Gut bacteria facilitate leaf beetles in adapting to dietary specialization by enhancing larval fitness.

Dietary specialization between insect stages can reduce intraspecific food competition. The involvement of gut bacteria and the mechanisms underlying this phenomenon received limited attention. Plagiodera versicolora is a pest harming Salicaceae trees. Here, we confirmed dietary specialization in P. versicolora, wherein adults prefer new leaves, while larvae predominantly consume mature leaves when both types are available. We demonstrated the larval preference for mature leaves confers ecological advantages by promoting growth, development and immunity and this advantage is contingent upon the presence of gut bacteria. Gut microbiota in larvae revealed a significant enrichment of Pantoea when feeding new leaves, with P. anthophila exhibiting the most pronounced inhibitory effect on larval development. Further exploration identified specific metabolites, such as Tyrosyl-valine, with higher content in new leaves, which serve as substrates for the entomopathogenic gut bacterium to facilitate its proliferation. This study provides a fresh perspective on the ecological role of gut bacteria.

Pinewood nematode induced changes in the assembly process of gallery microbiomes benefit its vector beetle's development.

Microbiomes play crucial roles in insect adaptation, especially under stress such as pathogen invasion. Yet, how beneficial microbiomes assemble remains unclear. The wood-boring beetle Monochamus alternatus, a major pest and vector of the pine wilt disease (PWD) nematode, offers a unique model. We conducted controlled experiments using amplicon sequencing (16S rRNA and ITS) within galleries where beetles and microbes interact. PWD significantly altered bacterial and fungal communities, suggesting distinct assembly processes. Deterministic factors like priority effects, host selection, and microbial interactions shaped microbiome composition, distinguishing healthy from PWN-infected galleries. Actinobacteria, Firmicutes, and Ophiostomataceae emerged as potentially beneficial, aiding beetle's development and pathogen resistance. This study unveils how nematode-induced changes in gallery microbiomes influence beetle's development, shedding light on microbiome assembly amid insect-pathogen interactions. Insights gleaned enhance understanding of PWD spread and suggest novel management strategies via microbiome manipulation.IMPORTANCEThis study explores the assembly process of gallery microbiomes associated with a wood-boring beetles, Monochamus alternatus, a vector of the pine wilt disease (PWD). By conducting controlled comparison experiments and employing amplicon approaches, the study reveals significant changes in taxonomic composition and functional adaptation of bacterial and fungal communities induced by PWD. It identifies deterministic processes, including priority effects, host selection, and microbial interactions, as major drivers in microbiome assembly. Additionally, the study highlights the presence of potentially beneficial microbes such as Actinobacteria, Firmicutes, and Ophiostomataceae, which could enhance beetle development and resistance to pathogens. These findings shed light on the intricate interplay among insects, microbiomes, and pathogens, contributing to a deeper understanding of PWD prevalence and suggesting innovative management strategies through microbiome manipulation.

Gut bacteria are essential for development of an invasive bark beetle by regulating glucose transport.

Insects and their gut bacteria form a tight and beneficial relationship, especially in utilization of host nutrients. The red turpentine beetle (RTB), a destructive and invasive pine pest, employs mutualistic microbes to facilitate its invasion success. However, the molecular mechanism underlying the utilization of nutrients remains unknown. In this study, we found that gut bacteria are crucial for the utilization of D-glucose, a main carbon source for RTB development. Downstream assays revealed that gut bacteria-induced gut hypoxia and the secretion of riboflavin are responsible for RTB development by regulating D-glucose transport via the activation of a hypoxia-induced transcription factor 1 (Hif-1α). Further functional investigations confirmed that Hif-1α mediates glucose transport by direct upregulation of two glucose transporters (ST10 and ST27), thereby promoting RTB development. Our findings reveal how gut bacteria regulate the development of RTB, and promote our understanding of the mutualistic relationship of animals and their gut bacteria.

HcGr76 responds to fructose and chlorogenic acid and is involved in regulation of peptide expression in the midgut of Hyphantria cunea larvae.

