Introduction

Lepidopteran pests are considered the most damaging to corn plants1,2,3. Among them, the Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae) is one of the most destructive pests affecting corn production worldwide, posing significant threats, especially in Asia, Africa, and Australia4,5. In China, despite various control measures, O. furnacalis causes estimated yield losses ranging from 10 to 80% during outbreak years6. The newly hatched larvae damage corn by attacking tender leaves, stalks, ears, and cobs, often boring into the cobs and stalks7. Moreover, the invasion of O. furnacalis also aggravates the epidemics of Fusarium ear rot of corn8. Current control methods primarily rely on synthetic chemicals and transgenic corn crops expressing Bacillus thuringiensis (Bt) toxins9. However, the extensive use of these chemicals and Bt varieties has led to significant issues, including deterioration of the natural environment, insecticide resistance, and resurgence of pest populations10. Therefore, it is critically important to find safe and efficient alternate approaches for Integrated Pest Management (IPM).

RNA interference (RNAi) has been widely utilized in the study of gene regulatory functions in insects and the biological control of pests since its initial discovery in Caenorhabditis elegans11,12,13,14. RNAi functions by suppressing the expression of key genes involved in insect growth and development, which may alter physiological functions or cause death12. Currently, Successful applications of RNAi have been reported in various insect species, such as Cnaphalocrocis medinalis15Plutella xylostella16Coridius chinensis17Rhynchophorus ferrugineus18and Spodoptera frugiperda19,20. Although RNAi is a conserved mechanism in different insect species, its efficacy can be diminished by factors such as the target site, target gene, tissue type, amount and length of double-stranded RNA (dsRNA), and delivery methods21,22,23,24,25,26. Ensuring the stability and penetration of dsRNA is critical to enhancing interference efficiency. Recently, an efficient nanocarrier (star polycation, SPc) -mediated dsRNA delivery system was developed to achieve efficient RNAi at all developmental stages of the lepidopteran insect S. frugiperda27. The nanomaterial can protect dsRNA from degradation by RNase A, insect gut fluids, and hemolymph, hereby significantly improving RNAi efficiency28,29,30. Moreover, this delivery system has demonstrated significant benefits for improving interference efficiency in many species, such as Drosophila melanogaster31Aphis glycines32Myzus persicae33Agrotis ypsilon34Aphis gossypii35. Therefore, nanocarrier-mediated RNAi deserves attention and serves as a valuable reference for studying gene function and developing novel pest control strategies.

Cathepsins are an important class of proteolytic enzymes that found in animals, plants, and viruses, playing pivotal role in various physiological and pathological processes within cells and tissues36,37,38. Based on their catalytic activity, cathepsins are classified into four families: cysteine proteases (cathepsins L, B, H, O, S, T, K, V, and F), aspartic proteases (cathepsins D and E), serine proteases (cathepsins A and G), and metalloproteases39,40. Cathepsin L (CTSL) is considered to play a significant role in the insect digestive system, immune system, embryonic development, metamorphosis, and reproduction41. For example, Cathepsin L functions as a digestive enzyme in the larvae of Diabrotica virgifera42while it is involved in innate immune responses in Diaphorina citri43Aedes aegypti44and Bombyx mori45. During the metamorphic development of Helicoverpa armigera, Cathepsin L induces midgut cell apoptosis by activating caspase-1, thereby promoting the midgut remodeling process46. Additionally, Cathepsin L participates in the degradation of the fat body and metamorphosis in Antheraea pernyi and B. mori47,48. Several studies have shown that silencing Cathepsin L can disrupt the molting and hinder the development and reproduction of aphid species49,50,51. These results suggest that Cathepsins L could serve as a promising target for pest control. However, the functional role of OfCTSL gene in O. furnacalis remains unclear.

