Zona pellucida proteins Piopio and Dumpy are essential for the apical extracellular matrix assembly and survival of Locusta migratoria

Abstract

BACKGROUND

The apical extracellular matrix (aECM) forms a critical interface between epithelial cells and the external environment. Its integrity, stability, and flexibility are crucial for the survival of organisms. However, the molecular mechanisms governing the correct assembly and maturation of the insect aECM remain poorly understood, impeding the development of pest control strategies.

RESULTS

The conserved zona pellucida (ZP) protein Piopio in Locusta migratoria (LmPio) was identified and characterized. LmPio is predominantly expressed in ectoderm‐derived tissues, and the protein localizes within the cuticle assembly zone in the apical region of epidermal cells. RNA interference (RNAi)‐mediated knockdown of LmPio resulted in 71.89% mortality, associated with cuticle thinning, reduced lamellae number, and impaired adhesion between the epidermis and cuticle. Furthermore, the transport of cuticular lipids and proteins to the cuticle was severely disrupted, although chitin content remained unchanged. In immunoprecipitation experiments we identified Dumpy (LmDpy), another ZP protein, as a potential partner of LmPio. Knockdown of LmDpy resulted in 100% mortality of locusts, with a phenotype highly similar to that induced by LmPio knockdown. Co‐immunoprecipitation and pull‐down assays confirmed that LmPio directly interacted with LmDpy to jointly regulate aECM assembly and maturation.

CONCLUSION

Our results demonstrate that LmPio and LmDpy are essential for the formation of the assembly zone and the maintenance of cuticle integrity. These findings reveal the critical role of ZP proteins in aECM assembly and cuticle morphogenesis. Knockdown of LmPio or LmDpy resulted in high mortality, validating these genes as promising targets for the RNAi‐based control of this pest. © 2026 Society of Chemical Industry.

Piopio in Locusta migratoria localizes to the assembly zone, mediates epidermis–cuticle adhesion, coordinates apical lipid and protein transport, and interacts with Dumpy to jointly regulate the apical extracellular matrix assembly and maturation.

1. INTRODUCTION

The apical extracellular matrix (aECM) is a protective layer covering the apical surface of epithelial cells, and its proper assembly and maturation are crucial for organismal survival. ¹ , ² , ³ In insects, the epidermal cuticle is an aECM that interfaces with the external environment. This structure not only provides a physical protective barrier against external cues (e.g., pathogens, pollution etc.) but also offers structural support and assists shaping of the body and appendages such as wings and legs. ⁴ , ⁵ , ⁶ From embryogenesis to adulthood, insects undergo complex metamorphic development, during which the aECM undergoes periodic degradation and reconstruction, called molting, to accommodate growth. ¹ , ⁷ While cuticle structure and its degradation during molting have been well described in a number of insect species, ⁵ , ⁸ , ⁹ , ¹⁰ , ¹¹ the molecular mechanisms responsible for the assembly and maturation of this aECM remain poorly understood. However, a full understanding of these mechanisms is crucial for the development of sustainable, species‐specific and eco‐friendly pest control strategies targeting the insect cuticle.

Nearly four decades ago, the assembly zone (also known as the deposition or adhesion zone) was identified as a narrow region between the cuticle and epidermis. It plays a central role in cuticle formation, as it is the site where cuticular proteins and putative enzymes are localized. ¹² Recent studies in Drosophila larvae have shown that DmObst‐A localizes to the apical end of epidermal cells and is specifically enriched in the assembly zone; it functions in concert with the deacetylases Serp and Verm, as well as the chitin protectant Knk, to promote aECM maturation and ensure its structural integrity. ¹ Chitinase 2 (Cht2) has also been implicated in this process. ¹³ , ¹⁴ Similarly, in Tribolium castaneum, TcCDA1 and TcCDA2 are located in the assembly zone, where they collaborate to maintain overall cuticle shape and structural integrity. ¹⁵ Collectively, these findings underscore the importance of chitin‐related factors in the proper assembly and maturation of the aECM. However, only a few proteins and enzymes of the assembly zone have been identified, and the molecular events occurring within this region remain largely unknown. Understanding these processes may provide valuable insights for the development of novel and intelligent pest control strategies.

In addition to the chitin‐related factors described above, zona pellucida (ZP) proteins are considered the most promising candidates for mediating aECM assembly. In multiple model systems, ZP proteins have been shown to be involved in the early stage of aECM formation. As key components of the apical cell membrane and aECM, they play crucial roles in organizing and shaping the highly specialized apical structures of epithelial cells. ² , ¹⁶ Piopio (Pio), initially identified in Drosophila, ¹⁷ is a transmembrane ZP protein localized to the apical surface of epithelial cells and participates in microtubule reorganization during wing and tracheal morphogenesis. During tracheal formation and muscle force transmission, Pio mediates adhesion between the apical membranes of tracheal and tendon cells and the overlying chitinous aECM in a Notopleural‐dependent manner, thereby ensuring cuticle integrity and stability. ⁴ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ Furthermore, in T. castaneum, Pio regulates metamorphosis and reproduction by modulating ecdysone and juvenile hormone (JH) metabolism, indicating its importance in developmental processes. ²¹ However, the function of Pio within the assembly zone of Locusta migratoria has not yet been investigated.

L. migratoria is a major agricultural pest worldwide, causing severe crop damage and substantial economic losses to global agriculture. ²² Currently, its control relies primarily on chemical insecticides, but long‐term overuse of traditional chemical control strategies has led to a triple predicament: escalating insecticide resistance, environmental pollution, and unintended harm to non‐target organisms. ²³ RNA interference (RNAi) is a promising eco‐friendly insecticidal strategy, ²⁴ and the key to its success lies in identifying genes essential for the survival of L. migratoria.