BACKGROUND: Sensing dietary components in the gut is important to ensure an appropriate hormonal response and metabolic regulation after food intake. The fall webworm, Hyphantria cunea, is a major invasive pest in China and has led to significant economic losses and ecosystem disruption. The larvae's broad host range and voracious appetite for leaves make H. cunea a primary cause of serious damage to both forests and crops. To date, however, the gustatory receptors (Grs) of H. cunea and their regulatory function remain largely unknown. RESULTS: We identified the fall webworm gustatory receptor HcGr76 as a fructose and chlorogenic acid receptor using Ca2+ imaging and determination of intracellular Ca2+ concentration by a microplate reader. Moreover, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis revealed that HcGr76 is highly expressed in the anterior and middle midgut. Knockdown of HcGr76 caused a significant reduction in the expression of neuropeptide F 1 (NPF1) and CCHamide-2, and led to a decrease in carbohydrate and lipid levels in the hemolymph. CONCLUSION: Our studies provide circumstantial evidence that HcGr76 expressed in the midgut is involved in sensing dietary components, and regulates the expression of relevant peptide hormones to alter metabolism in H. cunea larvae, thus providing a promising molecular target for the development of new insect-specific control products. © 2024 Society of Chemical Industry.

Distinct metabolites affect the phloem fungal communities in ash trees (Fraxinus spp.) native and nonnative to the highly invasive emerald ash borer (AGRILUS PLANIPENNIS).

Emerald ash borer (EAB, Agrilus planipennis) is an invasive killer of ash trees (Fraxinus spp.) in North America and Europe. Ash species co-evolved with EAB in their native range in Asia are mostly resistant, although the precise mechanism(s) remain unclear. Very little is also known about EAB or ash tree microbiomes. We performed the first joint comparison of phloem mycobiome and metabolites between a native and a nonnative ash species, infested and uninfested with EAB, in conjunction with investigation of larval mycobiome. Phloem mycobiome communities differed between the tree species, but both were unaffected by EAB infestation. Several indicator taxa in the larval gut shared a similarly high relative abundance only with the native host trees. Widely targeted metabolomics revealed 24 distinct metabolites in native trees and 53 metabolites in nonnative trees, respectively, that differed in relative content between infested and uninfested trees only in one species. Interestingly, four metabolites shared a strong relationship with the phloem mycobiomes, majority of which affected only the native trees. Collectively, our results demonstrate a complex interplay between host tree chemistry and mycobiome, and suggest the shared relationships between the mycobiomes of the native host tree and EAB may reflect their shared co-evolution.

A near-complete genome assembly of Monochamus alternatus a major vector beetle of pinewood nematode.

The Japanese sawyer beetle, Monochamus alternatus, is not only one of the most important wood boring pest itself, but also a major vector of the invasive pinewood nematode (PWN), which is the causal agent of the devastative pine wilt disease (PWD) and threats the global pine forest. Here, we present a near-complete genome of M. alternatus at the chromosome level. The assembled genome was 792.05 Mb with contig N50 length of 55.99 Mb, which is the largest N50 size among the sequenced Coleoptera insects currently. 99.57% of sequence was anchored onto ten pseudochromosomes (one X-chromosome and nine autosomes), and the final genome harbored only 13 gaps. BUSCO evaluation revealed the presence of 99.0% of complete core genes. Thus, our genome assembly represented the highest-contiguity genome assembly as well as high completeness in insects so far. We identified 20,471 protein-coding genes, of which 20,070 (98.04%) were functionally annotated. The genome assembly of M. alternatus provides a valuable resource for exploring the evolution of the symbiosis between PWN and the vector insects.

G-Protein Coupled Receptor Gpr-1 Is Important for the Growth and Nutritional Metabolism of an Invasive Bark Beetle Symbiont Fungi Leptographium procerum.

Leptographium procerum has been demonstrated to play important roles in the invasive success of red turpentine beetle (RTB), one of the most destructive invasive pests in China. Our previous studies found that bacterial volatile ammonia plays an important role in the maintenance of the RTB-L. procerum invasive complex. In this study, we found a GPCR gene Gpr-1 that was a response to ammonia but not involved in the ammonia-induced carbohydrate metabolism. Deletion of Gpr-1 significantly inhibited the growth and pathogenicity but thickened the cell wall of L. procerum, resulting in more resistance to cell wall-perturbing agents. Further analyses suggested that Gpr-1 deletion caused growth defects that might be due to the dysregulation of the amino acid and lipid metabolisms. The thicker cell wall in the ΔGpr-1 mutant was induced through the cell wall remodeling process. Our results indicated that Gpr-1 is essential for the growth of L. procerum by regulating the nutritional metabolism, which can be further explored for potential applications in the management of RTB.

Emerald Ash Borer Management and Research: Decades of Damage and Still Expanding.