In the present study, we cloned and characterized the OfCTSL gene based on transcriptomic data of O. furnacalis52. We analyzed tissue-specific and developmental stages gene expression levels using RT-qPCR. Nanocarrier-mediated dsRNA was used to knockdown the OfCTSL gene, exploring its effects on the O. furnacalis. Our findings provide significant information into the role of Cathepsin L in the growth, development, and reproduction of O. furnacalis.

Materials and methods

Insect rearing

The larvae of Ostrinia furnacalis were collected from a corn field of Huaxi (107°0′ E, 27°17′ N), Guiyang, Guizhou, China. The larvae were reared in a control chamber at 26 ± 1 ℃ and 65–75% RH with light-dark photoperiod of 14:10 h. Both larvae and adults were fed an artificial diet and 10% honey solution, respectively as previously described53.

Sample preparation

We meticulously collected a substantial number of O. furnacalis samples to assess the expression levels of OfCTSL. Initially, we procured samples at various developmental stages of O. furnacalis, including eggs (n = 60), 1 st instar larvae (n = 40), 2nd instar larvae (n = 20), 3rd instar larvae (n = 15), 4th to 5th instar larvae (n = 10), 3-day pupae (n = 8), as well as male and female adults (n = 8). Subsequently, we dissected nine different tissues from 3-day-old 5th instar larvae on ice, namely hindgut, midgut, foregut, silk gland, head, epidermis, fat body, Malpighian tubule and hemolymph. Each treatment was replicated three times to ensure statistical robustness. To maintain sample integrity, all collected specimens were swiftly frozen in liquid nitrogen and stored at −80 ℃ until RNA extraction process.

RNA extraction and cDNA synthesis

Total RNA was isolated from 5th instar larvae using Eastep Super® Total RNA kit (Promega, Shanghai, China) in accordance with the manufacturer instructions. For tissue-specific and developmental stages expression pattern, total RNA from the different body parts and developmental stages was extracted and stored at −80℃ until required. The integrity and the purity of the isolated RNA were assessed by 1% agarose gel electrophoresis and the NanoDrop 2000 spectrophotometer (Thermo Fisher, Waltham, MA, USA), respectively. The first-strand cDNA was synthesized using StarScript II First-strand cDNA Synthesis Mix With gDNA Remover (GenStar, Beijing, China). The cDNA was then stored at −80℃ for future experiments.

Cloning of OfCTSL gene using RT-PCR

For reverse transcription- polymerase chain reaction PCR (RT-PCR), the specific primers were designed using NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) based on the transcriptomic database of O.furnacalis52. All primers synthesized by Sangon Biotech (Shanghai) were shown in Table S1. The PCR reaction mixture carried in a 25 µL reaction system, including of 12.5 µL PCR master mix. 1 µL each of the forward and reverse primers, 3 µL of cDNA, and 8 µL of ddH2O. The cycling conditions were as follows: pre-denaturation at 95 ℃ for 3 min; 30 cycles of denaturation at 95 ℃ for 30 s, annealing at 56 ℃ for 30 s, extension at 72 ℃ for 1 min; final extension at 72 ℃ for 10 min. The expected size of the OfCTSL gene fragment was detected by 1% agarose gel electrophoresis (130 V, 25 min) and purified using the FastPure Gel DNA Extraction Mini Kit (Vazyme Biotech, Nanjing, China). The purified product incubated at 16 ℃ using pMD® 19-T Vector kit (TaKaRa, Dalian, China), and then transformed into competent Escherichia coli DH5α cells (Sangon Biotech, Shanghai, China). The vector colonies were picked and sent to Sangon Biotech (Shanghai, China) for sequencing.

