Plant pathogenic nematode exosomes remodel vector tracheae to enhance pathogen transmission

Significance

Pathogen-driven manipulation of vector physiology represents a fundamental yet understudied survival strategy for optimizing transmission efficiency. This study reveals a sophisticated mechanism whereby the pinewood nematode utilizes exosome-mediated cross-kingdom transmission of miRNA to remodel the tracheal development of its carrier. Activation of the vector’s Notch-Mpm3 pathway promotes extracellular matrix accumulation and tracheal enlargement, thereby overcoming structural bottlenecks to enhance nematode carrying capacity. These findings reveal a paradigm of interkingdom developmental reprogramming, offering profound insights into the vector-hijacking strategies underpinning the ecological dominance of invasive species. This work not only expands our understanding of pathogen–vector interactions but also identifies the exosome-miRNA pathway as a prime target for innovative biocontrol.

Abstract

Pathogens frequently employ vector-manipulation strategies to enhance their transmission efficiency. Exosomes are increasingly recognized as mediators of interspecific communication between pathogens and their vectors. However, the mechanisms by which plant pathogenic nematode exosomes mediate cross-kingdom manipulation of vector development remain largely unexplored. Here, we demonstrate that the plant pathogenic nematode (Bursaphelenchus xylophilus), transmitted by the vector beetle (Monochamus alternatus), secretes exosomes containing microRNAs (miRNAs) that remodel the tracheal development of its vector. Upon nematode entry into the trachea, the beetle’s tracheal diameter was markedly enlarged. Notably, exosome-like vesicles released from dispersal nematodes were internalized by the tracheal epithelial cells. Moreover, exosome-derived Bx-miR-71-5p directly activates Notch expression, a key regulator of cell proliferation and differentiation. Notch suppresses the expression of matrix metalloproteinases 3 (Mmp3), a critical enzyme for extracellular matrix (ECM) degradation, thereby promoting continuous ECM accumulation. Nanomaterial-mediated delivery of Bx-miR-71-5p to the vector beetle trachea upregulates the Notch gene, leading to significant increases in ECM thickness and tracheal diameter, which consequently enhances nematode load. Collectively, this study identifies nematode exosomes as the delivery vehicle for Bx-miR-71-5p and defines a Notch–Mmp3–ECM axis through which pathogen signals remodel tracheal architecture to enhance vector competence and nematode load. These findings highlight that exosomes-mediated miRNA delivery may present a conserved “toolkit” that can tune vector traits, and ultimately facilitates pathogen transmission efficiency during invasion.

Vector-borne pathogens are responsible for many of the most devastating infectious diseases affecting plant, animal, and human health (1–3). These pathogens have evolved direct and indirect effects on their vectors, thereby enhancing their transmission efficiency. However, significant gaps remain in our understanding of the putative manipulation mechanisms across taxa of vector-borne plant pathogens and the evolutionary implications of these patterns. Extensive studies have documented pathogen-induced alterations in vector or host sensory perception (4), reproductive output (5), immune function (6), behavior (7, 8), and environmental stress tolerance (9, 10) that facilitate pathogen survival and transmission. Nevertheless, although vector carrying capacity is a central determinant of transmission dynamics, it remains largely unexplored whether pathogens can actively manipulate vector’s developmental programs to remodel organ structures for enhanced dissemination. A mechanistic understanding of such pathogen-driven remodeling would both inform control strategies and illuminate coevolutionary dynamics between pathogens and their vectors.