Since the discovery of the ash tree (Fraxinus spp.) killer emerald ash borer (EAB; Agrilus planipennis) in the United States in 2002 and Moscow, Russia in 2003, substantial detection and management efforts have been applied to contain and monitor its spread and mitigate impacts. Despite these efforts, the pest continues to spread within North America. It has spread to European Russia and Ukraine and is causing sporadic outbreaks in its native range in China. The dynamics of EAB's range expansion events appear to be linked to the lack of resistant ash trees in invaded ranges, facilitated by the abundance of native or planted North American susceptible ash species. We review recently gained knowledge of the range expansion of EAB; its ecological, economic, and social impacts; and past management efforts with their successes and limitations. We also highlight advances in biological control, mechanisms of ash resistance, and new detection and management approaches under development, with the aim of guiding more effective management.

Effects of simulated gut pH environment on bacterial composition and pheromone production of Dendroctonus valens.

Bark beetles are an economically and ecologically important insect group, with aggregation behavior and thus host colonization success depends on pheromone-mediated communication. For some species, such as the major invasive forest pest in China, red turpentine beetle (Dendroctonus valens), gut microbiota participates in pheromone production by converting tree monoterpenes into pheromone products. However, how variation in gut microenvironment, such as pH, affects the gut microbial composition, and consequently pheromone production, is unknown. In this study, we fed wild caught D. valens with 3 different pH media (main host diet with natural pH of 4.7; a mildly acidic diet with pH 6 mimicking the beetle gut pH; and highly acidic diet with pH 4), and measured their effects on the gut pH, bacterial community and production of the main aggregation and anti-aggregation pheromone (verbenone). We further tested the verbenone production capacity of 2 gut bacterial isolates in different pH environments (pH 6 and 4). Compared to natural state or main host diet, feeding on less acidic diet (pH 6) diluted the acidity of the gut, whereas feeding on highly acidic diet (pH 4) enhanced it. Both changes in gut pH reduced the abundance of dominant bacterial genera, resulting in decreased verbenone production. Similarly, the highest pheromone conversion rate of the bacterial isolates was observed in pH mimicking the acidity in beetle gut. Taken together, these results indicate that changes in gut pH can affect gut microbiota composition and pheromone production, and may therefore have the potential to affect host colonization behavior.

Plasticity of aggregation pheromones in insects.

Pheromone plasticity is widely observed in insects and enhances their survival, adaptation, and reproductive success. Aggregation pheromones, which cause notable individual aggregation and consequently impact agriculture and human life, are renowned for their special function. Here, we present a review of research progress regarding pheromone plasticity in three typical aggregative insects: locusts, bark beetles, and cockroaches. These insects are major pest species with considerable impacts on the social economy and public health. Numerous studies have demonstrated the plasticity of aggregation pheromones in different populations of these insect species. Although pheromone chemicals and compositions vary across the three groups, the plasticity of aggregation pheromones is significantly impacted by population density, location, food resources, and gut symbiotic microorganisms, indicating the complexity of pheromone plasticity regulated by multiple factors. Finally, we discuss the potential application of pheromone plasticity in basic research and pest management.

Identification and functional analysis of odorant-binding proteins of the parasitoid wasp Scleroderma guani reveal a chemosensory synergistic evolution with the host Monochamus alternatus.

Scleroderma guani is a generalist ectoparasitoid of wood-boring insects. The chemosensory genes expressed in its antennae play crucial roles in host-seeking. In the present study, we identified 14 OBP genes for the first time from the antennae transcriptomes and genomic data of S. guani. The expression profiles of 14 OBPs were tested by RT-qPCR, and the RT-qPCR results showed that SguaOBP2/5/6/11/12/13 were specifically highly expressed in the female antennae. Then we performed ligand binding assays to test the interactions between six selected SguaOBPs with host specific chemical compounds from M. alternatus and pines. The binding results indicated that SguaOBP12 had a higher binding affinity with longifolene, ß-caryophyllene, α-pinene, ß-pinene, myrcene, butylated hydroxytoluene, and 3-carene. SguaOBP11 had a high or medium binding affinity with them. Furthermore, both SguaOBP11 and SguaOBP12 had a medium binding affinity with the aggregation pheromone of Monochamus species, 2-undecyloxy-1-ethanol. Finally, by using molecular docking and RNAi, we further explored the molecular interactions and behavioral functions of SguaOBP11 and SguaOBP12 with these vital odor molecules. Our study contributes to the further understanding of chemical communications between S. guani and its host, and further exploration for its role as a more effective biological control agent.

