Stocks were raised in an insectarium on an artificial diet at 27 ± 1°C and 60 ± 5% relative humidity in the dark, in accordance with a previous study (Keshavarz et al., 2019). Healthy, normal larvae at the 10th–12th instar (around 2.4 cm in length) were used for experiments (n = 20 per group).

Evidence of the constitutive expression of TmPGRP-LE during the developmental stages (Tindwa et al., 2013) prompted us to elucidate the tissue-specific pattern of TmPGRP-LE expression in the integument, fat body, hemocytes, gut, Malpighian tubules, ovary, and testes of larvae and adults.

Total RNA was extracted from all tissue samples using the LogSpin RNA isolation method with minor modifications (Yaffe et al., 2012). Briefly, the samples were homogenized in guanidine thiocyanate-based RNA lysis buffer mixed with 99% ethanol and transferred to silica spin columns (Bioneer, Korea, KA-0133-1). The resultant RNA was treated with DNase (Promega, M6101, United States) to eliminate the genomic DNA contamination for 15 min at 37°C. Preceding all steps each silica column was then washed twice with the supplied wash buffers using 3 M sodium acetate buffer and 80% ethanol and dried for 1 min. Total RNA was eluted with 30 μL of distilled water (Sigma, W4502-1L, United States). Next, 2 μg of total RNAs were converted to cDNA using the AccuPower^® RT PreMix (Bioneer, Korea) kit with an oligo-(dT) ^12–18 primer according to the protocol recommended by the manufacturer. The synthesized cDNAs (1:20 dilution with DNase/RNase free water) processed for quantitative reverse-transcription PCR (qRT-PCR) using AccuPower^® 2X GreenStar qPCR Master Mix (Bioneer, Korea). Data was normalized to T. molitor 60 S ribosomal protein L27a (TmL27a) transcripts and values were calculated using the comparative C_T method (2^–ΔΔCT method) (Schmittgen and Livak, 2008). Specific primers for qRT-PCR were designed using Primer 3.0 plus¹ and are listed in Table 2. For detailed information, including PCR conditions, see our previous paper (Keshavarz et al., 2019).

The gram-negative bacterium E. coli (starin K12) and gram-positive bacterium S. aureus (strain RN4220) were cultured in Luria-Bertani (LB) broth, and the fungus C. albicans was cultivated in Sabouraud dextrose broth at 37°C overnight. Cells from overnight microbial cultures were concentrated by centrifugation at 3,500 rpm for 15 min at room temperature (∼25°C). The supernatant was discarded, after which the pellet was washed (three times) and resuspended in phosphate-buffered saline (PBS; pH 7.0). The cell concentration was determined by optical density (OD) measurement at 600 nm. Based on OD₆₀₀ values, the microorganism suspensions were adjusted to 10⁶ cells/μL for E. coli and S. aureus, and 5 × 10⁵ cells/μL for C. albicans.

Microbial challenges were conducted on T. molitor larvae (10th–12th instar) by injecting 1 μL of E. coli (1 × 10⁶ cells/μL), S. aureus (1 × 10⁶ cells/μL), C. albicans (5 × 10⁴ cells/μL), or PBS control between the 3rd and 4th abdominal segment. For quantification of TmPGRP-LE mRNA expression, whole insects or dissected immune-related tissues (fat body, gut, and hemocytes) were collected at various time points (at 3, 6, 9, 12, and 24 h post-infection). Subsequently, total RNA extraction, cDNA synthesis, and qRT-PCR were performed as previously described.

Double-stranded RNA for use in RNAi experiments were synthesized as previously described (Tindwa et al., 2013). Concisely, the PCR product (636 bp sequence) tailed with T7 promotor sequence of TmPGRP-LE was amplified using AccuPower^® Pfu PCR PreMix with forward (dsTmPGRP-LE_Fw) and reverse (dsTmPGRP-LE_Rv) primers under the following conditions: denaturation at 95°C for 2 min, followed by 30 cycles of denaturation at 95°C for 20 s, annealing at 56°C for 30 s, and extension at 72°C for 5 min (Table 2). The synthesized dsTmPGRP-LE was purified using an AccuPrep^® PCR Purification Kit (Bioneer, Korea). Subsequently, the purified product was used as a template to synthesize dsTmPGRP-LE in vitro using an EZ^TM T7 High Yield in vitro Transcription Kit (Enzynomics, Korea) as per the manufacturer’s instructions. Then it was precipitated with 5 M ammonium acetate and washed with 70, 80, and 99.9% ethanol sequentially. After drying at room temperature, the precipitate was resuspended in 30 μL distilled water (Sigma, W4502-1L, United States) to obtain the final product.