To address the aforementioned knowledge gap and explore novel RNAi targets for L. migratoria control, we identified a ZP gene, Pio, in L. migratoria. We employed a combination of RT‐qPCR, immunohistochemistry, immunogold labeling, RNAi, hematoxylin–eosin (H&E) staining, transmission electron microscopy (TEM), cuticular component quantification, immunoprecipitation (IP), mass spectrometry (MS), AlphaFold 3 prediction, co‐immunoprecipitation (Co‐IP), and pull‐down assays to clarify its biological functions and underlying molecular regulatory mechanisms. Our results demonstrate that LmPio and its interacting protein Dumpy (Dpy) are essential for aECM assembly and cuticle morphogenesis in L. migratoria. RNAi‐mediated knockdown of these two genes disrupts these processes, leading to high mortality. The results of this study validate that LmPio and LmDpy are promising RNAi targets for locust control.

2. MATERIALS AND METHODS

2.1. Insect rearing

Eggs of L. migratoria were purchased from a locust breeding center in Cangzhou, Hebei Province, China. Newly hatched nymphs were reared in a growth chamber maintained at 30 ± 2 °C, 40–60% relative humidity, with a 14: 10 h light:dark photoperiod. The insects were fed fresh wheat seedlings. Third‐ and fifth‐instar nymphs were used for RNAi.

2.2. Identification and bioinformatics analysis of LmPio

Using the sequence of Drosophila melanogaster Pio as a query, the cDNA sequence of LmPio was identified by querying the whole‐body transcriptome (GEZB00000000) and genome databases (AVCP00000000) of L. migratoria. ²⁵ , ²⁶ Amino acid sequences were deduced using the translation tool ExPASy (http://www.expasy.org/tools/dna.html) and the corresponding protein domains were analyzed using SMART (http://smart.embl-heidelberg.de). The molecular weights and isoelectric points of the amino acid sequences were predicted using the ExPASy proteomics server (http://www.expasy.org). The furin cleavage site was predicted using ProP 1.0 (https://services.healthtech.dtu.dk/services/ProP-1.0). Orthologous sequences were retrieved using BLASTP, and multiple sequence alignments were performed using GENEDOC software. A phylogenetic tree was constructed using the neighbor‐joining method in MEGA 7.0. All the amino acid sequences used are listed in Supporting Information, Table S1.

2.3. Total RNA isolation and cDNA synthesis

To analyze developmental expression patterns, integument samples (the second segment) were dissected from 1‐day‐old second‐instar nymphs (N2D1) to 3‐day‐old fourth‐instar nymphs (N4D3). For tissue‐specific expression profiling, eight tissues were dissected from 4‐day‐old third‐instar nymphs (N3D4): antennae (AN), integument (IN), wing pads (WP), foregut (FG), gastric caeca (GC), midgut (MG), hindgut (HG), and legs (LE). Each sample consisted of pools of five nymphs, with three biological replicates. Total RNA was extracted using RNAiso Plus (Takara, Kusatsu, Japan), quantified using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and assessed for quality on a 1% agarose gel. Total RNA (1 μg) was used as a template to synthesize cDNA by using a reverse transcription kit (Vazyme Biotech, Nanjing, China) and each cDNA sample was diluted fivefold for subsequent RT‐qPCR analysis.

2.4. RT‐qPCR analysis

The above‐diluted cDNA was used as a template and BlasTaq™ 2X qPCR MasterMix (Abm, Vancouver, Canada) was used for RT‐qPCR. The specific system and method were performed as described previously. ²⁷ EF‐1α was used as the reference gene owing to its stable expression across tissues and developmental stages in L. migratoria. ²⁸ , ²⁹ All reactions were performed with at least three independent biological replicates. The primers used in this study are listed in Supporting Information, Table S2.

2.5. Antibody preparation and immunohistochemistry

A specific peptide sequence (LVTNQGRGFH, amino acids 118–128) in the ZP domain of LmPio was used to generate a mouse monoclonal antibody (prepared by the International Research Laboratory of Prevention and Control of Important Animal Infectious Diseases and Zoonotic Diseases of Jiangsu Higher Education Institutions, Yangzhou University, Yangzhou, China). For immunohistochemistry, integument tissues from 2‐ to 4‐day‐old third‐instar nymphs (N3D2–N3D4) were fixed and embedded in paraffin, and sections (5 μm) were prepared as previously described. ³⁰ The sections were incubated with the anti‐LmPio mouse monoclonal antibody (1:50) overnight at 4 °C and washed three times with 1 × PBS for 5 min each. A fluorescent secondary antibody (1:200) was applied and incubated at 37 °C for 2 h in the dark. After washing three times with 1 × PBS, chitin was stained with 1 mg mL⁻¹ Fluorescent Brightener 28 (Sigma, USA) for 30 s, and nuclei were labeled with SYTOX™ Green Nucleic Acid Stain (1:5000, Life Technologies, USA) for 15 min. The slides were mounted with antifade mounting medium (Beyotime, Shanghai, China) and imaged using an LSM 880 confocal laser‐scanning microscope (Zeiss, Oberkochen, Germany) at 100× magnification.