Palmitoleic acid as a coordinating molecule between the invasive pinewood nematode and its newly associated fungi.

Symbiotic microorganisms are ubiquitous on the body surface or internal tissues of invertebrates, providing them with benefits. Developing symbiotic relationships requires synchronization of developmental stages and physical proximity of partners. Therefore, the identification of metabolites that coordinate the reproduction of symbiotic partners is essential. This study demonstrates that palmitoleic acid (C16: 1) coordinates bilateral propagation by regulating the synchronization of reproduction between the invasive pinewood nematode (PWN) and its newly associated blue-stain fungus, Sporothrix sp.1. When the PWN fed on Sporothrix sp.1, there was a significant increase in lipid metabolism gene expression and metabolite abundance. Through further investigations, it highlighted a significant enhancement in the reproduction of the PWN through direct acquisition of C16: 1, which was abundantly present in Sporothrix sp.1. Furthermore, the PWN biosynthesized C16: 1 through the involvement of the stearoyl-CoA 9-desaturase gene fat-5 and its hormone nuclear receptor nhr-80, which was clarified to promote the egg-laying capacity of females. Moreover, it is worth noting that the production of C16: 1 was significantly higher by the associated fungus Sporothrix sp.1 to enhance sporulation during the spore formation phase compared to the hypha growth phase. Thus, by coordinating the fecundity and spore production, the key lipid metabolite C16: 1 facilitates the rapid and successful colonization of a mutually beneficial symbiotic relationship between the invasive PWN and the native Sporothrix sp.1 within the host. This finding emphasizes the significant role of metabolite sharing and its function in promoting partner synchronization within symbiotic relationships.

Gut Bacteria of Wood-Boring Streltzoviella insularis and Their Involvement in Pinoresinol Degradation of Ash Tree (Fraxinus sp.) Host.

Insect gut microbiota have been widely reported to help the insects to overcome host tree defense. Streltzoviella insularis (Lepidoptera: Cossidae) is one of the most common wood borers in China, attacking various hosts, including ash trees (Fraxinus sp.), but little is known about its gut microbial associates and their involvement in host tree defense. We isolated gut bacteria of S. insularis larvae, analyzed their ability to degrade pinoresinol (a defense compound of ash trees) and cellulose, and identified pinoresinol degradation products. Larval mortality increased with increasing pinoresinol concentration (reflecting natural variation observed in the host trees). All the five detected gut bacteria isolates were able to degrade pinoresinol, two of which were also capable of cellulose degradation. Furthermore, gut bacteria were also shown to degrade pinoresinol via the gluconeogenesis pathway. These results suggest that S. insularis-associated microorganisms help to overcome host pinoresinol defense and possibly contribute to insects or gut microbial nutrition via carbohydrate synthesis.

A chemical signal that promotes insect survival via thermogenesis.

Cold-activated thermogenesis of brown adipose tissues (BAT) is vital for the survival of animals under cold stress and also inhibits the development of tumours. The development of small-molecule tools that target thermogenesis pathways could lead to novel therapies against cold, obesity, and even cancer. Here, we identify a chemical signal that is produced in beetles in the winter to activate fat thermogenesis. This hormone elevates the basal body temperature by increasing cellular mitochondrial density and uncoupling in order to promote beetle survival. We demonstrate that this hormone activates UCP4- mediated uncoupled respiration through adipokinetic hormone receptor (AKHR). This signal serves as a novel fat-burning activator that utilizes a conserved mechanism to promote thermogenesis not only in beetles, nematode and flies, but also in mice, protecting the mice against cold and tumor growth. This hormone represents a new strategy to manipulate fat thermogenesis.

Hypoxia-induced tracheal elasticity in vector beetle facilitates the loading of pinewood nematode.

Many pathogens rely on their insect vectors for transmission. Such pathogens are under selection to improve vector competence for their transmission by employing various tissue or cellular responses of vectors. However, whether pathogens can actively cause hypoxia in vectors and exploit hypoxia responses to promote their vector competence is still unknown. Fast dispersal of pinewood nematode (PWN), the causal agent for the destructive pine wilt disease and subsequent infection of pine trees, is characterized by the high vector competence of pine sawyer beetles (Monochamus spp.), and a single beetle can harbor over 200,000 PWNs in its tracheal system. Here, we demonstrate that PWN loading activates hypoxia in tracheal system of the vector beetles. Both PWN loading and hypoxia enhanced tracheal elasticity and thickened the apical extracellular matrix (aECM) of the tracheal tubes while a notable upregulated expression of a resilin-like mucin protein Muc91C was observed at the aECM layer of PWN-loaded and hypoxic tracheal tubes. RNAi knockdown of Muc91C reduced tracheal elasticity and aECM thickness under hypoxia conditions and thus decreasing PWN loading. Our study suggests a crucial role of hypoxia-induced developmental responses in shaping vector tolerance to the pathogen and provides clues for potential molecular targets to control pathogen dissemination.