Extracellular vesicles (EVs), including exosomes, microvesicles, and bacterial outer membrane vesicles (OMVs), have emerged as versatile mediators of intercellular and interspecies communication (11–18). EVs provide a protected transport route for proteins, lipids, and diverse RNA species, enabling signals to cross tissue barriers and even species boundaries. In vector-borne systems, EVs have been implicated in multiple cross-kingdom processes: Vector-derived EVs can promote plant virus transmission to host (19, 20), host-derived EVs can convey defense signals to pathogens (14), and pathogen-derived EVs can reprogram host immunity to favor persistent infection (17, 18, 21–25). Similarly, bacterial OMVs and nematode exosomes can deliver effector molecules and small RNAs that modulate host gene expression (11, 26, 27). Despite these advances, prior work has mainly documented the cross-kingdom immunomodulatory effects of EVs. However, it remains unknown how pathogen-derived EVs cross-kingdom reprogram vector development to markedly alter transmission capacity.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causative agent of pine wilt disease and represents one of the most significant global threats to forest ecosystems (28–31). PWNs are predominantly transmitted via the tracheal systems of their vector beetles, such as Monochamus alternatus, although they can also be found in reproductive organs and beneath the elytra (32–34). This transmission relies on a close ecological association: The number of dispersal fourth-stage juveniles (J_IV) of PWN increases after the emergence of M. alternatus. Because the adult beetles reside within the pupal chamber for a period after emergence, they encounter a critical window during the latter part of the residence period to acquire massive J_IV loads (35–39). Upon beetle emergence, these nematodes reside in the tracheae until the beetle feeds on a new host tree, where they detach from the vector beetle and initiate a new round of infection in the host tree (40). Despite the fundamental importance of this dispersal stage, the molecular mechanisms by which PWNs influence vector tracheal development to enhance pathogen transmission remain elusive.

In this study, we demonstrate that B. xylophilus actively manipulates the respiratory architecture of its vector to facilitate its own transmission. We show that J_IV nematodes, upon entering the beetle trachea, secrete exosomes that induce significant tracheal expansion. Mechanistically, these exosomes deliver a specific microRNA, Bx-miR-71-5p, which modulates the Notch signaling pathway, a key regulator of cell proliferation—thereby inducing extracellular matrix (ECM) thickening and subsequent tracheal dilation. Our findings reveal a paradigm in vector–pathogen interactions, where a pathogen exploits exosomal miRNA signaling to remodel the vector’s developmental programs, ultimately optimizing its own dispersal efficiency.

Results

Elevated Nematode Burden in Beetles Correlates with Increased Pine Wilt Severity and Tracheal Diameter.

Field investigation (2020 to 2022 in Zhejiang, JiangSu, and ShanXi provinces) established a strong positive correlation between the average nematode burden in vector beetles and the clinical severity of pine wilt disease. Linear regression analysis confirmed a significant association between the number of B. xylophilus individuals per vector beetle and both the total diseased pine forest area (R² = 0.8414, P < 0.001; Fig. 1A) and the quantity of infested trees (R² = 0.7302, P < 0.01; Fig. 1B). These data suggest that transmission efficiency of beetles directly facilitates the expansion of B. xylophilus. Logarithmic regression analysis revealed a significant positive correlation between individual PWNs loading and trachea diameter (R² = 0.8366; Fig. 1C), supporting the idea that physical expansion of the tracheal system is a primary determinant of the beetle’s carrying capacity. The beetle tracheal diameter exhibited a substantial expansion of approximately 40 μm as the PWN load increased up to 10,000 individuals. Within the range of 10,000 to 20,000 PWNs, the rate of tracheal expansion slowed markedly. No significant variation in tracheal diameter was observed once nematode loads exceeded 20,000 to 25,000 individuals (Fig. 1D), indicating an apparent upper limit to tracheal dilation. This plateau suggests structural or ventilatory constraints on the tracheal capacity for PWN loading.

Fig. 1.

Correlation between PWN load in vector beetles and Pine Wilt Disease severity and vector’s tracheal structural modifications. (A) Correlation analysis showing a linear relationship between the PWN load in vector beetles and the diseased pine forest area (hm²). Data represent regions from Zhejiang (ZJ), Jiangsu (JS), and Shaanxi (SX) provinces over a 3-y period (2020 to 2022), with each point representing a region-year combination (n = 9). Linear regression: R² = 0.8414, P < 0.001. (B) Correlation analysis illustrating the linear relationship between the PWN load in vector beetles and the number of diseased pine trees, using the same spatiotemporal dataset as in (A) (n = 9). Linear regression: R² = 0.7302, P < 0.01. (C) Scatter plot depicting the logarithmic correlation between the PWN load in individual vector beetles and their tracheal diameter (μm). Each data point represent an individual beetle (n = 103). The relationship is modeled by the logarithmic regression: Y = 24.39 × log₁₀(X) + 527.3 (R² = 0.8366). (D) Comparison of tracheal diameter (μm) across vector beetles categorized by PWN load thresholds. Individual beetles (n = 103) were grouped based on naturally occurring PWN load ranges (1 to 5,000; 5,000 to 10,000; 10,000 to 15,000; 15,000 to 20,000; 20,000 to 25,000) to assess the impact of nematode density on tracheal expansion. Data are mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s HSD post hoc test (**P < 0.01, ***P < 0.001, ****P < 0.0001; n.s., not significant).

