The initial C. punctiferalis population was collected from maize fields in Tai’an City, Shandong province, China. All C. punctiferalis larvae were reared on fresh maize grains and adult moths were fed with a 10% (v/v) honey solution in an artificial climate incubator set at 25 ± 1 °C, 70 ± 5% relative humidity, and a photoperiod 14:10 (L:D) h.

The cultivation of B. bassiana strain (ACCC30107), preparation of conidial suspension, and method of C. punctiferalis larvae infected with B. bassiana were performed in accordance with our previous study (37). Then, the samples of larvae were frozen in liquid nitrogen at 12 h post injection (hpi) based on previous findings for further use (38).

The total RNA of non-infected and B. bassiana-infected larvae was extracted using TRNzol Universal Reagent (Tiangen, Beijing, China). Each control or treatment group included nine larvae, and each bioassay was conducted in triplicate. High-quality RNA samples were used to construct libraries that were sequenced on an Illumina NovaSeq 6000 platform at Beijing Novogene Co., Ltd., China. Quality control, de novo transcriptome assembly, and gene functional annotation were conducted as previously described (39). The differentially expressed genes (DEGs) were identified by DESeq2 software. The screening criteria of significantly differential genes were set as p adj < 0.05 and |log₂(fold change, FC)| > 1. GO and KEGG pathway enrichment analyses of DEGs were performed.

The C. punctiferalis larvae samples used for proteome sequencing were the same as those for transcriptome sequencing. Protein extraction, iTRAQ labeling, and LC-MS/MS analysis were performed as previously described (37). Proteome sequencing analysis was also conducted using the iTRAQ technique at Beijing Novogene Co., Ltd., China. The raw data were analyzed by the Proteome Discoverer 2.2 software. The results of protein quantitation were statistically analyzed by t-test and the proteins with significant differences between the treatment group and the control group were selected (the up-regulated expression: FC ≥ 1.2 and p ≤ 0.05; the down-regulated expression: FC ≤ 0.83 and p ≤ 0.05) and defined as differentially expressed proteins (DEPs). Functional annotation and enrichment analysis of DEPs were performed as previously described (37).

All CTLs of C. punctiferalis were retrieved and identified based on the transcriptome and proteome data. The open reading frame (ORF) of the CTLs was obtained using ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/). The theoretical molecular weight (MW) and isoelectric point (pI) of the mature protein were calculated using ExPASy (http://web.expasy.org/compute_pi/). Predictions of the signal peptide, conserved domains, and transmembrane regions were made using SignalP 5.0 (https://services.healthtech.dtu.dk/service.php?SignalP-5.0), SMART (http://smart.embl-heidelberg.de/), and TMHMM (https://services.healthtech.dtu.dk/services/TMHMM-2.0/), respectively. NetNGlyc 1.0 (https://services.healthtech.dtu.dk/services/NetNGlyc-1.0/) and NetOGlyc 4.0 (https://services.healthtech.dtu.dk/services/NetOGlyc-4.0/) were used to predict the N-glycosylation and O-glycosylation sites, respectively. A three-dimensional (3D) structure model was predicted using COFACTOR and COACH based on the I-TASSER server (https://zhanggroup.org/I-TASSER/) (40). The results of generated PDB files were visualized using the PyMOL Molecular Graphics System 2.6 software.

The amino acid sequences of the CTLs were retrieved and downloaded from GenBank. The CTLs from B. mori, Helicoverpa armigera, Spodoptera litura, M. sexta, Galleria mellonella, Ostrinia furnacalis, Drosophila melanogaster, Anopheles gambiae, Aedes aegypti, Rhynchophorus ferrugineus, Sitophilus oryzae, Dendroctonus ponderosae, Rhopalosiphum maidis, Rhopalosiphum padi, Bemisia tabaci, and Macrobrachium rosenbergii were selected for multiple sequence alignment and phylogenetic analysis. Multiple amino acid sequences were aligned using ClustalW (https://www.genome.jp/tools-bin/clustalw) and decorated with ESPript 3.0 (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Phylogenetic trees were constructed using MEGA 11.0 software through the neighbor-joining method (bootstrap = 1,000 replications).