Various parasites, bacteria and other disease-causing pathogens are transmitted by insects. A tiny worm called the pine wood nematode, for example, is spread by pine sawyer beetles which can carry up to 280,000 worms in their trachea, the network of tubes they use to breathe. This has resulted in millions of hectares of pine forests in Asia and Europe becoming infected with the deadly disease caused by the nematodes. Pine wood nematodes, as well as other pathogens, can exploit the biological processes of the insects carrying them to make the insects transmit them more effectively. Precisely how nematodes and other disease-causing agents do this is unclear. One possibility is that they reduce the amount of oxygen being supplied to the trachea ­ a phenomenon known as hypoxia ­ which occurs naturally at specific stages in the life of an insect, and during infections. To test this theory, Tang, Zhou, Koski et al. used genetics and imaging approaches to study how pine wood nematodes affect the trachea of pine sawyer beetles. The experiments found that when the nematodes infected the beetles, their trachea did indeed develop hypoxia. This, in turn, made the beetles' airways more elastic and made the layer of structure lining the trachea, known as the apical extracellular matrix, thicker. These changes increased the amount of pinewood nematodes the trachea could hold, allowing the beetle to spread more worms from tree to tree. Further experiments revealed that hypoxia in the trachea increased the levels of a protein called Muc91C in the apical extracellular matrix. When the levels of Muc91C were artificially decreased in the beetles, this made their airways less elastic and the apical extracellular matrix thinner. This work suggests that pine wood nematodes exploit the beetles' normal responses to loss of oxygen supply to make the beetles more effective at transmitting the nematodes between pine trees. Other pathogens carried by insects may also use this strategy to help increase their transmission. Further studies on the Muc91C protein may provide clues for potential drug targets to control pine wood nematodes and protect pine trees from disease.

The effect of inactivation of aldehyde dehydrogenase on pheromone production by a gut bacterium of an invasive bark beetle, Dendroctonus valens.

Semiochemical-based management strategies are important for controlling bark beetles, such as invasive Red Turpentine Beetle (Denroctonus valens), the causal agent for mass mortality of pine trees (Pinus spp.) in China. It has been previously shown that the pheromone verbenone regulates the attack density of this beetle in a dose-dependent manner and that the gut bacteria of D. valens are involved in verbenone production. However, molecular functional verification of the role of gut bacteria in the pheromone production of D. valens is still lacking. To better understand the molecular function of gut bacterial verbenone production, we chose a facultative anaerobic gut bacterium (Enterobacter xiangfangensis) of D. valens based on its strong ability to convert cis-verbenol to verbenone, as shown in our previous study, and investigated its transcriptomics in the presence or absence of cis-verbenol under anaerobic conditions (simulating the anoxic environment in the beetle's gut). Based on this transcriptome analysis, aldehyde dehydrogenase (ALDH1) was identified as a putative key gene responsible for verbenone production and was knocked-down by homologous recombination to obtain a mutant E. xiangfangensis strain. Our results show that these mutants had significantly decreased the ability to convert the monoterpene precursor to verbenone compared with the wild-type bacteria, indicating that ALDH1 is primarily responsible for verbenone conversion for this bacterium species. These findings provide further mechanistic evidence of bacterially mediated pheromone production by D. valens, add new perspective for functional studies of gut bacteria in general, and may aid the development of new gene silencing-based pest management strategies.

Symbiotic microbes aid host adaptation by metabolizing a deterrent host pine carbohydrate d-pinitol in a beetle-fungus invasive complex.

The red turpentine beetle (RTB) is one of the most destructive invasive pests in China and solely consumes pine phloem containing high amounts of d-pinitol. Previous studies reported that d-pinitol exhibits deterrent effects on insects. However, it remains unknown how insects overcome d-pinitol during their host plant adaptation. We found that d-pinitol had an antagonistic effect on RTB, which mainly relied on gallery microbes to degrade d-pinitol to enhance host adaptation with mutualistic Leptographium procerum and two symbiotic bacteria, Erwinia and Serratia, responsible for this degradation. Genomic, transcriptomic, and functional investigations revealed that all three microbes can metabolize d-pinitol via different branches of the inositol pathway. Our results collectively highlight the contributions of symbiotic microbes in RTB's adaptation to living on pine, thereby facilitating outbreaks of RTB in China. These findings further enrich our knowledge of symbiotic invasions and contribute to the further understanding of plant-insect interactions.