Tracing tracheal development during metamorphosis, we observed that diameter expansion primarily occurs between the late pupal (P3) and early adult (A1) stages (SI Appendix, Fig. S2A). While the thicknesses of the chitinous intima and epithelial layer (SI Appendix, Fig. S2C) were synchronized with this initial expansion phase (P3–A1), the outer ECM exhibited a delayed thickening profile, remaining thin during eclosion and only expanding significantly from A3 to A7 (SI Appendix, Figs. S1 and S2B). This temporal decoupling identifies the ECM as a critical component in the postemergence remodeling of the Monochamus tracheal system.

PWNs Induce Tracheal ECM Remodeling in Vector Beetles.

We further verified the impact of nematode loading on the vector tracheal development using indoor-reared beetles. Nematodes were experimentally inoculated, and dissections were performed at different developmental stages. Nematode acquisition intensifies during the beetle’s normal residence in the pupal chamber and reaches its highest levels toward the latter part of this residence period (SI Appendix, Fig. S3). The tracheal morphology of beetles with and without PWN carriage was compared. Light microscopy showed that in beetles without nematode loading, the tracheal system appeared translucent and pale yellow. In contrast, the trachea of nematode-harboring beetles exhibited a distinctly opaque and significantly expanded diameter (Fig. 2 A and D). Furthermore, transmission electron microscopy (TEM) revealed that the tracheal ECM layer was significantly thicker in nematode-carrying beetles than in nematode-free controls (Fig. 2 B–D). Expression of ECM-related genes in the beetle trachea was continuously upregulated following nematode entry (SI Appendix, Fig. S4). These results suggest that nematode loading induced tracheal diametric expansion in the vector beetle by promoting sustained ECM thickening, even after ECM deposition had ceased in nematode-free controls.

Fig. 2.

PWN carriage induces structural remodeling of the vector trachea. (A) Light microscopy visualization of the tracheal system anatomy in beetles carrying PWNs (+PWN) and nematode-free beetles (−PWN). (B and C) TEM analysis of tracheal ultrastructure in +PWN and −PWN beetles. Representative images show the overall tracheal ultrastructure (B) and detailed organization of the tracheal wall (C). (D) Quantification of PWN-induced tracheal morphological changes, including tracheal diameter (Left) and ECM thickness (Right), in −PWN and +PWN beetles (n = 10, Student’s t test). Data are mean ± SEM. Significant differences between the two treatments are indicated by asterisks (***P < 0.001, Student’s t test). The middle image (+PWN) was reproduced from Tang et al. (41), with permission/under the Creative Commons Attribution License.

Characteristics of Exosomes Derived from J_IV Nematodes.

TEM of beetle tracheae identified an abundance of vesicle-like structures in the nematode-carrying vectors (Fig. 2B). To determine the origin of these vesicles, scanning electron microscopy (SEM) was performed on the cuticle of dispersal fourth-stage juveniles (J_IV) nematodes. Numerous EVs, with diameter ranging from 30 to 200 nm, were distributed across the J_IV surface (Fig. 3A). EVs were extracted from the culture medium of J_IV nematodes. These vesicles appeared as cup-shaped or spherical morphology under the TEM (Fig. 3B) (13). Nanoparticle tracking analysis (NTA) revealed a particle concentration of approximately 5 × 10⁸ particles/mL, with a peak diameter of 93.0 nm (Fig. 3C). Additionally, Western blot confirmed the presence of conserved exosomal markers, including TSG101, CD81, and CD63 (42, 43). Crucially, the absence of Calnexin and Histone H3 excluded cell contamination (Fig. 3D). Immunoelectron microscopy (IEM) further localized the molecular chaperone HSP70 within the vesicles (Fig. 3E) (44, 45). To further confirm their identity as exosomes, nanoflow cytometry (nFCM) was employed to verify the enrichment of canonical exosomal surface markers CD81, CD9, and CD63 (Fig. 3F) on the isolated vesicles. Collectively, these characterizations unequivocally establish that exosomes are actively secreted by J_IV nematodes.