An additional dsRNA stock of EGFP (dsEGFP) was generated using the PCR product (546 bp sequence) of the enhanced green fluorescent protein (EGFP) gene derived from the plasmid EGFP-C1. dsEGFP was used as a negative control in subsequent RNAi experiments.

A key question concerning TmPGRP-LE is its role in combatting larval infection; to address this question, we examined the viability of larvae exposed to microbes after silencing TmPGRP-LE expression. However, it is crucial to verify that merely TmPGRP-LE depletion do not affect the survival percent. To quantitatively address this question, we injected 1 μL (1 μg) of dsTmPGRP-LE into 10th–12th instar T. molitor larvae. The knockdown efficiency for the target gene (TmPGRP-LE) was measured on the 6th day post-treatment. After confirmation of silencing, dsRNA-injected larvae (n = 10 per group) were challenged with E. coli (1 × 10⁶ cells/μL), S. aureus (1 × 10⁶ cells/μL), or C. albicans (5 × 10⁴ cells/μL). PBS were injected into dsRNA-injected larvae as a control. Survivors were counted daily for a duration of 10 days. The experiments were repeated three times, with 10 larvae per group for each experiment.

To understand the function of TmPGRP-LE in regulating AMP genes, we examined the gene expression profiles of 14 AMPs, namely TmTenecin-1 (TmTene1), TmTenecin-2 (TmTene2), TmTenecin-3 (TmTene3), TmTenecin-4 (TmTene4), TmAttacin-1a (TmAtt1a), TmAttacin-1b (TmAtt1b), TmAttacin-2 (TmAtt2), TmDefensin-1 (TmDef1), TmDefensin-2 (TmDef2), TmColeoptericin-1 (TmCole1), TmColeoptericin-2 (TmCole2), TmCecropin-2 (TmCec2), TmThaumatin-like protein-1 (TmTLP1), and TmThaumatin-like protein-2 (TmTLP2) in the TmPGRP-LE-silenced larvae after microbial challenges. In addition to AMP gene expression profiling, the mRNA levels of three previously identified transcription factors composed of TmRelish, TmDorsal X1 isoform (TmDorX1), and TmDorsal X2 isoform (TmDorX2) were also measured. dsEGFP was used as a negative control, and PBS served as a wound control. Knowledge of the immunological role of TmPGRP-LE in different tissues is valuable; thus, we dissected the fat body, gut, and hemocytes of experimental samples 24 h post-injection. Each experiment was independently repeated trice (n = 20 per group). Samples were processed for cDNA synthesis, and qRT-PCR analysis was conducted using AMP-specific primers (Table 2).

Three independent biological replicates were used for all experiments. Values were reported as mean ± SE. Differences between groups were analyzed using one-way statistical analysis of variance (ANOVA) and Tukey’s test; p < 0.05 were considered significant. The results for the mortality assay were analyzed using the Kaplan-Meier plot (log-rank Chi-square test) in Excel.²

As an intracellular receptor, cytoplasmic DmPGRP-LE was previously detected in hemocytes and Malpighian tubules. However, as an extracellular PGN receptor, it was previously detected in the fat body and hemocytes; in these tissues, DmPGRP-LE can enhance DmPGRP-LC-mediated recognition of PGN and activate the Imd signaling pathway (Takehana et al., 2004; Kaneko et al., 2006). Similar to other long PGRPs (excluding PGRP-LB), TmPGRP-LE lacks a signal peptide. Thus, it is constitutively expressed in the cytoplasm during all developmental stages and acts as an intracellular scavenger of PGN (Tindwa et al., 2013).

In this study, we employed qRT-PCR to investigate the tissue distribution of TmPGRP-LE transcripts (Figure 1). TmPGRP-LE mRNA was detected in all T. molitor larval tissues, with the highest expression level observed in the gut, followed by that in hemocytes and Malpighian tubules, while the lowest mRNA quantities were found in the integument and fat body (Figure 1A). Similarly, there was high expression of TmPGRP-LE in the gut and hemocytes of 5-day-old adults, with lower expression found in the integuments. The transcription of TmPGRP-LE was weakly detected in the fat body, Malpighian tubules, and ovary. The lowest expression of TmPGRP-LE was found in the testis (Figure 1B).