Bioinformatic analyses

We identified the open reading frame (ORF) of OfCTSL gene using ORF finder (https://www.ncbi.nlm.nih.gov/orffinder). The molecular weight and isoelectric point (pI) of the OfCTSL gene were analyzed with ProtParam (https://web.expasy.org/protparam). The Signal peptide of OfCTSL was predicted with SignalP-6.0 (https://services.healthtech.dtu.dk/services/SignalP-6.0/). A phylogenetic tree was constructed using MEGA-X based on the neighbor-joining (NJ) method and bootstrap test was carried out with 1,000 replicates and visualized in iTOL(https://itol.embl.de/) (Letunic & Bork, 2021). The three-dimensional (3D) structural homology modeling of OfCTSL was predicted with SWISS-MODEL (https://swissmodel.expasy.org), and visualized using PyMOL 2.5.2 (Schrodinger, New York, NY, USA). The homology model was analyzed using the ConSurf software (https://consurf.tau.ac.il/) and optimized with PyMOL 2.5.2.

OfCTSL expression different developmental stages and tissues

The above test samples were synthesized into cDNA following the method described previously and then diluted to a consistent concentration of 500 ng/µL for follow-up reserve. We used NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) online tool to design qPCR primers (Table S1) based on the OfCTSL sequence obtained from the previous cloning and identification experiments. qPCR analysis was performed on a CFX96 Real-Time PCR System (Bio-Rad, USA) using TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, Dalian, China) to detect the expression levels of OfCTSL in the samples collected above. The RT-qPCR reaction was performed in a final volume of 20 µL containing 1 µL of sample cDNA, 1 µL of forward and reverse primer, 10 µL of TB Green® Premix Ex Taq™ II and 7 µL of ddH2O. The RT-qPCR reaction program was as follows: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5 s, annealing at 56–58 ℃ for 30 s, extension at 65 ℃ for 30 s, for a total of 40 cycles. The Ribosomal Protein L8 (RPL8) and Actin were used as internal reference genes, and the geometric mean of these two genes was applied for normalization calibration54,55. Three biological replicates and three technical replicates were performed for each developmental stages and tissues.

The synthesis of dsRNA

The dsRNA target sequence of OfCTSL was selected from the ORF based on designed primers using online RNAi design tools (https://www.flyrnai.org/cgi-bin/RNAi_find_primers.pl). The forward (OfCTSL-F1) and reverse (OfCTSL-R1) primers were designed to amplify the dsRNA target sequence (Table S1). The purified PCR product was cloned into DH5α competent cells and sequenced by Sangon Biotech (Shanghai, China). The extracted plasmid was used to amplify the correct target fragments using dsOfCTSL-F/R primers. The dsRNA of OfCTSL was synthesized and purified using the MEGAscript® Kit (Thermo Fisher, USA), and MEGAclear™ Purification Kit (Thermo Fisher, USA), respectively. The quality and integrity of the purified dsRNA was assessed as described above and stored at −80 ℃ for future experiments. The green fluorescent protein gene (GFP) was as used as control56.

Preparation of dsRNA/nanocarrier complex and bioassay

For the RNAi experiment, a star-shaped polycation (SPc) was used as the nanocarrier for dsRNA, as described by Yan et al.32 and Li et al.34. The dsRNA was mixed with nanomaterials at a mass ratio of 1:1, with the final concentration of dsCTSL being 900 ng/µL, followed by the addition of a detergent solution at 0.5% of the total volume to form the dsRNA (dsCTSL and dsGFP)/nanocarrier/detergent complex27,57,58. 1.5 µL of the synthesized complexes was injected into the 4th instar final larvae, and 7-day-old 5th instar larvae, respectively. 25 individuals were used for each treatment, with three biological replicates. Phenotypic deformities, molting and, pupation was observed with the help of stereomicroscope (SMZ25 Nikon Corporation, Tokyo, Japan). For the evaluation of female adults: To minimize potential physical harm to the female adults, we specifically selected female pupae that were on the verge of emerging and injected them with 1.5 µL of the dsRNA (dsCTSL and dsGFP)/nanocarrier/detergent complex. Within 48 h of emergence, we individually paired each female adult with two male adults and housed them in disposable plastic cups. The total number of eggs laid by each female adult of O. furnacalis in the cup was meticulously recorded (n = 20). To assess the RNAi efficiency, 48 h after all dsRNA injection experiments, 6 live samples were randomly collected for the detection of gene expression levels, with three biological replicates performed.