The total RNA of 5^th-instar larvae (3-day-old) was extracted and reverse transcribed into cDNA according to the manufacturer’s instructions. The coding sequence (CDS) of CpIML4 from the transcriptome data was employed to design CDS-specific primers (IML4-F and IML4-R) using Primer Premier 6 software (Supplementary Table 1). The sequence of CpIML4 was amplified by PCR using above primers. The PCR products were purified and ligated into the pMD™ 18-T Vector (TaKaRa, Japan). The positive clones were selected by PCR and confirmed by sequencing at Beijing Liuhe BGI Co., Ltd., China.

Samples of the developmental stages, including eggs (1-day-old, 300 eggs), 1^st-instar larvae (1-day-old, 100 individuals), 2^nd-instar larvae (1-day-old, 80 individuals), 3^rd-instar larvae (1-day-old, 40 individuals), 4^th-instar larvae (1-day-old, 20 individuals), 5^th-instar larvae (1-day-old, 10 individuals), pupae (1-day-old, 10 individuals), and adults (1-day-old, 10 individuals) were collected. Samples of different tissues, including the head (20 individuals), midgut (20 individuals), fat body (20 individuals), hemolymph (20 individuals), and cuticle (20 individuals), were collected from 3-day-old 5^th-instar larvae. Three biological replicates were analyzed for each sample. The methods of total RNA extraction and cDNA synthesis were the same as those described above. Specific primers (qIML4-F and qIML4-R) for CpIML4 were designed using Primer Premier 6 software (Supplementary Table 1). The C. punctiferalis ribosomal protein 49 (RP49) was used as an internal control in all trials. A reaction system of 20 μL was prepared including 10 μL of SuperReal PreMix Plus (2×), 1 μL of cDNA, 1 μL of upstream and downstream primer, and 7 μL of RNase-free ddH₂O. The relative expression level of CpIML4 was analyzed by qRT-PCR using a Bio-Rad CFX96 Touch Real Time PCR Detection System (Bio-Rad, USA) with the following setting: 95 °C for 15 min, 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. All samples were analyzed in triplicate and procedures were repeated thrice as independent biological replicates. The 2^−ΔΔCT method (41) was used to determine the relative expression levels of CpIML4.

The double-stranded RNA (dsRNA) of CpIML4, dsIML4, was synthesized using the T7 RiboMAX™ Express RNAi System (Promega, USA). Green fluorescent proteins (GFP) were employed to generate the dsRNA (dsGFP).Specific primers for the target gene (dsIML4-F, dsIML4-R, dsIML4-T7F, and dsIML4-T7R) and for the GFP gene (dsGFP-F, dsGFP-R, dsGFP-T7F, and dsGFP-T7R) were designed using Primer Premier 6 software (Supplementary Table 1). The dsRNA Off-target Minimization Generator (dsOMG) (https://dsomg.sysu.edu.cn) (42) was used to select dsIML4 fragments with a low risk of off-target effects. The integrity of dsRNA was checked by 1% agarose gel electrophoresis. The NanoDrop One spectrophotometer (Thermo Fisher Scientific, USA) was used to measure the concentration of dsRNA, which was subsequently diluted with nuclease-free water to a final concentration of 1 μg/μL. A total of 2 μg of dsIML4 was injected into the hemocoel of 5^th-instar larvae (3-day-old) using a microinjector (Hamilton, Switzerland). Larvae injected with the same volume of dsGFP were used as controls. The larvae from the treatment and control groups were collected at 12, 24, and 36 hpi. The relative expression levels of CpIML4 were validated by qRT-PCR.

The 5^th-instar larvae (3-day-old, 120 individuals) were divided into four groups to evaluate their survival rates after CpIML4 RNAi: (I) dsGFP+PBS, 30 larvae were injected with 2 μL sterile PBS after injection with dsGFP (as control); (II) dsIML4+PBS, 30 larvae were injected with 2 μL sterile PBS after CpIML4 RNAi; (III) dsGFP+Bb, 30 larvae were injected with 2 μL B. bassiana conidial suspension (5 × 10⁴ conidia/μL) after injection with dsGFP (as a control); (IV) dsIML4+Bb, 30 larvae were injected with 2 μL B. bassiana conidial suspensions (5 × 10⁴ conidia/μL) after CpIML4 RNAi. Three independent replicates were used in each trial. All larvae were maintained in the above-mentioned normal rearing conditions. The survival rates and phenotypic changes of larvae were recorded every 12 h until pupation or death.

