶ cells/µl), or C. albicans (5 × 10⁴ cells/µl) in triplicate experiments. The challenged larvae were maintained for 10 days, and the number of surviving larvae was recorded. The survival rates of TmSpz5-silenced larvae were compared with those of the control larvae. Relevant analysis was performed using Kaplan–Meier plots (51).

To evaluate the functional properties of TmSpz5 in the regulation of AMP gene expression in response to pathogens, RNAi was used for TmSpz5 gene silencing, followed by the injection of larvae with E. coli, S. aureus, or C. albicans. dsEGFP and PBS were used as the negative and injection controls, respectively. At 24 h postinjection, the hemocytes, fat body, gut, and MTs were dissected, total RNA was extracted from each tissue, and cDNA was synthesized as described above. Next, qRT-PCR was performed with specific primers ( Table 1 ) to analyze the temporal expression patterns of 14 AMP genes: TmTenecin-1, -2, -3, and -4 (TmTene1, 2, 3, and 4), TmAttacin-1a, -1b, and -2 (TmAtt1a, 1b and 2), TmDefensin (TmDef), TmDefensin-like (TmDef-like), TmColeoptericin-A and -B (TmColeA and B), TmCecropin-2 (TmCec-2), and TmThaumatin like protein-1 and -2 (TmTLP1 and 2).

To study the effects of dsTmSpz5 on the expression of NF-κB genes, including TmDorsal isoform X2 (TmDorX2) and TmRelish (TmRel), TmSpz5 was silenced in early-instar larvae and E. coli, S. aureus, and C. albicans were injected at 4 days post-double-strand treatment. At 24 h after pathogen injection, the MTs, hemocytes, gut, and fat bodies were dissected. Total RNA extraction and cDNA synthesis were performed as described above.

The AMPs and NF-κB gene expression patterns led us to perform colony-forming unit (CFU) assay to assess the in vitro AMP activity against Gram-negative bacteria. Therefore, TmSpz5 dsRNA-treated young instar larvae of T. molitor were injected with E. coli (10⁶ cells). At 48 h post–pathogen injection, the hemolymph, midgut, hindgut, and Malpighian tubules were isolated in 100 μl 1× PBS. PBS and dsEGFP were injected as uninfected and dsRNA control groups, respectively. Tissue samples were homogenized and centrifuged at 15,000 rpm at 4°C for 10 min, and then the supernatants were boiled at 100°C for 10 min and centrifuged again at 15,000 rpm at 4°C for 10 min. Consequently, the protein content of extracted peptides has been measured by an Epoch machine and 50 ng of tissue samples was assayed with 10⁶ cells of E. coli in 1× PBS at 37°C for 2 h (52). Eventually, 2,000-fold serial dilutions were performed, and 100 μl of the resulting mixture was plated onto LB agar, followed by incubation at 37°C for 16 h. The colony numbers of assayed plates were then counted.

Statistical analyses were performed using SAS 9.4 (SAS Institute, Inc., Cary, NC, USA), and cumulative survival was analyzed by Tukey’s multiple-comparison test, with a significance level of p < 0.05. Fold change in expression of the AMP genes compared to the levels of the internal control (TmL27a) and external control (PBS) was calculated using the 2^-ΔΔCt method.

To acquire the full-length cDNA sequence of TmSpz5, a local blast search of the T. molitor RNAseq database was performed using the T. castaneum Spätzle5 protein sequence as the query. The TmSpz5 full-length ORF consisted of 1,062 bp, encoding a polypeptide of 353 amino acid residues ( Figure 1 ). As determined using InterProScan, the TmSpz5 amino acid sequence contained a cystine-knot domain at the C-terminus (which binds to the Toll receptor), one cleavage site predicted to be processed by SPE, and a predicted signal peptide at the N-terminus ( Figure 1 ). Additionally, the conserved domains in TmSpz5 were compared at the amino acid level using ClustalX 2.1 and multiple-sequence alignment. TmSpz5 sequences were conserved at the protein level among insect species ( Figure 2A ). A phylogenetic analysis illustrated that TmSpätzle5 in the order Coleoptera formed a group with other isoforms of Spätzle5 from T. castaneum ( Figure 2B ).

TmSpz5 mRNA expression patterns were evaluated by qRT-PCR at different developmental stages and in various tissues in larvae and adults. TmSpz5 was observed at essentially all developmental stages ( Figure 3A ). However, the highest expression levels were seen in embryos and pupae. The mRNA levels decreased at the larval stage, and in late larvae, it shows the lowest expression. We observed fluctuations in the expression pattern during pupal stages with a plateau phase in late pupae. Overall, increased TmSpz5 mRNA levels were observed during molting and each ecdysis, with a gradual fall across each individual stage.

With respect to tissue expression patterns ( Figures 3B, C ), TmSpz5 expression levels were highest in MTs, followed by (in decreasing order) the hemocytes, fat bodies, integument, and gut in larvae. Contrarily, in adults, the mRNA expression of TmSpz5 was low in MTs and highest in the gut.

TmSpz5 expression in immune-challenged T. molitor larvae was examined after E. coli, S. aureus, and C. albicans injections ( Figure 4 ), using PBS injection as the control. Four tissues, including the fat bodies ( Figure 4A ), hemocytes ( Figure 4B ), gut ( Figure 4C ), and MTs ( Figure 4D ), were collected at 3, 6, 9, 12, and 24 h post-pathogen injection for total RNA extraction. TmSpz5 expression was considerably upregulated in response to bacterial and fungal infections. TmSpz5 expression varied in tissue- and time-dependent manners. The highest expression levels were seen in the gut at 12 and 24 h and in the fat bodies at 9 and 24 h after infection (in that order), in response to all three pathogens. Of note, in the fat bodies, the expression of TmSpz5 was lowest at 12 h, possibly due to fluctuations in mRNA expression after infection as also reported earlier (35, 44, 49, 53). In MTs, there was also a noticeable upregulation in response to C. albicans and E. coli at 3 h post injection and in response to S. aureus at 9 h postinjection. C. albicans also induced TmSpz5 expression in the hemocytes at 12 h postinjection.