2.6. RNA interference

Primers containing T7 promoter sequences (Supporting Information, Table S3) were designed to synthesize double‐stranded RNA (dsRNA) targeting GFP (dsGFP, control), LmPio (dsLmPio), and LmDpy (dsLmDpy). DsRNA was synthesized using the T7 RiboMAX™ Express RNAi system (Promega, Madison, WI, USA), quantified by NanoDrop 2000, and its integrity confirmed by 1% agarose gel. The concentration of dsRNA was adjusted to 2 μg μL⁻¹. Using a microinjector, 6 μg of dsLmPio or dsLmDpy was injected into the hemocoel between the second and third abdominal segments of 1‐day‐old third‐instar nymphs (N3D1), 10 μg of dsLmPio was injected into the same site of 1‐day‐old fifth‐instar nymphs (N5D1), and equal amounts of dsGFP were injected as a control. Three biological replicates were performed. After injection of dsRNAs, integument tissues (of the second segment) were dissected at 48 and 96 h post‐injection, and the silencing efficiency of LmPio or LmDpy was determined by RT‐qPCR as described above. L. migratoria were continuously fed, and their daily growth and development were observed and recorded until they molted into fourth‐instar nymphs or adults.

2.7. Hematoxylin–eosin staining

Microscopic sectioning and H&E staining were performed to further explore the effects of LmPio and LmDpy on cuticle development. Integument samples (of the second dorsal segment) from dsRNA‐treated nymphs (N3D2–N3D4) were dissected and fixed with 3% glutaraldehyde solution at 4 °C. Paraffin sectioning and H&E staining were performed as previously described. ³⁰ The sections were observed under an Olympus BX51 microscope, and images were captured using an Olympus digital camera.

2.8. Transmission electron microscopy and immunogold labeling

To further observe the effects of LmPio and LmDpy on the development of the cuticle and assembly zone at the ultrastructural level, integument samples (from the dorsal region of the third segment) from dsRNA‐treated and control nymphs were collected as described above. Ultra‐thin sections (80 nm) were prepared as previously described. ³⁰ To determine the precise location of LmPio in the cuticle, immunogold labeling was performed using the LmPio‐specific antibody as previously described. ³¹ The primary and secondary antibodies (all mouse‐derived) were diluted 1:50. Images were captured using a JEM‐1200EX transmission electron microscope (JEOL, Tokyo, Japan).

2.9. Quantitative analysis of cuticular components

The contents of the main components in the cuticle of L. migratoria were compared. Cuticular lipid, chitin, and protein contents were measured in the dsGFP‐ and dsLmPio‐treated groups at N3D2, N3D3, and N3D4, whereas those in the dsLmDpy‐treated group were only measured at N3D2 and N3D3. Integument tissues (from the second to eighth segments) from five locusts were collected per replicate, with at least three biological replicates. Measurements were performed as previously described. ³² The expression of lipid transport‐related genes (LmABCH‐9B and LmABCH‐9C) was detected by RT‐qPCR. The primers used are listed in Supporting Information, Table S2.

2.10. Immunoprecipitation, mass spectrometry, and AlphaFold 3 prediction

To identify the proteins that may interact with LmPio, IP and MS analyses were conducted. Proteins from the integument of N3D4 nymphs were extracted for IP using the LmPio mouse monoclonal antibody. Supernatant from SP2/0 cells served as a negative control. Protein A+G magnetic beads (Beyotime) were washed three times with 1 × TBS before use. The eluted proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and analyzed by liquid chromatography–tandem mass spectrometry (Fanxing Boao, Beijing, China). Potential interacting proteins were further evaluated using AlphaFold 3 for structure‐based interaction prediction, and the results were visualized using PyMOL software.

2.11. Co‐immunoprecipitation and pull‐down assays

Co‐IP and pull‐down assays were conducted to verify the relationship between LmPio and LmDpy. The cDNA sequences of LmPio (amino acids 30–337, and the Furin protease cleavage site sequence RRRR was mutated to AAAA) and LmDpy (amino acids 20 284–20 884) were ligated into pEZT‐BM plasmids (From Defen Lu's lab, Taiyuan, China) to generate LmPio‐His and LmDpy‐HA constructs. The recombinant LmPio‐His was co‐expressed with LmDpy‐HA in HEK293T cells at 37 °C in Dulbecco's modified eagle medium (Gibco, Grand Island, NY, USA). LmPio‐His and LmDpy‐HA were expressed separately in HEK293T cells, with untransfected HEK293T cells serving as controls. Cells were harvested by centrifugation at 48 h post‐infection, and their total lysates were prepared for subsequent experiments. Protein A+G magnetic beads and anti‐His/HA antibodies (Bioss, Beijing, China) were used for Co‐IP assays. The experiments were performed in accordance with the manufacturer's instructions. Finally, the harvested magnetic beads were boiled with 1 × SDS‐PAGE loading buffer (Bioss) before SDS‐PAGE and western blotting (WB) using anti‐His/HA antibodies.

The purified LmPio‐His fusion protein was prepared. Lysates of HEK293T cells transfected with LmDpy‐HA were enriched using anti‐HA‐Protein A+G magnetic beads, followed by elution via peptide competition to obtain micropurified LmDpy‐HA protein for subsequent pull‐down experiments. The purified LmPio‐His protein (30 μg) was mixed with Ni²⁺‐NTA agarose beads (50 μL), and the mixture was supplemented with pre‐chilled binding buffer to a final volume of 500 μL. The mixture was incubated on a horizontal rotator at 4 °C for 2 h for conjugation, with a blank control set in parallel. After incubation, the mixture was centrifuged at 3000 × g for 3 min at 4 °C, the supernatant was discarded, and the agarose beads were washed twice with binding buffer. Subsequently, the micro‐purified LmDpy‐HA protein (250 μg) was added and the mixture was supplemented with binding buffer to a final volume of 1 mL. The mixture was then incubated on a horizontal rotator at 4 °C overnight. After incubation, the mixture was centrifuged as described above, and the resulting precipitate was washed four times with washing buffer. Finally, the harvested agarose beads were boiled with 1 × SDS‐PAGE loading buffer before performing SDS‐PAGE and WB using anti‐His or anti‐HA antibodies.