Chromosome-level genome assembly and population genomic analyses provide insights into adaptive evolution of the red turpentine beetle, Dendroctonus valens.

BACKGROUND: Biological invasions are responsible for substantial environmental and economic losses. The red turpentine beetle (RTB), Dendroctonus valens LeConte, is an important invasive bark beetle from North America that has caused substantial tree mortality in China. The lack of a high-quality reference genome seriously limits deciphering the extent to which genetic adaptions resulted in a secondary pest becoming so destructive in its invaded area. RESULTS: Here, we present a 322.41 Mb chromosome-scale reference genome of RTB, of which 98% of assembled sequences are anchored onto fourteen linkage groups including the X chromosome with a N50 size of 24.36 Mb, which is significantly greater than other Coleoptera species. Repetitive sequences make up 45.22% of the genome, which is higher than four other Coleoptera species, i.e., Mountain pine beetle Dendroctonus ponderosae, red flour beetle Tribolium castaneum, blister beetle Hycleus cichorii, and Colorado potato beetle Leptinotarsa decemlineata. We identify rapidly expanded gene families and positively selected genes in RTB, which may be responsible for its rapid environmental adaptation. Population genetic structure of RTB was revealed by genome resequencing of geographic populations in native and invaded regions, suggesting substantial divergence of the North American population and illustrates the possible invasion and spread route in China. Selective sweep analysis highlighted the enhanced ability of Chinese populations in environmental adaptation. CONCLUSIONS: Overall, our high-quality reference genome represents an important resource for genomics study of invasive bark beetles, which will facilitate the functional study and decipher mechanism underlying invasion success of RTB by integrating the Pinus tabuliformis genome.

Microhabitat Governs the Microbiota of the Pinewood Nematode and Its Vector Beetle: Implication for the Prevalence of Pine Wilt Disease.

Our understanding of environmental acquisition of microbes and migration-related alteration of microbiota across habitats has rapidly increased. However, in complex life cycles, such as for many parasites, exactly how these microbes are transmitted across multiple environments, such as hosts and habitats, is unknown. Pinewood nematode, the causal agent of the globally devastating pine wilt disease, provides an ideal model to study the role of microbiota in multispecies interactions because its successful host invasion depends on the interactions among its vector insects, pine hosts, and associated microbes. Here, we studied the role of bacterial and fungal communities involved in the nematode's life cycle across different micro- (pupal chamber, vector beetle, and dispersal nematodes) and macrohabitats (geographical locations). We identified the potential sources, selection processes, and keystone taxa involved in the host pine-nematode-vector beetle microbiota interactions. Nearly 50% of the microbiota in vector beetle tracheae and ~60% that of third-stage dispersal juveniles were derived from the host pine (pupal chambers), whereas 90% of bacteria of fourth-stage dispersal juveniles originated from vector beetle tracheae. Our results also suggest that vector beetles' tracheae selectively acquire some key taxa from the microbial community of the pupal chambers. These taxa will be then enriched in the dispersal nematodes traveling in the tracheae and hence likely transported to new host trees. Taken together, our findings contribute to the critical information toward a better understanding of the role of microbiota in pine wilt disease, therefore aiding the knowledge for the development of future biological control agents. IMPORTANCE Our understanding of animal microbiota acquisition and dispersal-mediated variation has rapidly increased. In this study, using the model of host pine-pinewood nematode-vector beetle (Monochamus sp.) complex, we disentangled the routes of microbial community assembly and transmission mechanisms among these different participants responsible for highly destructive pine wilt disease. We provide evidence that the microhabitat is the driving force shaping the microbial community of these participants. The microbiota of third-stage dispersal juveniles (LIII) of the nematodes collected around pupal chambers and of vector beetles were mainly derived from the host pine (pupal chambers), whereas the vector-entering fourth-stage dispersal juveniles (LIV) of the nematodes had the simplest microbiota community, not influencing vector's microbiota. These findings enhanced our understanding of the variation in the microbiota of plants and animals and shed light on microbiota acquisition in complex life cycles.