Considering our observation that TmSpz5 expression is induced by different pathogens, we further examined the survival rate of TmSpz5-silenced larvae using the RNAi technique. TmSpz5 mRNA levels were decreased by 86% 4 days after dsTmSpz5 injection ( Figure 5A ), confirming the efficiency of the RNAi.

Subsequent to confirmation of RNAi efficiency, pathogens of interest were injected. Survival rates of TmSpz5-silenced larvae were then evaluated over 10 days following microbial infection. dsEGFP was used as the control group for dsTmSpz5. PBS-injected larvae showed no statistically significant differences in survival between the dsTmSpz5 and dsEGFP groups (data not shown). dsTmSpz5 larvae showed considerable reductions in survival in response to E. coli and S. aureus (survival rates of approximately 33% and 58%, respectively) ( Figures 5B, C ). Interestingly, C. albicans-injected larvae showed similar survival rates to those of the PBS group ( Figure 5D ).

The survival analysis indicated that TmSpz5 gene silencing accelerated the vulnerability of larvae challenged with E. coli and S. aureus, but not C. albicans. We further evaluated the induction of AMPs following challenge with E. coli, S. aureus, and C. albicans in TmSpz5-silenced T. molitor larvae. In particular, we knocked down TmSpz5 and evaluated the levels of 14 AMP genes 24 h after the microbial challenge.

According to the results of the survival analysis, we expected TmSpz5 silencing to lead to AMP downregulation in response to E. coli and S. aureus. Our data illustrated that following confirmation of the TmSpz5 knockdown efficiency ( Supplementary Figure 1 ), 10 out of 14 AMP genes were significantly downregulated in the MTs of TmSpz5-silenced larvae after E. coli and S. aureus injections but not after fungal infection. In particular, the E. coli challenge resulted in reductions in the levels of TmTene1, TmTene2, TmTene3, TmTene4, TmColeA, TmColeB, TmAtt1a, TmAtt1b, TmAtt2, TmTLP1, and TmTLP2 and the S. aureus challenge resulted in substantial reductions in the levels of TmTene2, TmTene4, TmColeA, TmColeB, TmAtt1a, TmAtt1b, TmAtt2, TmTLP1, and TmTLP2 ( Figure 6 ). In the gut, silencing of TmSpz5 suppressed the E. coli-induced upregulation of TmColeA, TmAtt1a, and TmAtt1b as well as the S. aureus-induced regulation of TmTene2, TmTene4, TmColeB, TmAtt1a, and TmAtt1b ( Figures 7B, D, H–K ). In the hemocytes, TmTene1, TmDef, and TmAtt2 were downregulated in response to E. coli and TmDef and TmAtt2 were downregulated in response to S. aureus ( Figures 8A, E, L ). Moreover, in response to C.albicans, mRNA levels of TmTen3 and TmCec2 were downregulated ( Figures 8C, G). In the fat bodies, only the levels of TmTene4, TmDef, and TmTLP1 were reduced in response to E. coli infection ( Figures 9D, E, M ). Surprisingly, dsTmSpz5 elevated the mRNA levels of some AMPs in response to pathogens in all dissected tissues, particularly the levels of the Cecropin, Attacin, and Tencin families in the gut, fat bodies, and hemocytes ( Figures 7A, C, E–G , 8B, D, F , and 9A–C, F–L, N ). Finally, mRNA levels of almost all AMPs did not differ between the dsTmSpz5 group and the control group in response to C. albicans.

Following the same protocol used to evaluate the expression of AMP genes following knockdown, the NF-κB pathway genes TmDorX2 and TmRelish were examined ( Figure 10 ). dsTmSpz5 considerably depleted TmDorX2 expression levels in MTs following E. coli and S. aureus infection ( Figure 10A ). A less substantial reduction in TmRelish expression was observed in MTs ( Figure 10B ). Moreover, following the microbial challenge, TmDorX2 was upregulated in the fat bodies and gut and TmRelish was upregulated in the fat bodies.

The AMP assay result clearly demonstrated that E. coli and S. aureus infection in dsTmSpz5-treated larvae induced AMP expression significantly in Malpighian tubules and partially in the gut. Thus, we examined whether this suppression would affect bacterial growth in the hemolymph, MTs, midgut, and hindgut by CFU assay. Following dsEGFP and dsTmSpz5 injection, larvae were exposed to E. coli, and the aforementioned tissues were dissected 48 h postinfection and cultured with E. coli on LB agar plates. Elevated antimicrobial activity was observed in all dissected tissues in E. coli-injected larvae compared with the PBS-injected group ( Figures 11A, C, E, G ). Moreover, it was found that E. coli growth was hindered in the dsEGFP-injected gut, compared to the dsTmSpz5-injected larvae in the MTs, hindgut, and midgut (in decreasing order) ( Figures 11D, F, H ). In contrast, in the hemolymph, no significant difference in proliferation inhibition was observed between the dsEGFP- and dsTmSpz5-injected larvae ( Figure 11B ). These results imply that the effect of TmSpz5 knockdown on AMP gene depletion in MTs causes suppressed antimicrobial activity against Gram-negative bacteria. Additionally, while antimicrobial activity in hemolymph remained indifferent, downregulation of AMP genes in MTs subsequent to TmSpz5 knockdown exhibits reduced antimicrobial activity in the hindgut.