2.12. Statistical analysis

The relative mRNA levels of target genes were calculated using the 2^−∆∆Ct method. ³³ Significant differences among developmental stages and different tissues were analyzed using Tukey's HSD multiple comparison test (different letters represent significant differences). All other data were statistically analyzed using independent sample Student's t‐test. Asterisks indicate significant differences (*P < 0.05, **P < 0.01, *** P < 0.001). All data were analyzed using SPSS 22 software (IBM Corp., Armonk, NY, USA).

3. RESULTS

3.1. Identification and characterization of LmPio

To analyze the molecular characteristics of LmPio, its cDNA sequence was obtained from the transcriptome of L. migratoria. Comparison with the genomic sequence revealed that LmPio consists of eight exons, with an open reading frame of 1026 bp (Fig. 1(A)). The LmPio gene encodes a protein consisting of 401 amino acids, with a predicted signal peptide domain (amino acids 1–21), a ZP domain (amino acids 41–285), and a transmembrane domain (amino acids 349–371). The predicted molecular weight was 43.91 kDa and the isoelectric point was 7.02. In addition, a putative furin cleavage site (RRRR, amino acids 288–291) was identified, indicating potential proteolytic processing and secretion of the extracellular domain (Fig. 1(B)).

Figure 1.

Bioinformatics analysis of LmPio. (A) Structure of LmPio. The blue box indicates the exon and the black line represents the intron. (B) Schematic of the predicted protein structure of LmPio. The red, green, and orange boxes indicate the signal peptide, zona pellucida, and transmembrane domain, respectively. (C) Multiple sequence alignments of Pio in insects. The red, green, and orange underlines represent the SP (signal peptide), ZP (zona pellucida), and TM (transmembrane) domain regions, respectively. The red asterisk indicates the conserved cysteine. Lm, Locusta migratoria; Sa, Schistocerca americana; Dm, Drosophila melanogaster; Aa, Aedes albopictus; Sf, Spodoptera frugiperda; Bm, Bombyx mori; Mp, Myzus persicae; Tc, Tribolium castaneum; Ci, Chelonus insularis; Cs, Coccinella septempunctata; Cc, Chrysoperla carnea. (D) A phylogenetic tree of Pio in insects was constructed using the neighbor‐joining method.

Direct homologs of LmPio from other insect species were obtained through BLAST analysis to reveal the conservation and evolutionary relationship of Pio. No direct homologs were found in species other than insects, suggesting that it may perform insect‐specific functions. Multiple sequence alignment analysis indicated that Pio is conserved among insects, with a ZP domain containing eight strictly conserved cysteine residues (Fig. 1(C)). Phylogenetic analysis indicated that LmPio is closely related to its putative ortholog from Schistocerca americana and more distantly related to orthologs from lepidopteran species (Fig. 1(D)).

3.2. Expression pattern and tissue localization of LmPio

The spatiotemporal expression characteristics of LmPio were analyzed to explore its potential physiological functions. The expression level of LmPio was low on the first day of each instar, then gradually increased during the instar, and peaked just before molting (Fig. 2(A)). Tissue‐specific analysis showed that LmPio was highly expressed in the antennae, integument, wing pads, foregut, hindgut, and legs, with lower expression levels in the gastric caeca and midgut (Fig. 2(B)). Immunohistochemical results showed that LmPio was localized apically in epidermal cells, appearing as a continuous thin signal at mid‐instar (N3D3). In late third‐instar nymphs (N3D4), a discontinuous thin signal was detected beneath the old cuticle and between the new cuticle and epidermis (Fig. 2(C)). An immunogold labeling technique was applied to precisely determine the localization of LmPio in the cuticle. As shown in Fig. 2(D), LmPio was predominantly localized to the assembly zone at the apical cell surface of N3D3 nymphs, suggesting that it may play an important role in this region.

Figure 2.

Expression pattern of LmPio and localization of LmPio protein. (A) Relative expression of LmPio in nymphs at different days was determined by RT‐qPCR. N2D1–N4D3, 1‐day‐old second‐instar nymphs to the 3‐day‐old fourth‐instar nymphs. (B) Relative expression of LmPio in different tissues of 4‐day‐old third‐instar nymphs was determined by RT‐qPCR. AN, antenna; IN, integument; WP, wing pads; FG, foregut; GC, gastric caeca; MG, midgut; HG, hindgut; LE, legs. All data are shown as mean ± SD. Different letters above the bars represent statistically significant differences based on one‐way ANOVA analysis followed by Tukey's tests. (C) Localization of LmPio in the integument (N3D2–N3D4) by immunohistochemistry. Chitin, nucleus, and LmPio were detected by blue, green, and red signals, respectively. Cut, cuticle; OC, old cuticle; NC, new cuticle; EC, epidermal cell. The white arrows indicate the LmPio signal. Scale bar indicates 10 μm. (D) Immunogold localization of LmPio. Endo, endocuticle; AZ, assembly zone; Mv, microvilli; EC, epidermal cell. The blue dashed boxes indicate the magnified view of the local area, the cyan dashes indicate the apical cell surface, the yellow dashes mark the basal limit of the endocuticle, with the intervening region being the assembly zone, and the white arrows point to immunogold labeling of LmPio. Scale bars indicate 500 and 250 nm, respectively.