Statistical analysis was conducted using IBM SPSS Statistics 26 software and GraphPad Prism 9.0 software. The temporal and spatial expression data were examined through a one-way ANOVA; RNAi efficiency was determined through an unpaired t-test; and the Kaplan-Meier survival rates were performed using the log-rank (Mantel-Cox) test. All data were evaluated for mean ± standard error (SE), and significant differences between values were defined as p < 0.05.

A total of 132,834,380 raw reads were generated, and 127,808,299 clean reads were obtained after quality filtration (Supplementary Table 2). In total, 135,925,102 transcripts and 45,921,295 unigenes were obtained using the Trinity 2.5.1 software and the Corset 4.6 software (Supplementary Table 3). A total of 33,648 unigenes were annotated in seven databases, including NR, NT, KEGG, SwissProt, Pfam, GO, and KOG (Supplementary Table 4). Among these, 11,978 unigenes were annotated and grouped into three GO categories (Supplementary Figure 1A). 6,000 unigenes were annotated and distributed across 26 KOG categories (Supplementary Figure 1B). 7,080 unigenes were annotated and clustered into five KEGG metabolic categories (Supplementary Figure 1C). According to the homology analysis, the majority of unigenes mapped to O. furnacalis (8,359 unigenes), followed by Chilo suppressalis (1,851 unigenes), and H. armigera (754 unigenes) (Supplementary Figure 2).

According to the differential gene screening criteria, a total of 114 DEGs were identified, of which 76 were up-regulated and 38 were down-regulated (Figure 1A). A total of 54 DEGs were annotated to 452 GO terms based on GO enrichment analysis. Among these, the most enriched GO terms were “pathogenesis” (GO 0009405) in biological process, “extracellular region” (GO 0005576) in cellular component, and “hydrolase activity” (GO 0016787) in molecular function (Figure 1B). Based on the KEGG pathway enrichment analysis, 16 DEGs annotated to 34 KEGG terms. The most enriched KEGG pathways were “protein processing in endoplasmic reticulum” (ko 04141), “antigen processing and presentation” (ko 04612), “estrogen signaling pathway” (ko 04915), and “longevity regulating pathway-multiple species” (ko 04213) (Figure 1C).

To improve the accuracy of the proteome data, protein quality controls were conducted (Supplementary Figure 3). In total, 67,130 spectra were matched to 583,724 total spectra, and 31,653 peptides, 3,544 identified proteins and 3,431 quantifiable proteins were checked and identified (Supplementary Table 5). In total, 1,468 proteins were functionally annotated in GO, COG, KEGG, and IPR databases (Supplementary Figure 4). Among these, a total of 2,336 proteins were annotated and grouped into three GO categories (Supplementary Figure 5A). A total of 1,832 proteins were annotated and distributed across 25 COG categories (Supplementary Figure 5B). A total of 3,399 proteins were annotated and clustered into five KEGG metabolic categories (Supplementary Figure 5C). A total of 3,088 proteins were annotated in the IPR analysis (Supplementary Figure 5D). Subcellular localization showed that 2,051 proteins were classified into 14 categories (Supplementary Figure 5E).

According to the differential protein screening criteria, a total of 3,431 proteins were identified, of which 197 were DEPs, 109 were up-regulated proteins, and 88 were down-regulated proteins (Figure 2A). In total, 117 DEPs were annotated to 132 GO terms on the basis of GO enrichment analysis. Among these, the most enriched GO terms were “aminoglycan metabolic process” (GO 0006022) in biological process and “serine-type peptidase activity” (GO 0008236) in molecular function (Figure 2B). According to the KEGG pathway enrichment analysis, 80 DEPs annotated to 150 KEGG maps. The most enriched KEGG pathways were “glutathione metabolism” (map 00480), “fatty acid metabolism” (map 01212), “ras signaling pathway” (map 04014), “epithelial cell signaling in Helicobacter pylori infection” (map 05120), and “PPAR signaling pathway” (map 03320) (Figure 2C).

A scatter plot of transcriptome and proteome expression levels showed a Pearson correlation coefficient of 0.055, indicating a positive correlation between protein and corresponding gene expression (Supplementary Figure 6). The corresponding relationship between the transcriptome and proteome was shown in a Venn diagram through the integration of mRNA and protein information. Of the 3,431 identified proteins, 3,377 had corresponding transcripts in the transcriptome data. A total of 114 DEGs and 197 DEPs were identified, of which only three DEGs (DEPs) were screened together (Figure 3A).