Statistical analysis

Excel 2019 was used for data sorting, and the relative quantitative analysis of OfCTSL expression levels was conducted using the 2−ΔΔCT method59. SPSS 27.0 (IBM Corporation, Armonk, NY, USA) was used for variance homogeneity test and one-way analysis of variance (ANOVA), and Tukey’s HSD test applied for multiple comparisons. Significant differences were indicated by different letters (p < 0.05). Independent samples t-test performed to analyze the efficiency of RNAi, with different significance levels denoted by * (p < 0.05), ** (p < 0.01), and *** (p < 0.001).

Results

Molecular characterization of OfCTSL gene

The cloned length of the OfCTSL cDNA was 1653 bp (GenBank accession no.: OL744079), containing a complete open reading frame (ORF) of 1026 nucleotides (nt) that encodes 341 amino acids (aa). The molecular formula of OfCTSL protein is C1649H2504N448O492S25, with molecular weight of 38.2 kDa and a theoretical isoelectric point 6.23, respectively. Signal peptide prediction revealed that OfCTSL contains 16 amino acid signal peptide sequence (1–16 aa). Conserved structural domain analysis identified an I29 inhibitor structural domain at the N-terminus of OfCTSL, as well as the scattered conserved sequence motif ERFNIN (EX3RX2(V/I) FX2NX3IX3N). A papain family cysteine protease peptidase_C1 domain was found at the C-terminus, with four conserved active site residues Gln142, Cys148, His287 and Asn308 identified in this structural domain, as well as the S2 subsite consisting of six residues Leu192, Met193, Ala259, Leu285, Gly288 and Ala335 (Fig. 1). The 3D structural analysis revealed that the OfCTSL protein folds into two subdomains (L and R) separated by an active site cleft (Fig. 2A). The L subdomain is primarily composed of α-helices, while the R subdomain comprises both α-helices and β-sheets. Homology of OfCTSL reveals that the dark red conserved amino acids region is critically important for its structure and function (Fig. 2B). The multiple sequence alignment results show that OfCTSL is highly conservation with Galleria mellonella, Plutella xylostella, Spodoptera frugiperda, Spodoptera litura, and Helicoverpa armigera (Fig. 3). Moreover, the phylogenetic tree results revealed that OfCTSL of O. furnacalis clustered with 11 Lepidopteran insects (Fig. 4).

Fig. 1
figure 1

Nucleotide sequence and putative amino acid sequence of OfCTSL. The start codon (ATG) of the open reading frame is indicated in bold font, and the stop codon (TAG) at the end of the open reading frame is marked with an asterisk. The signal peptide sequence is indicated by a dotted line. The underlined sequence represents the I29 inhibitor structural domain. The papain family cysteine protease peptidase_C1 domain is shown in shadow. The conserved active site and the S2 subsite indicated by boxes and circles, respectively.

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Fig. 2
figure 2

The 3D molecular structure of OfCTSL. (A) This graphic was generated using PyMOL 2.5.2. Red indicates α-helices, yellow denotes β-pleated sheets, and green represents random coils. The four conserved active site residues highlighted in purple. (B) The highly conserved regions shown in dark red.

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Fig. 3
figure 3

Multiple sequence alignment of OfCTSL with other insect species. OfCSTL, Ostrinia furnacalis (OL744079.1); GmCTSL: Galleria mellonella (XP_026748905.2); PxCSTL: Plutella xylostella (KAG7298922.1); SfCTSL: Spodoptera frugiperda (ADN19567.1); SlCTSL: Spodoptera litura (XP_022827903.1); HaCTSL: Helicoverpa armigera (AAQ75437.1). The ERFNIN motif of Cathepsin L indicated by a black box. The I29 inhibitor structural domain labelled with a red line. The papain family cysteine protease peptidase_C1 domain labelled with a blue line. The four conserved active sites indicated by red triangles.