3.3. LmPio is crucial for development and molting of L. migratoria

The effects of LmPio on the growth and development of L. migratoria were investigated using RNAi. Equal amounts of dsGFP and dsLmPio were injected into N3D1 nymphs, and the silencing efficiency was determined by RT‐qPCR. The transcription level of LmPio in the dsLmPio‐treated group was significantly reduced by 90.21% compared with that in the dsGFP‐control group (Fig. 3(A)). Detailed phenotypic observations and recordings were conducted daily during the growth and development of L. migratoria. The dsGFP‐control nymphs molted successfully to the fourth instar. However, the dsLmPio‐treated nymphs exhibited 15.63% pre‐ecdysial mortality and 56.26% mortality during ecdysis, resulting in a total mortality of 71.89%, whereas some surviving individuals displayed abnormally erect wing phenotypes (Fig. 3(B),(C)). Similar results were observed following RNAi in fifth‐instar nymphs (Supporting Information, Fig. S1). These results indicate that LmPio is critical for the normal growth, development, and molting of L. migratoria.

Figure 3.

Effects on the growth and development of Locusta migratoria after injection of dsLmPio in 1‐day‐old third‐instar nymphs. (A) Relative expression of LmPio after injection of dsLmPio was determined by RT‐qPCR. All data are shown as mean ± SD. Statistical significance was analyzed with Student's t‐test. Asterisks indicate significant differences (**P < 0.01). (B) Statistical analysis of mortality after injection of dsLmPio. All data are shown as mean ± SD. Statistical significance was analyzed with Student's t‐test. Asterisks indicate significant differences (***P < 0.001). (C) Phenotypes in growth and development of L. migratoria after injection of dsLmPio. BE, before ecdysis; EC, ecdysis. The red slashes indicate lethality and the yellow boxes indicate the magnified view of the wings. Scale bar indicates 5 mm.

3.4. LmPio is essential for aECM formation and epidermis–cuticle attachment

To further clarify the role of LmPio, integument sections from different developmental stages were analyzed by H&E staining and TEM. H&E staining showed that the cuticle was significantly thinner in the dsLmPio‐treated group than in the dsGFP‐control group at N3D2 and N3D3, with reductions of 57.05% and 27.83%, respectively, mainly due to thinning of the endocuticle. At a later developmental stage (N3D4), a new cuticle was formed in both groups, although it was thinner in the dsLmPio‐treated group than in the dsGFP‐control group (Fig. 4(A),(B)). TEM observations confirmed these results (Fig. 5(A),(B)). To further analyze the effect of LmPio on cuticular thickness, the cuticular lamellar structure was observed at the ultrastructural level and the number of lamellae was assessed. The results showed that the number of cuticular lamellae was significantly reduced by 33.33% in the dsLmPio‐treated group at N3D3 but not at N3D2 (Fig. 5(A),(C)). These results indicate that LmPio is essential for normal cuticle formation and deposition.

Figure 4.

Effects on the microstructure of the cuticle in Locusta migratoria after injection of dsLmPio. (A) Microstructural analysis of the cuticle by H&E after injection of dsLmPio. Exo, exocuticle; Endo, endocuticle; OC, old cuticle; NC, new cuticle. The yellow I‐shaped lines represent cuticular thickness, the red arrows indicate the exocuticle, the green arrows indicate the endocuticle, and the black triangles indicate the old and new cuticle. Scale bar indicates 20 μm. (B) Quantification analysis of cuticular thickness changes after injection of dsLmPio at the microscopic level. All data are shown as mean ± SD. Statistical significance was analyzed with Student's t‐test. Asterisks indicate significant differences (**P < 0.01, ***P < 0.001).

Figure 5.

Effects on the ultrastructure of the cuticle and assembly zone in Locusta migratoria after injection of dsLmPio. (A) Ultrastructural analysis of the cuticle by TEM after injection of dsLmPio. Epi, epicuticle; Exo, exocuticle; Endo, endocuticle; OC, old cuticle; NC, new cuticle; AZ, assembly zone; Mv, microvilli. The orange I‐shaped line represents cuticular thickness, the black triangles indicate the old and new cuticle, the blue dashed boxes indicate the magnified view of the local area, the cyan dashes indicate the apical cell surface, the yellow dashes mark the basal limit of the endocuticle, with the intervening region being the assembly zone, the white double arrows represent the thickness of the assembly zone, the red arrows point to the microvilli, and the green arrows indicate the space between the epidermis and the cuticle. Scale bars indicate 1 μm and 250 nm, respectively. (B) Quantification analysis of cuticular thickness changes after injection of dsLmPio at the ultrastructural level. (C) Quantification analysis of cuticular lamellae changes after injection of dsLmPio at the ultrastructural level. All data are shown as mean ± SD. Statistical significance was analyzed with Student's t‐test. Asterisks indicate significant differences (*P < 0.05, **P < 0.01).

Since the assembly zone is critical for cuticular fiber assembly and LmPio localizes to this region, we examined its ultrastructure. In the controls, the assembly zone thickened progressively during the instar, with microvilli arranged in an orderly manner and closely associated with the cuticle. However, the assembly zone was thinner in the dsLmPio‐treated nymphs than in the controls, although the microvillus structure appeared normal. As development proceeded, the assembly zone gradually thickened, but it was still thinner than in respective dsGFP‐control nymphs. We also observed the detachment of the cuticle from the epidermis (Fig. 5(A)). These results indicate that LmPio is critical for the normal formation of the assembly zone and for the maintenance of adhesion between the epidermis and cuticle.