In total, 314 immunity-related genes and proteins were identified from the transcriptome and proteome data. These genes and proteins were grouped according to their functions, including 83 pathogen recognition molecules, 53 extracellular signal modulation, 66 intracellular signal transduction, and 112 immune response effectors (Figure 3B).

The transcripts (proteins) with common significant differences were selected based on GO function and KEGG pathway enrichment analysis of the transcriptome and proteome. GO functional enrichment analysis included binding, structural constituent of cuticle, and catalytic activity. In KEGG pathway enrichment analysis, Toll and Imd signaling pathways (map 04624) and metabolic pathways (map 01100) were identified (Supplementary Table 6). In the Toll and Imd signaling pathways, a total of 15 proteins were identified in the KEGG pathway, of which two proteins were up-regulated differential proteins, namely ModSP (modular serine protease) and Relish (a nuclear factor kappa B, NF-κB) (Figure 3C).

A total of 14 CTLs of C. punctiferalis were identified, four of which were found in the transcriptome and 12 in the proteome (Figure 4A). Based on CRD organization, six CTLs with a single CRD belonged to CTL-S, and the remaining eight CTLs with a dual CRD belonged to IMLs. CTL-X containing a single CRD and other functional domains were not found in the CTLs of C. punctiferalis. CTL-S1−S5 and IML-1−7 contained an N-terminal signal peptide, indicating the potential for secretion into the plasma. CTL-S6 was likely located in the cytoplasm due to the lack of N-terminal secretion signals. IML-8 contained a transmembrane region, suggesting a potential location on the cell membrane (Figure 4B). In total, 22 CRDs were identified from all CTLs, five of which contained the EPN (Glu-Pro-Asn) motif, six contained the QPD (Gln-Pro-Asp) motif, and 11 contained atypical motifs, such as EPD (Glu-Pro-Asp) (Table 1).

The cluster heatmap showed that CTL-S1 and IML-4 were identified in both the transcriptome and proteome data. Notably, the expression of IML-4 (named CpIML4) was increased after B. bassiana infection compared to the control group. The expression of CpIML4 exhibited significant up-regulation, especially in the proteome (Figure 4A). Subsequent trials were conducted to investigate this phenomenon.

The C. punctiferalis larval transcriptome data showed the full-length cDNA sequence of CpIML4 was 987 bp. The ORF of CpIML4 was 963 bp and a protein of 320 amino acids was encoded with a theoretical MW of 35.32 kDa and pI of 5.84. An N-terminal signal peptide (amino acid residues 1 to 24) and two conserved CRDs, CRD1 (amino acid residues 37 to 159) and CRD2 (amino acid residues 169 to 309), were predicted in CpIML4 (Figure 5A; Supplementary Figure 7). Ten conserved cysteine (Cys) residues within the amino acid sequence (Cys₅₈, Cys₁₃₆, Cys₁₅₀ and Cys₁₅₈ in CRD1, and Cys₁₆₉, Cys₁₈₃, Cys₂₀₀, Cys₂₈₅, Cys₂₉₉, and Cys₃₀₈ in CRD2) generated at least four disulfide bonds (Cys₅₈-Cys₁₅₈, Cys₁₃₆-Cys₁₅₀, Cys₁₆₉-Cys₂₀₀, and Cys₂₈₅-Cys₂₉₉) (Figure 5A). CpIML4 contained two motifs: EPD (Glu₁₂₇-Pro₁₂₈-Asp₁₂₉) in CRD1 and QPD (Gln₂₇₂-Pro₂₇₃-Asp₂₇₄) in CRD2 (Figure 5A; Supplementary Figure 7). The glycosylation site analysis showed that CpIML4 contained two N-glycosylation sites (Asn₁₀₇ and Asn₁₆₆) and three O-glycosylation sites (Ser₂₆₉, Ser₂₇₀, and Thr₂₇₆) (Figure 5A; Supplementary Figure 7). Moreover, predictions made through TMHMM analysis suggested that CpIML4 may constitute an extracellular protein because of the lack of typical transmembrane domains (Supplementary Figure 8).