Fig. 3.

Characterization of PWN-derived exosomes and their effects on vector tracheal structure. (A) SEM images of the nematode surface showing EVs (arrows). (B) TEM of purified PWN-derived exosomes. (C) NTA showing size distribution and concentration of PWN-derived exosomes, with a major peak at 93 nm and a secondary peak at 70 nm. (D) Western blot analysis of exosomal markers (TSG101, CD81, CD63). Absence of the cellular markers Calnexin (ER) and Histone H3 (nucleus) confirms exosome purity (n = 3). (E) IEM localization of Hsp70 on PWN-derived exosomes. NC, negative control. (F) Nanoflow cytometric detection of exosomal surface markers CD9, CD63, and CD81. Representative dot plots display side scatter area (SS-A) vs. fluorescence intensity height (FITC-H). The blank panel serves as a negative control. (G) Confocal microscopy showing internalization of PKH67-labeled PWN-derived exosomes (green) by tracheal epithelial cells. (H) Quantification of PWN load in beetle tracheae following exosome injection (n = 20). (I) Effects of PWN-derived exosomes on vector tracheal morphology compared with PBS control (n = 10). (J) TEM analysis of tracheal ECM thickness following exosome treatment, with PBS as a negative control. Data are mean ± SEM. Significant differences between the two treatments are indicated by asterisks (*P < 0.05, ***P < 0.001, Student’s t test).

Nematode Exosomes Induce Tracheal Enlargement and PWN Transmission.

To determine whether the nematode-derived exosomes can enter beetle cells, the nematode exosomes labeled with PKH67 were incubated in vitro with beetle tracheal epithelial cells (11), green fluorescent signal was observed to be localized in the cytoplasm (Fig. 3G), confirming the internalization of nematode exosomes by beetle cells. The physiological impact of these exosomes on tracheal architecture was further investigated in vivo. Nematode-derived exosomes were injected into the beetle tracheae at 3 d posteclosion—a stage characterized by significant diameter sensitivity to exosome treatment (SI Appendix, Fig. S5). Notably, 4 d postinjection, the tracheal diameter increased by 1.12%, while the thickness of the ECM layer was approximately 1.9-fold that of the PBS-injected control (Fig. 3 I and J). These exosome-induced structural modifications mirror the morphology observed in beetles naturally loaded with nematodes, strongly suggesting that nematode-derived exosomes promote tracheal diameter expansion in the vector beetle by remodeling the tracheal ECM layer. Furthermore, the injection of exosomes resulted in an increased load of PWN within the tracheae of vector beetles (Fig. 3H), demonstrating that nematode-derived exosomes promote PWN transmission.

Bx-miR-71-5p Targets and Regulates Notch.

The molecular mechanism of tracheal ECM thickening was further investigated. The transcript expression levels of ECM-related genes were analyzed in the vector beetle before and after PWN entry. Notably, genes encoding regulators of ECM degradation, such as matrix metalloproteinases (Mmps), were downregulated in the vector beetle trachea after PWN loading. Conversely, genes associated with ECM-affiliated transmembrane proteins and receptors (Notch, mucins, integrins, cadherins), as well as ECM renewal, were upregulated in beetle trachea carrying PWN (Fig. 4A). These results suggest that nematode entrance leads to the positive regulation of beetle tracheal ECM synthesis genes. Building on this, this study further explored the specific tracheal ECM genes in the vector beetle that are targeted by PWN exosomes. Four miRNAs from nematode exosomes were identified that shared identical seed sequences with those in vector beetles: Bx-miR-7-5p, Bx-miR-1-3p, Bx-miR-71-5p, and Bx-miR-81-3p (SI Appendix, Fig. S6). Among these, Bx-miR-71-5p exhibited the most significant upregulation (6.8-fold) in dispersal-stage J_IV nematodes compared to reproductive-stage nematodes and was predicted to target the Notch gene (Fig. 4 B and C). Further validation confirmed a significant upregulation of Notch at both the transcript and protein expression levels in the trachea of beetles carrying nematodes, concurrent with a downregulation of Mmp3 expression, relative to beetles without nematode loading (Fig. 4D). The direct interaction between Bx-miR-71-5p and the Notch gene was verified using a dual-luciferase assay in Drosophila S2 cells. Luciferase activity of the construct with Notch target sites was upregulated by Bx-miR-71-5p. Conversely, mutations in the binding site of the Bx-miR-71-5p seed sequence abolished the up-regulation effect of Bx-miR-71-5p on reporters with target sites from Notch (Fig. 4E) These findings demonstrate that Bx-miR-71-5p directly regulates Notch expression at the posttranscriptional level. Moreover, exosome injection into the tracheae mirrored the effects of nematode infestation, leading to increased Notch levels and decreased Mmp3 levels (Fig. 4 F and G). Mmps which are conserved zinc-dependent endopeptidases that regulate the ECM, are notably subject to inhibitory control by Delta/Notch signaling (46). These results indicate that nematode-derived Bx-miR-71-5p functions as a key modulator of Notch expression.