3.5. LmPio is crucial for the transport of lipids and proteins in the cuticle

The cuticle is primarily composed of lipids, chitin, and proteins. We hypothesized that assembly zone defects could impair the transport of cuticular components. Quantitative analyses revealed that compared with the dsGFP‐control group, lipid content was significantly reduced in the dsLmPio‐treated group at N3D2 and N3D3, as well as in the new cuticle of N3D4 nymphs, by 59.18%, 70.93%, and 72.09%, respectively (Fig. 6(A)). Chitin content showed no significant changes at any developmental stage (Fig. 6(B)). Protein content was significantly reduced only at N3D3, by 27.78% (Fig. 6(C)). Collectively, these results demonstrate that LmPio is indispensable for the transport of lipids and proteins into the cuticle. Given the significant reduction in lipid content, we further analyzed two well‐characterized lipid transport genes (ABCH‐9B and ABCH‐9C), which mediate lipid transport from epidermal cells to the cuticle. ³⁴ , ³⁵ Knockdown of LmPio significantly down‐regulated the expression of LmABCH‐9B and LmABCH‐9C by 71.82% and 58.96%, respectively (Supporting Information, Fig. S2), suggesting that lipid trafficking was disrupted, which consequently decreased cuticular lipid content. Together, these findings confirm our initial hypothesis that LmPio‐mediated aECM assembly is critical for the transport of lipids and proteins in the cuticle.

Figure 6.

Changes in substance content of the cuticle on different days after injection of dsLmPio in Locusta migratoria. (A) Changes in cuticular lipid content in pools of five insects on different days (N3D2–N3D4) after dsLmPio injection. (B) Changes in cuticular chitin content in pools of five insects on different days (N3D2–N3D4) after dsLmPio injection. (C) Changes in cuticular protein content in pools of five insects on different days (N3D2–N3D4) after dsLmPio injection. OC, old cuticle; NC, new cuticle. All data are shown as mean ± SD. Statistical significance was analyzed with Student's t‐test. Asterisks indicate significant differences (**P < 0.01, ***P < 0.001).

3.6. LmPio interacts with LmDpy

To identify potential LmPio‐interacting proteins, we performed IP followed by MS using the LmPio‐specific antibody (Fig. 7(A)). Sixty proteins were specifically precipitated in the anti‐LmPio group compared with the SP2/0 control group (Supporting Information, Table S4). Given that ZP proteins often form filaments or matrices through homo‐ or heteropolymerization, ³⁶ we focused on ZP family candidates. Among these proteins, Dumpy (Dpy), another large ZP domain protein with a furin cleavage site, was identified as a candidate interactor (Fig. 7(B) and Supporting Information, S3A).

Figure 7.

Analysis of the interaction between LmPio and LmDpy. (A) Schematic of the immunoprecipitation protocol for LmPio. (B) Tandem mass spectrum of the peptide fragments of LmPio and LmDpy. (C) Predicted model of LmPio and LmDpy complex and key interaction sites. (D) Ultrastructural analysis of the cuticle and assembly zone by TEM after injection of dsLmDpy. Epi, epicuticle; Exo, exocuticle; Endo, endocuticle; OC, old cuticle; AZ, assembly zone; Mv, microvilli. The orange I‐shaped line represents cuticular thickness, the blue dashed boxes indicate the magnified view of the local area, the cyan dashes indicate the apical cell surface, the yellow dashes mark the basal limit of the endocuticle, with the intervening region being the assembly zone, the white double arrows represent the thickness of the assembly zone, the red arrows point to the microvilli, and the green arrows indicate the space between the epidermis and the cuticle. Scale bars indicate 1 μm and 500 nm, respectively. (E) Quantification analysis of cuticular thickness changes after injection of dsLmDpy at the ultrastructural level. (F) Quantification analysis of cuticular lamellae changes after injection of dsLmDpy at the ultrastructural level. All data are shown as mean ± SD. Statistical significance was analyzed with Student's t‐test. Asterisks indicate significant differences (**P < 0.01, ***P < 0.001). (G) Co‐immunoprecipitation (Co‐IP) assay of LmPio and LmDpy. LmPio‐His and LmDpy‐HA expressed in HEK293 cells to confirm their binding. Western blots of input cell lysates (top) and IP‐HA elution (bottom) detected with anti‐HA and anti‐His antibodies are shown. LmPio‐His was only detectable in IP elution when co‐expressed with LmDpy‐HA. (H) Pull‐down assay of LmPio and LmDpy. Western blots of input cell lysates (top) and pull‐down products (bottom) detected with anti‐HA and anti‐His antibodies are shown. LmDpy‐HA was pulled down by LmPio‐His.

In Drosophila, Pio and Dpy interact to organize the luminal ZP matrix during tracheal development. ²⁰ We investigated whether this interaction is conserved in L. migratoria and what role LmDpy plays. Expression analysis showed that LmDpy was highly expressed at the end of each instar and in tissues, including the antennae, integument, wing pads, foregut, hindgut, and legs (Supporting Information, Fig. S3B,C), resembling the expression pattern of LmPio. Protein structure prediction using AlphaFold 3 suggested extensive polar contacts at the LmPio–LmDpy interface, with 87 significant interaction sites (Fig. 7(C)). RNAi knockdown of LmDpy resulted in 100% mortality at N3D3–N3D4 (Supporting Information, Fig. S4A,B). Phenotypic analysis revealed cuticle thinning, a reduced number of lamellae, a thinner assembly zone, abnormal microvilli, and cuticle–epidermis detachment (Supporting Information, Figs S4C and S7D–F). Additionally, cuticular lipid and protein contents were significantly decreased (Supporting Information, Fig. S5), closely resembling the phenotype observed in LmPio knockdown nymphs. These results indicate that LmDpy and LmPio likely function in the same pathway and strongly support physical and functional interactions between them.