The Drosophila PGRP-LE protein serves as a master bacterial peptidoglycan-sensing molecule in the gut that mediates NF-κB-induced responses to infectious pathogens (Bosco-Drayon et al., 2012). Thus, to elucidate how whole organs and tissues of T. molitor (including fat body, gut, and hemocytes) respond to various microbes, we evaluated changes in the transcriptional abundance of TmPGRP-LE after challenging hosts with E. coli, S. aureus, and C. albicans at various time points (3, 6, 9, 12, and 24 h post-infection) (Figure 2). In the whole body, fat body, and gut tissues, E. coli infection led to a gradual but significant upregulation in transcription of TmPGRP-LE, resulting in an up to threefold increase in expression over the PBS-injected controls by 9 h post-infection (p < 0.05). Levels of TmPGRP-LE in the whole body and fat body increased at early time points (3, 6, and 9 h) but did not persist at 12 and 24 h post-infection. Similarly, TmPGRP-LE expression levels were significantly increased in the gut, but there was a slight decrease (p < 0.05) in expression after 9 h (Figure 2A). These results indicate that the T. molitor larval gut responds to infection by E. coli and that the responses involve TmPGRP-LE.

As shown in Figure 2B, S. aureus challenge primarily induced TmPGRP-LE expression at 6 h post-infection in the whole body and fat body, whereas its expression dropped noticeably at later time points. In the gut, S. aureus exposure resulted in weak expression of TmPGRP-LE and its expression remained mostly unchanged at 3, 6, and 9 h post-infection (p < 0.05) (Figure 2B). Similar results were observed for TmPGRP-LE transcription following exposure to C. albicans as were found after infection with E. coli. The fold-increase in TmPGRP-LE mRNA levels was highest at 9 h post-infection with C. albicans in the fat body and gut in comparison to mock controls (p < 0.05) (Figure 2C).

Although microbial infection (E. coli, S. aureus, and C. albicans) within hemocytes was different in T. molitor larvae, the mRNA expression patterns of TmPGRP-LE were extremely similar. These results show a significant increase in TmPGRP-LE expression 6 h after the onset of infection followed by a dramatic decline in expression during later time points (p < 0.05) (Figures 2A–C). In hemocytes, the expression level of TmPGRP-LE after E. coli infection was more potent than it was after infection with S. aureus and C. albicans (Figure 2A).

In insects, the conserved RNAi pathway has been widely described, and RNAi techniques using dsRNA injection have been applied for gene function studies for over a decade (Brown et al., 1999; Amdam et al., 2003). To assess the response of the T. molitor young larvae (10th–12th instar) to dsTmPGRP-LE injection compared to dsEGFP control injection, the knockdown efficiency was measured by qRT-PCR. The transcription of TmPGRP-LE was significantly reduced (80%) 6 days after dsRNA injection (p < 0.05) (Figure 3A). As expected, silencing of TmPGRP-LE had no effect on PBS-injected larvae mortality (Supplementary Figure S1). Furthermore, in order to determine the necessity of TmPGRP-LE in larval survival, we challenged dsRNA-treated larvae with E. coli, S. aureus, and C. albicans and assessed insect mortality over 10 days. The survival rate of TmPGRP-LE-silenced larvae that were infected with E. coli dropped dramatically to 30% compared to >60% survival of the controls (p < 0.05) (Figure 3B). The percent survival resulting from S. aureus (50%, Figure 3C) and C. albicans (30%, Figure 3D) challenges in the TmPGRP-LE-silenced groups were significantly lower than that in the dsEGFP-injected controls (p < 0.05).

