The construction and evaluation of secretory expression engineering bacteria for the trans-Cry3Aa-T-HasA fusion protein against the Monochamus alternatus vector.

Pine wood nematode disease is currently the most deadly forest disease in China, and the Monochamus alternatus is its primary vector. Controlling the M. alternatus is crucial for managing pine wood nematode disease. This study, based on the selected HasA (pGHKW4) secretory expression vector, used electroporation to combine the genetically modified high-toxicity toxin Cry3Aa-T with the entomopathogenic bacterium Yersinia entomophaga isolated from the gut of the M. alternatus. The SDS-PAGE and Western blotting techniques were employed to confirm the toxin protein's secretion capability. The engineered bacteria's genetic stability and effectiveness in controlling M. alternatus were assessed for their insecticidal activity. The results of the SDS-PAGE and Western blotting analyses indicate that the HasA system effectively expresses toxin protein secretion, demonstrates certain genetic stability, and exhibits high insecticidal activity against M. alternatus. This study constructed a highly toxic entomopathogenic engineered bacterial strain against M. alternatus larvae, which holds significant implications for controlling M. alternatus, laying the foundation for subsequent research and application of this strain.

Assessing the impact of pine wilt disease on aboveground carbon storage in planted Pinus massoniana Lamb. forests via remote sensing.

The continuous spread of Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle, commonly known as the organism that causes pine wilt disease (PWD), has become a notable threat to forest security in East Asia and southern Europe, and an assessment of the carbon loss caused by PWD damage is important to achieving carbon neutrality. This study used satellite remote sensing and 15-year ground monitoring data to measure the impact of PWD on the carbon storage of Pinus massoniana Lamb. (P. massoniana), the conifer with the largest planted area in southern China. This study showed that the occurrence of PWD had an impact on the increase in carbon storage of P. massoniana. The infected and dead P. massoniana trees accounted for only 1.46 % of the total number of trees but caused a carbon storage loss of 1.99 t/ha, which accounted for 6.23 % of the total carbon sink in healthy P. massoniana forests over the last 15 years. The most pronounced decline in carbon storage occurred in the first five years of PWD invasion. After 10 years of clearcutting and replanting of Schima superba Gardn. et Champ., the increase in carbon storage of the reformed forest far exceeded that of the healthy forest during the same period, which was 2.04 times (10 years) and 1.56 times (15 years) that of the healthy P. massoniana forest. In addition, our study found that during the 15-year period (from the forest age of 22 to the forest age of 37), the average carbon storage of P. massoniana forest was 31.9 t/ha. This study helps to evaluate the impact of PWD on the carbon sink of pine forests and provides methodological references for analyzing the impact of biological disturbances on the carbon cycle.

Calcium stress reduces the reproductive capacity and pathogenicity of the pine wood nematode (Bursaphelenchus xylophilus) by inhibiting oxidative phosphorylation reaction.

The continuous use of chemical pesticides to control nematodes could result in the developing of pesticide-resistant nematodes. Novel nucleic acid pesticides are becoming the focus of pesticide research due to their strong specificity, high efficiency, and environmental friendliness. However, the limited known biochemical targets restrict the development of target pesticides for nematodes. The calcium stress experiments on pine wood nematodes (PWN) showed that 100 mmol/L Ca2+ resulted in longitudinal depression on the PWN body wall, reduced oviposition, and increased corrected mortality. To enrich the biological targets of nematode pesticides, we further investigated the response mechanism of PWN to calcium stress at the molecular level. Differentially expressed gene analysis showed that genes involved in the oxidative phosphorylation (OXPHOS) pathway were significantly enriched. RNA interference results of 6 key genes belonging to four mitochondrial complex I (BXNDUFA2), III (BXQCR8), IV (BXCOX17), V (BXV-ATPaseB, BXV-ATPaseE, BXV-ATPaseε) in non-stressed nematodes showed reduction in PWN oviposition, population size, feeding ability, and pathogenicity. The BXNDUFA2 gene interference had the highest inhibitory impact by decreasing the oviposition from 31.00 eggs to 6.75 eggs and PWN population size from 8.27 × 103 nematodes to 1.64 × 103 nematodes, respectively. Interestingly, RNA interference of these 6 key genes in calcium-stressed nematodes also led to increased mortality and decreased oviposition of PWN. In summary, calcium stress inhibited the reproductive capacity of PWN by down-regulating key genes BXNDUFA2, BXQCR8, BXV-ATPaseB, BXV-ATPaseE, BXV-ATPaseε, and BXCOX17, thereby reducing the pathogenicity. The current results enrich the RNAi targets in PWN and provide a scientific basis for developing novel nucleic nematicides.

Discovery and Characterization of MaK: A Novel Knottin Antimicrobial Peptide from Monochamus alternatus.