To confirm the interaction between LmPio and LmDpy, we performed a Co‐IP assay by co‐expressing LmPio‐His and LmDpy‐HA fusion proteins in HEK293T cells. LmPio‐His co‐immunoprecipitated with antibodies bound to LmDpy‐HA (Fig. 7(G)), indicating that LmPio interacts with LmDpy. Additionally, two distinct bands were observed in the WB analysis of LmDpy‐HA due to cleavage at the furin protease recognition site (Supporting Information, Fig. S3A). To further validate the interaction between LmPio and LmDpy, we performed an in vitro pull‐down assay using purified LmPio‐His and LmDpy‐HA proteins. The results showed that LmDpy‐HA was pulled down by LmPio‐His (Fig. 7(H)). Taken together, these results suggest that LmPio directly interacts with LmDpy.

4. DISCUSSION

4.1. LmPio exhibits unique molecular properties

Various ZP proteins, including ZP2, ZP3, and NompA, have been confirmed to be cleaved at conserved furin cleavage sites. ³⁷ , ³⁸ LmPio contains both a putative furin cleavage site and a transmembrane domain. In Drosophila, this cleavage site is functional, ¹⁸ suggesting that LmPio may act both as a secreted protein and a transmembrane protein. Consistent with previous reports, our phylogenetic analysis revealed that Pio is conserved among insects but has no homologs outside this class, supporting the hypothesis that it exerts insect‐specific functions. ¹⁸ , ²¹

In Drosophila, Pio is expressed in the foregut, hindgut, salivary gland, tracheal system, posterior spiracle, and epidermis. ¹⁷ In T. Castaneum, it is highly expressed in the larval epidermis and head. ²¹ Similarly, we found that LmPio is highly expressed in the antennae, integument, wing pads, foregut, hindgut, and legs of L. migratoria, with lower expression in the gastric caeca and midgut. These results indicate that Pio is predominantly expressed in ectoderm‐derived tissues and may play important roles in multiple ectoderm‐derived tissues. At the protein level, Pio localizes to the apical membrane and tracheal lumen in Drosophila embryos and shows a punctate distribution at the apical side of epithelial cells in the pupal wing. ¹⁸ , ²⁰ In L. migratoria, LmPio is localized to the assembly zone in the apical region of epidermal cells, further supporting its role in apical cellular functions.

4.2. LmPio serves specific functions in aECM assembly and maturation

The loss of Pio in Drosophila leads to the failure of the trachea to form narrow tubes with self‐cell connections, resulting in severe disruption of tracheal branching patterns. ¹⁷ Its absence also leads to the loss of pupal wing‐specific microtubule bundles and defects in the innermost layer of the embryonic cuticle. ¹⁸ These defects disrupt epidermal–tendon cell attachment to the cuticle, ultimately affecting cuticle stability, flexibility, and organismal function. ⁴ Moreover, Pio disruption causes wing blistering in D. melanogaster, B. mori, T. castaneum, and L. migratoria, ³⁹ and its knockdown inhibits pupation and eclosion in T. castaneum. ²¹ In this study, RNAi‐mediated silencing of LmPio impaired aECM integrity, leading to thinning of the assembly zone and cuticle, reduced the number of cuticular lamellae, decreased cuticular lipid and protein contents, and disrupted the adhesion between the epidermis and cuticle. Although TEM does not always clearly distinguish the exocuticle from the endocuticle, H&E staining and cuticle developmental patterns suggest that the inner endocuticle is primarily affected. ³¹ This finding is consistent with the fact that the exocuticle is already formed prior to dsRNA injection at N3D1, whereas the endocuticle continues to thicken during the instar (Supporting Information, Fig. S6). Notably, chitin content remained unchanged despite the reduction in the number of lamellae, which may reflect distinct transport mechanisms for chitin deposition, lipid delivery, and protein secretion. ⁴⁰ Together, these results indicate that LmPio is essential for aECM assembly and maturation, and its disruption leads to lethality in L. migratoria, highlighting its role during insect molting.

4.3. The interaction between Pio and Dpy may be conserved across species

Dpy is a large protein containing numerous repetitive domains that may provide interaction sites for binding to other aECM components. ⁴¹ , ⁴² During tracheal system development in Drosophila, Pio and Dpy interact to form a ZP matrix, which mediates the adhesion between the tracheal cells and the aECM. When Pio is mutated, Dpy fails to localize apically in tracheal cells, indicating that Pio supports Dpy secretion. ²⁰ This interaction mechanism may be highly conserved among insect species. Our findings support this view for the following reasons: (i) both Pio and Dpy are highly conserved among insect species (Fig. 1) ⁴³ ; (ii) LmPio and LmDpy exhibit similar spatio‐temporal expression patterns and RNAi‐induced phenotypic defects, indicating that they may function in the same pathway; (iii) IP and MS analyses identified LmDpy as a potential interacting partner of LmPio, and structural prediction using AlphaFold 3 revealed numerous polar interaction sites between LmPio and LmDpy (Fig. 7(A)–(C)), further supporting a direct interaction; and (iv) Co‐IP and pull‐down assays revealed a direct interaction between LmPio and LmDpy. Collectively, we propose that this mechanism is likely conserved across insect species, providing a foundation for understanding aECM assembly in insects.