Among the previously described receptors of the Imd signaling pathway, DmPGRP-LE senses bacteria-derived PGN either independently or synergistically with DmPGRP-LC depending on its localization. It eventually conveys the signal transduction to the NF-κB transcription proteins and in-turn regulates immunity effector genes, i.e., AMPs (Takehana et al., 2004). Considering the significant mortality ratios observed in TmPGRP-LE-silenced larvae groups after microbial invasion, we reasoned that these phenotypes could be due to TmPGRP-LE knockdown which would subsequently impair the antimicrobial responses. These observations prompted us to investigate the expression levels of 14 AMP genes in larvae with depleted levels of TmPGRP-LE. Therefore, we injected 1 μL (1 μg) of dsTmPGRP-LE and dsEGFP into two sets of T. molitor larvae and confirmed the knockdown efficiency (80%) of the target genes after 6 days. Given that immune tissues might be variably tolerant to infection (Neyen et al., 2012), we examined the larval fat body, gut, and hemocytes of dsTmPGRP-LE- and dsEGFP-treated cohorts 24 h after challenge with E. coli, S. aureus, and C. albicans.

In dsEGFP-injected controls, E. coli infection induced the expression of TmTene1 (Figures 4A, 5A), TmTene2 (Figures 4B, 5B), TmTene4 (Figures 4D, 5D), TmAtt1a (Figures 4E, 5E), TmAtt1b (Figures 4F, 5F), TmAtt2 (Figures 4G, 5G), TmCole1 (Figures 4H, 5H), TmCole2 (Figures 4I, 5I), TmDef1 (Figures 4J, 5J), TmDef2 (Figures 4K, 5K), and TmCec2 (Figures 4L, 5L) in both the fat body and gut (Figures 4, 5). In dsTmPGRP-LE-injected groups, E. coli-induced gut expression of all 11 AMP genes was significantly downregulated (Figure 5), while fat body expression of TmTene1 (Figure 4A), TmTene2 (Figure 4B), TmAtt1b (Figure 4F), TmCole1 (Figure 4H), and TmDef1 (Figure 4J) was upregulated.

In the fat body, C. albicans-induced expression of almost all AMPs was decreased in TmPGRP-LE-silenced larvae with the exception of four AMPs: TmTene3 (Figure 4C), TmCec2 (Figure 4L), TmTLP1 (Figure 4M), and TmTLP2 (Figure 4N). This suggests that TmPGRP-LE is crucial for the production of AMPs after C. albicans infection (Figure 4). In contrast, S. aureus-dependent expression of AMPs was dramatically increased in TmPGRP-LE-silenced larvae with the exception of TmAtt1a (Figure 4E), TmCec2 (Figure 4L), and TmTLP2 (Figure 4N). Furthermore, TmPGRP-LE knockdown did not affect the mRNA levels of TmDef1 (Figure 4J) and TmTLP1 (Figure 4M) after S. aureus infection and of TmCec2 (Figure 4L) and TmTLP1 (Figure 4M) following C. albicans challenge.

Notably, AMP genes were upregulated dramatically in the gut of dsEGFP-injected larvae infected with E. coli (Figure 5). The upregulation of TmTene1 (Figure 5A), TmTene2 (Figure 5B), TmTene4 (Figure 5D), TmAtt1a (Figure 5E), TmAtt1b (Figure 5F), TmAtt2 (Figure 5G), TmCole1 (Figure 5H), TmCole2 (Figure 5I), TmDef1 (Figure 5J), and TmDef2 (Figure 5K) was notably suppressed in dsTmPGRP-LE-treated larvae. This result highlights the critical role of TmPGRP-LE in the humoral immune response of the gut.

However, in contrast to the strong AMP induction observed in the gut following E. coli challenge, AMP expression was comparatively mild in S. aureus and C. albicans infections.

Consistently, silencing of PGRP-LE decreased the expression of S. aureus-induced AMP genes, including TmTene1 (Figure 5A), TmTene4 (Figure 5D), TmAtt1b (Figure 5F), TmAtt2 (Figure 5G), TmCole1 (Figure 5H), TmCole2 (Figure 5I), TmDef1 (Figure 5J), and TmCec2 (Figure 5L) in comparison with their levels in dsEGFP-injected larvae. Compared with the prominent effect of TmPGRP-LE on AMPs expression upon E. coli challenge, TmPGRP-LE acts as a positive regulator during S. aureus infection, where it conveys the signal to produce AMPs. As shown Figure 5, silencing TmPGRP-LE had surprising effects on the induction of gut AMPs by C. albicans. More precisely, the mRNA levels of 11 AMP genes, namely TmTene1 (Figure 5A), TmTene4 (Figure 5D), TmAtt1a (Figure 5E), TmAtt1b (Figure 5F), TmAtt2 (Figure 5G), TmCole1 (Figure 5H), TmCole2 (Figure 5I), TmDef1 (Figure 5J), TmDef2 (Figure 5K), and TmCec2 (Figure 5L), were significantly decreased.