Fig. 4.

PWN-derived exosomal microRNAs remodel the tracheal ECM via Notch signaling. (A) Transcriptomic profile and KEGG enrichment analysis of tracheal ECM-related genes in PWN-free (Tr-CK) and PWN-loaded (Tr-Bx) vector tracheae. (B) Regulatory network of exosomal miRNAs and their predicted tracheal targets. The Sankey diagram (Left) links specific PWN exosome miRNAs (e.g., Bx-miR-71-5p) to target genes involved in ECM formation (Right), including Notch. Heatmap columns indicate relative expression levels. (C) Differential expression of exosomal miRNAs across PWN developmental stages. qPCR analysis compares the dispersal stage (J_IV) and reproductive stage (L_n) (n = 3). (D) qPCR analysis of Notch and Mmp3 mRNA levels in vector tracheae under nematode-carrying (+PWN) and nematode-free (−PWN) conditions (n = 3). (E) Dual-luciferase reporter assay validating the direct interaction between Bx-miR-71-5p and the Notch 3’ UTR. Top: Sequence alignment of the Bx-miR-71-5p seed region (pink), the target site in wild-type (WT) Notch (red), and the mutated bases in mutant (MT) Notch (blue). Bottom: Relative luciferase activity in Drosophila S2 cells cotransfected with the reporter plasmids and Bx-miR-71-5p mimics (n = 3). (F) Western blot analysis of Notch protein levels in tracheae under PWN-carried or exosome treatment conditions. Vinculin serves as the loading control (n = 3). (G) Representative qPCR quantification of Notch and Mmp3 mRNA levels following exosome treatment, with PBS as a negative control (n = 3). Data are mean ± SEM. Significant differences between the two treatments are indicated by asterisks (*P < 0.05, **P < 0.01, ****P < 0.0001, n.s., not significant; Student’s t test).

Bx-miR-71-5p-Mediated Notch Promotes Tracheal Enlargement and Improves Nematode Transmission.

Further investigation using nano-chitosan encapsulated Bx-miR-71-5p, agomir-71-5p, and antagomir-71-5p treatment confirmed that Bx-miR-71-5p modulates both tracheal diameter and ECM thickness. RT-qPCR validation revealed that Bx-miR-71-5p and its agomir significantly upregulated Notch expression while suppressing the transcript levels of Mmp3, a key enzyme involved in ECM degradation (SI Appendix, Fig. S7 A–D). In contrast, silencing Bx-miR-71-5p with an antagomir downregulated Notch and triggered a robust induction of Mmp3 (SI Appendix, Fig. S7 E and F). TEM analysis revealed that tracheal ECM thickness in beetles injected with Bx-miR-71-5p was approximately 1.94-fold that of the control group (Fig. 5 A and B). Concurrently, Bx-miR-71-5p injection led to a 3.14% increase in the tracheal diameter relative to the control (Fig. 5A). Agomir-71-5p injection significantly increased ECM thickness to 1.47-fold of the control and expanded tracheal diameter by 3.72% (Fig. 5 C and D). Conversely, antagomir-71-5p treatment reduced ECM thickness to 0.64-fold that of the control group and resulted in a 2.07% reduction in tracheal diameter (Fig. 5 E and F). These findings further demonstrate that Bx-miR-71-5p positively upregulates Notch to promote tracheal diameter expansion, mediated by thickening of the ECM layer.