4.4. Evaluation and application prospects of targets

Our study showed that LmPio functions within the assembly zone. It not only mediates the adhesion between the epidermis and cuticle but also coordinates the transport of apical lipids and proteins. Furthermore, it interacts with Dpy to regulate the assembly and maturation of the aECM (Fig. 8). This process is critical for the molting of L. migratoria, an arthropod‐specific biological phenomenon. Thus, these targets identified from this unique pathway inherently minimize potential risks to non‐target organisms. ¹¹ To our surprise, the more severe phenotypic defects were observed in LmDpy‐knockdown nymphs, which may be attributed to the significant structural advantages of LmDpy (Supporting Information, Fig. S3A), which forms large fibrous structures and plays a dominant role in aECM assembly. ⁴¹ Therefore, our results demonstrate that both LmPio and LmDpy are promising RNAi‐based targets for L. migratoria control.

Figure 8.

LmPio organizes the aECM at the apical cell surface. Pio is specifically enriched in the assembly zone, where it mediates adhesion between the epidermis and the cuticle, and coordinates the transport of lipids and proteins at the apical surface of cells. Pio interacts with Dpy to jointly regulate the proper assembly and maturation of the aECM.

Although an injection approach was used in this study, oral delivery is a more viable strategy for pest control in the field. Currently, dsRNA stability remains a critical issue for practical applications. In recent years, nanomaterials have been used as delivery vectors for dsRNAs to boost RNAi efficiency in L. migratoria. ⁴⁴ , ⁴⁵ In future studies, we will develop more efficient RNA delivery systems to facilitate the field application of RNA pesticides.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest regarding the content of this article.

Supporting information

Figure S1. Effects on the growth and development of L. migratoria after injection of dsLmPio in 1‐day‐old fifth‐instar nymphs. (A) Relative expression of LmPio after injection of dsLmPio was determined by RT‐qPCR. All data are shown as mean ± SD. Statistical significance was analyzed with Student's t‐test. Asterisks indicate significant differences (**P < 0.01). (B) Phenotypes in the growth and development of L. migratoria after injection of dsLmPio. BE, before ecdysis; EC, ecdysis; AE, after ecdysis. The red slashes indicate lethality. Scale bar indicates 1 cm.

Figure S2. RT‐qPCR analysis shows the relative expression level of LmABCH‐9B and LmABCH‐9C at 48 h post‐dsLmPio injection (N3D3). Statistical significance was analyzed with Student's t‐test. Asterisks indicate significant differences (*P < 0.05, ***P < 0.001).

Figure S3. Domain characterization and the expression pattern of LmDpy. (A) Schematic of the predicted protein structure of LmDpy. (B) RT‐qPCR analysis shows the relative expression of LmDpy in nymphs at different days. N2D1–N4D3, 1‐day‐old second‐instar nymphs to the 3‐day‐old fourth‐instar nymphs. (C) RT‐qPCR analysis shows the relative expression of LmDpy in different tissues of 4‐day‐old third‐instar nymphs. AN, antenna; IN, integument; WP, wing pads; FG, foregut; GC, gastric caeca; MG, midgut; HG, hindgut; LE, legs. All data are shown as mean ± SD. Different letters above the bars represent statistically significant differences based on one‐way ANOVA analysis followed by Tukey's tests.

Figure S4. Effects on the growth and development of L. migratoria after injection of dsLmDpy in 1‐day‐old third‐instar nymphs. (A) Relative expression of LmDpy after injection of dsLmDpy was determined by RT‐qPCR. All data are shown as mean ± SD. Statistical significance was analyzed with Student's t‐test. Asterisks indicate significant differences (***P < 0.001). (B) Phenotypes in growth and development of L. migratoria after injection of dsLmDpy. The red slashes indicate lethality. Scale bar indicates 5 mm. (C) Microstructural analysis of the cuticle by H&E after injection of dsLmDpy. The yellow I‐shaped lines represent cuticular thickness, the red arrows indicate the Exo (exocuticle), the green arrows indicate the Endo (endocuticle), and the black triangles indicate the OC and NC (old and new cuticle). Scale bar indicates 20 μm.

Figure S5. Changes in substance content of the cuticle on different days after injection of dsLmDpy in L. migratoria. (A) Changes in cuticular lipid content in pools of five insects on different days (N3D2 and N3D3) after dsLmDpy injection. (B) Changes in cuticular chitin content in pools of five insects on different days (N3D2 and N3D3) after dsLmDpy injection. (C) Changes in cuticular protein content in pools of five insects on different days (N3D2 and N3D3) after dsLmDpy injection. OC, old cuticle. All data are shown as mean ± SD. Statistical significance was analyzed with Student's t‐test. Asterisks indicate significant differences (**P < 0.01, ***P < 0.001).

Figure S6. The cuticle structure of 1‐day‐old third‐instar nymphs of L. migratoria. Epi, epicuticle; Exo, exocuticle; Endo, endocuticle; AZ, assembly zone; Mv, microvilli. The yellow arrows point to the microvilli. Scale bar indicates 1 μm.

Table S1. Species and GenBank accession number for phylogenetic tree.

Table S2. Primers used in RT‐qPCR.

Table S3. Double‐stranded RNA primers.

Table S4. Specific protein mass spectrometry analysis results.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.