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Fig. 4
figure 4

The phylogenetic tree of insect Cathepsin L constructed by using neighbor-joining method based on the amino acid sequence. The location of OfCTSL labeled by a red triangle. The Cathepsin L of Mus musculus were used as the outgroup. Numbers at the branches indicate the percentage of 1000 bootstrap replicates obtained using MEGA-X.

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Developmental stages and tissue-specific expression pattern of OfCTSL

The relative expression level of OfCTSL in different development stages and various body tissues was determined using RT-qPCT. The developmental stages expression profiling of the O. furnacalis reveals that the OfCTSL gene was highly expressed in pupae, followed by 5th instar larvae, and in eggs (Fig. 5A). The tissue-specific expression profile of 5th instar larvae shows the highest expression level in the hemolymph, followed by Malpighian tubule, and then the epidermis and fat body. (Fig. 5B).

Fig. 5
figure 5

Expression profiles of OfCTSL in all development stages (A) and 5th instar larval tissues (B) of O. furnacalis. Developmental stages include eggs (E), 1 st to 5th instar nymphs (L1–L5), 3-day-old pupae (P), and 3-day-old adult female (F) and male (M). And larval tissues include Hindgut (Hg), Midgut (Mg), Foregut (Fg), Silk gland (Sg), Head (He), Epidermis (Ep), Fat body (Fb), Malpighian tubule (Mt) and Hemolymph (Hl). Each bar represents the mean ± SE. Different letters above bars indicate significant differences (p < 0.05, Tukey).

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Effects of RNAi on larval molting, pupation, and female reproduction

To investigate the role of OfCTSL in larval molting, pupation, and female reproduction of O. furnacalis, dsCTSL/nanocarrier/detergent complex was synthesized and injected into the 4th instar larvae, 5th instar larvae, and pupae. The transcription level of OfCTSL in O. furnacalis 4th instar larvae was significantly reduced by 65.22% 48 h after injection (Fig. 6A) and the larvae exhibited an abnormal phenotype in 84% of cases as compared to control (Fig. 6B). Additionally, the larvae failed to molt, leading to mortality in some individuals (Fig. 6C). After 48 h of injection into 5th instar larvae, the expression OfCTSL was significantly reduced by 31.55% as compared to control (Fig. 7A). Silencing OfCTSL affects the pupation of larvae and can even cause abnormal pupae, with an abnormal phenotype rate of 58.67% (Fig. 7B, C). 48 h after injecting the final pupae, the expression level of OfCTSL in the emerged adult female significantly decreased by 48.12% (Fig. 8A), and the number of females laying eggs dropped by 44.17% compared to the control group (Fig. 8B).

Fig. 6
figure 6

Effects of knockdown of the OfCTSL gene on larval molting in O. furnacalis. (A) Expression levels of OfCTSL 48 h after dsRNA injection in 4th instar larvae. (B) Abnormal rates of larvae. (C) Abnormal phenotype of larvae. Data shown mean ± SE. P-value were calculated by independent samples t-test, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

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Fig. 7
figure 7

Effects of knockdown the OfCTSL gene on pupation in O. furnacalis. (A) Expression levels of OfCTSL 48 h after dsRNA injection in 7-day-old 5th instar larvae. (B) Abnormal rates of pupae. (C) Abnormal phenotype of pupae.

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Fig. 8
figure 8

Effects of knockdown the OfCTSL gene on female reproduction in O. furnacalis. (A) Expression levels of OfCTSL 48 h after dsRNA injection in final pupae. (B) Analysis of female fecundity after dsCTSL or dsGFP treatment (n = 20).

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Discussion

Lepidoptera is the third largest order within the class Insecta, characterized by complex and diverse metamorphosis throughout their developmental stages60. They progress from eggs to larvae, which undergo multiple molts to become pupae and eventually emerge as moths or butterflies. After mating, they fulfill their reproductive role before dying. Numerous studies have shown that Cathepsin L plays a crucial role throughout the insect developmental process61,62. However, the biological function of OfCTSL in O. furnacalis remains unknown. In this study, we identified the OfCTSL gene in O. furnacalis and performed functional characterization.