Fig. 5.

Bx-miR-71-5p directly targets and modulates Notch expression, resulting in structural remodeling of the vector tracheae. (A) Quantification of tracheal ECM thickness (Left) and tracheal diameter (Right) following injection of native Bx-miR-71-5p (n = 6). (B) TEM analysis of tracheal ECM thickness after Bx-miR-71-5p manipulation. (C and D) Effects of nematode-derived Bx-miR-71-5p agomir on tracheal diameter (C) and ECM thickness (D) (n = 6). (E and F) Impact of Bx-miR-71-5p antagomir treatment on beetle tracheal morphology, including tracheal diameter (E) and ECM thickness (F) (n = 6). Data are mean ± SEM. Significant differences between the two treatments are indicated by asterisks (* P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t test).

To further clarify the role of Notch in tracheal development and the capacity for nematode loading, we knocked it down by injecting double-stranded RNAs (dsRNAs) in the tracheal system of beetles. Injection of dsNotch significantly reduced Notch mRNA and protein levels (Fig. 6 A–C). Conversely, Mmp3 mRNA and protein levels increased after injecting dsNotch (Fig. 6 A–C). Regarding phenotypic changes, dsNotch-injected beetles exhibited an effective abrogation of nematode-induced ECM thickening. Furthermore, tracheal diameter remained significantly smaller than that of dsGFP controls, regardless of whether nematodes were present (Fig. 6 E and F). Crucially, the nematode carrying capacity of beetles’ tracheae was reduced by 33.13% following Notch knockdown (Fig. 6D), highlighting the essential role of Notch in facilitating nematode transmission. Collectively, these results demonstrate that Notch is a key regulator of tracheal development and is necessary for nematode transmission by M. alternatus.

Fig. 6.

Notch is required for tracheal development and nematode loading capacity in beetles. (A) Representative qPCR quantification of Notch and Mmp3 mRNA levels in the tracheae of beetles following injection of Notch dsRNA. The control group was injected with GFP dsRNA (dsGFP) (n = 3). (B and C) Representative Western blot and quantification of Notch and Mmp3 protein levels in beetles after Notch dsRNA injection. The control group was injected with dsGFP (n = 3). (D) Number of nematodes carried by beetles after injection of dsGFP or dsNotch (n = 20). (E) TEM micrographs of the tracheal ECM in beetles injected with dsGFP or dsNotch, with (+PWN) or without (−PWN) nematode carriage. (F) Quantification of tracheal diameter (Top) and ECM thickness (Bottom) in beetles injected with dsGFP or dsNotch under +PWN or −PWN conditions (n = 6). Data are mean ± SEM. Significant differences between the two treatments are indicated by asterisks (**P < 0.01, ***P < 0.001, Student’s t test).

Discussion

This study elucidates a mechanism by which nematode-derived miRNAs are transported into insect vector cells via exosomes that regulate vector genes associated with tracheal development. Specifically, the PWN secretes exosomes to deliver Bx-miR-71-5p into vector beetle cells, which subsequently targets the Notch gene to promote ECM thickening. These structural modifications enhance the capacity of the vector beetle trachea to accommodate greater nematode loading, ultimately facilitating their transmission. This study highlights a paradigm of exosome-mediated cross-kingdom miRNA trafficking that directly facilitates pathogen transmission. Notably, this mechanism is not mutually exclusive and may operate in concert with additional molecular or physiological strategies that collectively optimize vector-mediated transmission.

Vectors and pathogens are engaged in a constant antagonistic arms race, where each participant’s primary goal is to maximize its performance and fitness. EVs have evolved into a highly regulated system of communication with complex functions that transfer immune factors and regulatory RNAs across species (47). By facilitating the continuous transfer of molecular information within the pathogen–vector–host tripartite system, EV signaling can have evolutionary consequences within pathogen–vector–host networks (48, 49). Our results show that nematode-derived exosomes enhance vector competence not only through molecular regulation but also by remodeling vector architecture, revealing a direct route by which a pathogen can tune developmental programs in its vector to increase transmission efficiency.