The 3D structure of CpIML4 contained four α-helices and five β-stands. CRD1 included one patchy α-helix, one α-helix, and two β-stands, whereas CRD2 included two α-helices and two β-stands. The Ca²⁺/sugar binding sites of the 3D structural model were predicted using COACH based on the I-TASSER server. There was a potential melibiose (MLB) site in CRD1, a potential Ca²⁺ site and a raffinose (RAF) site in CRD2. The MLB site contained amino acid residues (Glu₁₂₇ and Asp₁₂₉) in the EPD motif, and the galactose-type RAF site contained amino acid residues (Gln₂₇₂ and Asp₂₇₄) in the QPD motif (Figure 5B).

The amino acid sequence alignment demonstrated extensive coverage (≥ 89%) and high similarity between CpIML4 and the CTLs sequences of other insects. CpIML4 had the closest identity with OfIML4 (50.51%, AIR96000.1), followed by BmIML (42.61%, XP_004922068.1), HaCTL6 (40.68%, AFI47451.1), GmIML (40.34%, XP_052759365.1), SlIML (37.70%, XP_022827254.1), and MsIML (30.89%, XP_030038662.1). In CRD1, all insects contained an EPD motif, except for MsIML, which contained a typical EPN motif. In CRD2, CpIML4, OfIML4, and GmIML all contained a typical QPD motif, whereas BmIML, HaCTL6, and SlIML all contained a typical EPN motif. Ten conserved Cys residues were identified in CpIML4, and the disulfide bridges formed by these residues may affect the stability of the protein structure (Supplementary Figure 9).

A phylogenetic tree was constructed employing amino acid sequences from a total of 15 insect species. The CTL sequence of M. rosenbergii was used as an outgroup. CpIML4 was clustered in a subgroup of lepidopteran CTLs and shared a branch with OfIML4. The results indicated that CpIML4 and OfIML4 were the most homologous and performed similar functions (Figure 5C).

The expression of CpIML4 was detected throughout all the developmental stages (egg, larva, pupa, and adult), showing the lowest expression level in eggs and the highest expression level in 5^th-instar larvae, followed by the other developmental stages. The relative expression level of CpIML4 in 5^th-instar larvae was 13.48 times higher than that in eggs (Figure 6A). Moreover, the expression levels of CpIML4 exhibited variation in different larval tissues (head, midgut, fat body, hemolymph, and cuticle), with the highest expression level in the hemolymph, followed by the fat body, and lower expression levels in the midgut and head. The relative expression levels of CpIML4 in the hemolymph and fat body were 11.85 and 6.66 times higher than those in the midgut and 10.66 and 5.99 times higher than those in the head, respectively (Figure 6B).

Larvae were injected with dsIML4 or dsGFP (control) and collected at different times to assess CpIML4 RNAi efficiency. Compared with the control group, the expression levels of CpIML4 were significantly reduced to 22.71%, 68.14%, and 68.43% at 12, 24, and 36 hpi, respectively (Figure 7A). The expression level of CpIML4 was highly significant (p < 0.001) compared to the control group at 24 hpi, indicating that CpIML4 RNAi was reliable for further study.

To explore the effect of CpIML4 RNAi on larval susceptibility to fungal infection, the survival rates of C. punctiferalis larvae were recorded every 12 h. The results indicated that the survival rate of (dsGFP+PBS)-treated larvae was 93.33% at 144 h, and larvae underwent no significant changes and survived normally (Figures 7B, C). The survival rate of (dsIML4+PBS)-treated larvae was 66.67% at 144 h, which differed significantly (p < 0.05) from the control group (dsIML4+PBS) (Figure 7B). The cuticle of (dsIML4+PBS)-treated larvae showed black spots and developmental malformation, and the dead larvae turned black and wrinkled (Figure 7C). The survival rate of (dsGFP+Bb)-treated larvae was 3.33% at 120 h, and the time of death ranged between 72 to 96 hpi (Figure 7B). The cuticle of (dsGFP+Bb)-treated larvae appeared as black spots, and the dead larvae became dark red, mummified, and overgrown with hypha and conidia of B. bassiana (Figure 7C). All (dsIML4+Bb)-treated larvae died at 72 h, and the time of death was concentrated between 12 to 36 hpi. The survival rates differed significantly (p < 0.01) from that of the control group (dsGFP+Bb) (Figure 7B). The cuticle of the larval body exhibited a mass of black spots that later spread throughout the body, and the dead larvae turned black, dried, and wrinkled (Figure 7C). The results showed that C. punctiferalis larvae with a low expression of CpIML4 were easily killed after B. bassiana infection and indicated that CpIML4 critically influenced anti-fungal immunity.