The antimicrobial responses to fungal and bacterial challenges in hemocytes of dsTmPGRP-LE-treated larvae were weaker compared to the other tissues studied (Figure 6). Similar to fat body and gut responses, when dsTmPGRP-LE-injected larvae were challenged with E. coli, the transcription levels of TmTene1 (Figure 6A), TmTene4 (Figure 6D), TmAtt1a (Figure 6E), TmAtt1b (Figure 6F), TmAtt2 (Figure 6G), TmCole1 (Figure 6H), TmCole2 (Figure 6I), TmDef1 (Figure 6J), and TmDef2 (Figure 6K) were downregulated compared to dsEGFP-treated controls. In contrast, however, TmTene1 (Figure 6A), TmTene2 (Figure 6B), TmTene4 (Figure 6D), TmAtt1a (Figure 6E), TmCole2 (Figure 6I), TmDef1 (Figure 6J), and TmDef2 (Figure 6K) gene expression following infection with S. aureus remained inducible in dsTmPGRP-LE-injected larvae, suggesting that TmPGRP-LE acts as a negative signal transducer in hemocytes following S. aureus challenge. Note that levels of expression of AMP genes in larval hemocytes after challenge with C. albicans were not significantly different in TmPGRP-LE-silenced larvae compared to dsEGFP controls (Figure 6).

Altogether, these results indicate that reducing TmPGRP-LE levels in the fat body, gut, and hemocytes led to a significant downregulation of AMP genes after E. coli infection. Similarly, TmPGRP-LE depletion was sufficient to suppress the expression of several AMP genes in the fat body and gut following C. albicans challenge, highlighting the critical role of TmPGRP-LE as a receptor of the NF-κB signaling pathway. In contrast to the responses to E. coli and C. albicans infection, no drastic reduction in the transcription of AMP genes was observed during S. aureus infections in the fat body of TmPGRP-LE-silenced larvae.

Previous studies in insect immunity have revealed that different transcription factors regulate the Toll and Imd immunity pathways. Relish and Dorsal, key downstream proteins in the Imd and Toll pathways, respectively, from dimers to participate in the precise control of the production of AMPs (Sagisaka et al., 2004; Shin et al., 2005; Schlüns and Crozier, 2007; Tanaka et al., 2007; Oeckinghaus and Ghosh, 2009; Keshavarz et al., 2019). We wanted to find out whether the reduction of TmPGRP-LE, which is a sensor of PGN (Tindwa et al., 2013), would have an effect on the expression levels of T. molitor transcription proteins. To address this question, we assessed the mRNA expression of TmRelish, TmDorsal X1 isoform (TmDorX1), and TmDorsal X2 isoform (TmDorX2) in the fat body, gut, and hemocytes of TmPGRP-LE-silenced larvae following infection with E. coli, S. aureus, and C. albicans.

After E. coli and C. albicans infections, depletion of TmPGRP-LE appeared to have a significant effect in downregulating TmRelish and TmDorX1 transcription in the larval fat body and gut (Figures 7A, B). TmPGRP-LE knockdown larvae showed slightly decreased TmDorX1 expression in the fat body and hemocytes after S. aureus infection (Figure 7B).

In dsTmPGRP-LE-injected larvae, expression of TmDorX2 was significantly decreased in both the fat body and gut following C. albicans challenge (Figure 7C). In response to E. coli infection, there was a moderate decline in the expression of TmDorX2 in the gut (Figure 7C) and of TmDorX1 in hemocytes (Figure 7B).

Collectively, all NF-κB genes were significantly downregulated in the larval gut of TmPGRP-LE knockdown groups after infection with either E. coli or C. albicans. Of note, a similar result was observed in the fat body of dsTmPGRP-LE-injected larvae following C. albicans challenge. It is plausible that the Toll and Imd pathways synergistically activate through TmPGRP-LE (Tanji et al., 2007). This conclusion is in agreement with our findings regarding the expression of AMP genes in the fat body and gut.