Bioinformatics analysis showed that OfCTSL encodes a protein of 341 amino acids, a which contains a signal peptide sequence and two conserved structural domains, the inhibitor structural domain (I29) and the peptidase_C1 domain, similar to those found in other Cathepsin L proteins from insects. The conserved ERFNIN motif, dispersed in the proregion of the protein, is the characteristic feature of insect Cathepsin L61. Phylogenetic tree analysis indicated that OfCTSL shares the closest evolutionary relationship with other Lepidopteran insects. The 3D structure shows that the structure of OfCTSL consists of an active site gap partition of four conserved catalytic residues Gln142, Cys148, His287 and Asn308 into two substructural domains L and R. It is highly similar to the structure of Cathepsin L from other insects, such as B. mori63H. armigera64 and Tribolium castaneum65. Cysteine Cathepsins are classified in the papain like enzymes family clan 1 (C1) due to the homology of the functional domain of the cysteine protease to the papain fold66. These results indicate that the Cathepsin L gene is highly conserved in Lepidoptera, suggesting that the OfCTSL gene may have a similar biological function to Cathepsin L in other insects.

To better understand the role of OfCTSL gene in the growth and development of O. furnacalis, we investigated its expression patterns using RT-qPCR. Our results revealed higher expression levels of OfCTSL in eggs, 5th instar larvae and pupal stage, with concentrated expression in the hemolymph. This suggests that OfCTSL may play a critical role in embryonic development, molting, and pupation. In insect eggs, the main developmental processes includes the specific protease degradation of vitellin and vitellogenin, and the acquisition of amino acids, energy and nutrients necessary for embryonic development67. For instance, elevated levels of Cathepsin L have been identified in the eggs of B. mori, where it plays a critical role in this process68. Additionally, Cathepsin L is involved in insect molting and metamorphosis. For example, Cathepsin L is significantly highly expressed in the 5th instar larvae of Hermetia illucens69. In H. armigera, Cathepsin L is specifically expressed in hemocytes of 5th instar larvae, with notably increased expression during larval molting and pupation, suggesting its association with insect developmental processes64.

It is well-known that hemocytes in insect hemolymph regulate innate immunity by phagocytosing pathogens70. A Cathepsin L-like cysteine protease specifically expressed in hemocytes of B. mori was found to significantly respond to bacterial infection71. Upon Escherichia coli invasion, the expression of Cathepsin L in the intestine of H. illucens larvae increases significantly69. Notably, tissue remodeling is a critical biological event during the metamorphic transition of holometabolous insects from larval molting to pupation, characterized by programmed cell death (PCD) of larval tissues and differentiation of adult tissues72,73,74. Studies have confirmed that hemocytes not only clear untransformed larval tissues via phagocytosis but also directly participate in the construction and remodeling of adult tissues75,76. For instance, approximately 20–30% of hemocytes undergo PCD during molting and metamorphosis in B. mori77. Cathepsin L is involved in and regulates these essential processes. HaCTSL is highly expressed during 5th to 6th instar molting stages and pre-pupation in H. armigera78It’s inhibition by either a cysteine protease inhibitor (E-64) or the Cathepsin L-specific inhibitor CLIK148 significantly delays the molting onset, suggesting that it plays a key role in the larval molting cycle. Zhang et al.79 further found that HaCTSL is primarily expressed in the fat bodies of final instar larvae and early pupae of H. armigera and participated in the process of dissociating fat body cells and releasing them into the hemolymph. Additionally, Cathepsin L can enter cells from the fat body to regulate PCD, contributing to the degradation of internal tissues during the larva-to-pupa transition48. Therefore, we speculate that OfCTSL may be involved in the ontogenesis and cellular immunity of O. furnacalis.