A central evolutionary question is whether this exosome-mediated control reflects recent innovation associated with invasion, or a conserved trait that predates the emergence of pine wilt disease. Addressing this point, we compared miRNA sequences between the invasive B. xylophilus and its closely related native congener B. mucronatus and found that miR-71-5p is identical in both species (SI Appendix, Fig. S8 A and B). This conservation argues against de novo sequence innovation as a prerequisite for exosome-mediated effects and instead supports a model in which at least part of the exosomal small-RNA repertoire represents an ancestral, conserved “toolkit.” Furthermore, the capacity to molecularly tune the physiology of a new vector species likely facilitates vector switching or host range expansion (50), serving as a major driver of global invasive success. To conclude, these insights suggest that the pathogen-driven remodeling of the vector’s respiratory architecture may therefore be a key determinant of its transmission and ecological dominance across evolutionary timescales.

The ECM has long been recognized as a foundational scaffold for maintaining tissue integrity and defining organ shape (51–55). Beyond its foundational role in tissue integrity, our results identify the ECM as a key physiological target for pathogen-mediated structural manipulation. Typically, tracheal tubes expand their diameters primarily during molts when the cuticle is replaced; in contrast to their continuous elongation throughout larval growth (56). However, our findings reveal a dramatic expansion of the tracheal lumen that occurs independently of the natural molting cycle, suggesting that the pathogen “hijacks” the vector’s growth signals to bypass developmental constraints. By establishing this link, we provide a definitive molecular explanation for the spatial constraints observed by Aikawa and Togashi (57), who reported the number of nematodes at the cut end of the trachea increased with increasing tracheal diameter, confirming that tracheal size acts as a physical bottleneck for nematode loading. The increase in the diameter of the trachea expanded the accommodation space for the nematode, and as ECM is an important component of the tube wall structure, its reaccumulation enhanced the stability of the tracheal structure, thereby preparing for the expansion of the trachea.

Our findings demonstrate that exosomes serve as key signaling vehicles in cross-kingdom miRNA trafficking between the pathogen and its vector beetle. Specifically, Bx-miR-71-5p targets the vector’s Notch receptor; given Notch’s established role in Mmp3-mediated ECM remodeling, this miRNA-induced Notch activation provides a direct mechanistic link between pathogen-derived miRNAs and tracheal remodeling in the vector. Although miRNAs are generally considered negative regulators of gene expression, accumulating evidence suggests that miRNAs can promote the expression of its target (58–60, 61). The Notch pathway is a highly conserved, contact-dependent signaling cascade that governs cell-fate determination and tissue remodeling (62). Notch signaling has been closely linked to Mmp regulation and ECM dynamics (46), providing a plausible mechanistic basis for the tracheal remodeling observed in our study.

In conclusion, we have identified a critical role of pathogen-derived exosomes in manipulating insect vector tracheal development. This finding opens broad avenues for understanding interspecies communication and pathogen transmission. Furthermore, the demonstrated function of exosomes in cross-kingdom RNAi and the successful use of nano-chitosan-delivered miRNA in our verification experiment may facilitate the development of effective delivery methods for artificial RNAs, offering a potential strategy for controlling plant diseases.

Materials and Methods

Methods included collection and rearing of nematodes and beetles, field investigation of pine wilt disease, nematode loading, tracheal dissection and sample preparation, purification of vesicles, fluorescent antibody labeling and nanoflow cytometry, electron microscopy (TEM and SEM), RNA interference, confocal analysis of uptake, immunogold labeling with TEM, digital western blotting, dual-luciferase assay, and nano-chitosan coating with Bx-miR-71-5p. Detailed protocols are provided in SI Appendix, SI Materials and Methods.

Statistical Analysis.

All data were analyzed using GraphPad Prism 9.0. Data comparison between two groups were performed using Student’s t test. Data comparisons among multiple groups were performed using one-way ANOVA with Tukey’s HSD post hoc test.

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

The RNA-seq data used in this study are from the China National Center for Bioinformation Database under project Accession Nos. CRA006464 (63), CRA040045 (64), and CRA040052 (65). All other data are included in the manuscript and/or supporting information.