Some studies have reported that Cathepsin L is involved in insect growth and development, as well as the regulation of female fecundity80,81,82. To clarify the definite effects of OfCTSL on O. furnacalis, we utilized nanocarrier-mediated RNAi to target the OfCTSL gene of O. furnacalis. The results showed that the interference efficiency of dsOfCTSL/nanocarrier/detergent complex ranged from 31.55 to 65.22% after injection into 4th instar larvae, 5th instar larvae and last instar pupae, respectively. This significantly led to the failure to molt or even death of larvae, pupal deformities, and a significant reduction in adult fecundity. Studies in other species have also demonstrated the function of Cathepsin L. For example, Cathepsin L is involved in fat body degradation and tissue remodeling by regulating hemocyte granulation and PCD, and silencing its expression leads to incomplete molting and abnormal pupation in H. armigera and B. mori48,64,78,83. Additionally, knockdown of MpCTSL and TsCTSL genes were significantly reduced fecundity of M. persicae and Trichinella spiralis51,84. As Cathepsin L localizes to the ovary and spermatheca, directly influencing female reproduction by regulating the expression of the Vitellogenin gene and ovarian development80,85. These results suggest that Cathepsin L plays a critical role in molting, pupation, and fecundity of O. furnacalis, suggesting its potential as an RNAi target for pest control strategies.

RNAi is a biological mechanism in which dsRNA regulates the expression of important genes in organisms, it can selectively silencing specific genes in pest species to cause loss of gene function and achieve the purpose of eliminating pests21,86,87. Although RNAi technology has been widely applied in the fields of gene function research and pest control, its effectiveness in some insects often faces significant challenges21,88. The main reasons are the rapid degradation of dsRNA by nucleases and the low efficiency of cellular dsRNA uptake, as the stability and delivery efficiency of dsRNA limit the technology’s application and development in insensitive insects89,90. In recent years, the development of nanotechnology has provided new solutions to overcome these obstacles28,29,30. For example, nanocarrier-based RNAi has achieved high gene silencing efficiency across all developmental stages of S. frugiperda27. Yan et al.32 constructed a nanocarrier-based transdermal dsRNA delivery system, which achieved the highest mortality rate when applied via spraying to A. glycines. Zhang et al.32 developed a broad-spectrum RNA nano-pesticide that effectively controls M. persicae and Acyrthosiphon pisum, while being safe for non-target predatory ladybird Harmonia axyridis. Ma et al.91 enhanced RNAi-based control of Sogatella furcifera using nanoparticle delivery, with no harmful effects on the natural enemy Cyrtorhinus lividipennis. A dual-target RNA biopesticide based on SPc nanocarriers was designed for neuropeptide F receptor (NPFR) and AMP-activated protein kinase (AMPK) in O. furnacalis57. After field spraying, this biopesticide achieved control efficiencies of 80% and 84% against newly hatched larvae and 3rd-instar larvae within 7 d, respectively, without significantly affecting the expression of AMPK genes in beneficial insects Orius sauteri and Apis mellifera. Thus, these results indicate that nanomaterial-based RNAi technology exhibits significant advantages in scientific research and pest control, effectively improving RNAi stability and delivery efficiency while reducing impacts on natural enemies and non-target organisms through precise targeting, providing a viable solution for green control of agricultural pests.

Conclusions

In summary, we identified and analyzed the molecular features of the O. furnacalis OfCTSL gene, and clarified its expression pattern at different developmental stages and in different tissues, and functional characterization of OfCTSL was performed using RNAi. The results showed that OfCTSL played a significant role throughout the metamorphic development of O. furnacalis, including larval molting, pupation, and female fertility. Furthermore, we demonstrated the feasibility of using nanomaterials to enhance RNAi efficiency in O. furnacalis. Our study not only highlights the essential of Cathepsin L for the ontogenesis and reproduction of O. furnacalis but also provides new insights for the development of novel pest control strategies and the identification of candidate gene targets.