Knottin-type antimicrobial peptides possess exceptional attributes, such as high efficacy, low vulnerability to drug resistance, minimal toxicity, and precise targeting of drug sites. These peptides play a crucial role in the innate immunity of insects, offering protection against bacteria, fungi, and parasites. Knottins have garnered considerable interest as promising contenders for drug development due to their ability to bridge the gap between small molecules and protein-based biopharmaceuticals, effectively addressing the therapeutic limitations of both modalities. This work presents the isolation and identification of a novel antimicrobial peptide derived from Monochamus alternatus. The cDNA encodes a 56-amino acid knottin propeptide, while the mature peptide comprises only 34 amino acids. We have labeled this knottin peptide as MaK. Using chemically synthesized MaK, we evaluated its hemolytic activity, thermal stability, antibacterial properties, and efficacy against nematodes. The results of this study indicate that MaK is an exceptionally effective knottin-type peptide. It demonstrates low toxicity, superior stability, potent antibacterial activity, and the ability to suppress pine wood nematodes. Consequently, these findings suggest that MaK has potential use in developing innovative therapeutic agents to prevent and manage pine wilt disease.

Antibacterial peptides from Monochamus alternatus induced oxidative stress and reproductive defects in pine wood nematode through the ERK/MAPK signaling pathway.

Pine wilt disease is a devastating disease of pine caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus. Long-term use of chemical nematicides leads to the development of resistance in nematodes and harms the environment. Evaluations for green environmental protection agents, identified the antibacterial peptide, MaltDef1, from Monochamus alternatus which had nematicidal effect. We studied its nematicidal activity and action against PWN. In this study, the antibacterial peptide S-defensin was synthesized from M. alternatus. The results showed that S-defensin caused mortality to the PWN, causing shrinkage, pore, cell membrane dissolution and muscle atrophy. In addition, PWN reproduction was also affected by S-defensin; it decreased in a concentration dependent manner with increasing treatment concentration. By contrast, reactive oxygen species (ROS) in vivo increased in a concentration-dependent manner. We applied transcriptome to analyze the changes in gene expressions in S-defensin treated PWN, and found that the most significantly enriched pathway was the ERK/MAPK signaling pathway. RNAi was used to validate the functions of four differential genes (Let-23, Let-60, Mek-2 and Lin-1) in this pathway. The results showed that knockdown of these genes significantly decreased the survival rate and reproductive yield of, and also increased ROS in PWN. The antibacterial peptide S-defensin had a significant inhibitory effect on the survival and reproduction of PWN, shown by cell membrane damage and intracellular biological oxidative stress via regulating the ERK/MAPK signaling pathway. This indicates that S-defensin has a target in B. xylophilus, against which new green target pesticides can be developed.

Double-strand RNAs targeting MaltRelish and MaltSpz reveals potential targets for pest management of Monochamus alternatus.

Overcoming the innate immunity of insects is a key process to improve the efficiency of biological control. Antimicrobial peptides (AMPs) are important effectors in insect innate immunity, usually mediating resistance to pathogenic microorganisms through Toll and IMD signaling pathways. This study investigated the effect of key genes on upstream immune recognition receptor (GNBP3) and downstream effectors (AMPs) by RNAi technology. The transcriptome KEGG enrichment analysis and differential gene annotation results showed that the immune response genes MaltSpz and MaltRelish are important regulators of Toll and IMD signaling pathways, respectively. Both dsSpz and dsRelish could affect AMP gene expression and increase the expression of the immune recognition receptor MaltGNBP3. Moreover, they significantly reduce the survival rate of Monochamus alternatus and promote hyphal growth after Beauveria bassiana infection. This helps to improve the biological control effect of B. bassiana, control the population of vector insects and cut off the transmission route of pine wood nematode. The combined MaltSpz and MaltRelish knockdown increased the infection rate of M. alternatus larvae from 20.69% to 83.93%, achieving the best efficiency in synergistic B. bassiana infection. Our results showed important roles of MaltRelish- and MaltSpz-mediated regulation of AMP genes function in insect entomopathogenic fungi tolerance and induced significant mortality in larvae. Based on this study, MaltSpz and MaltRelish could represent candidate gene targets for the biological control of M. alternatus by RNAi.

Pathogenicity virulence of Beauveria spp. and biosafety of the BbMQ strain on adult ectoparasitic beetles, Dastarcus helophoroides Fairmaire (Coleoptera: Colydiidae).

Introduction: Beauveria spp. and Dastarcus helophoroides Fairmaire adults were simultaneously released to attack elder larvae or pupae of Monochamus alternatus in pine forests in China. However, little is known about the pathogenicity virulence and biosafety of Beauveria spp. on beneficial adults of D. helophoroides, and specific Beauveria bassiana (Bb) strains should be selected for synthetic release together with D. helophoroides. Methods: A total of 17 strains of Beauveria spp. were collected, isolated, and purified, and then their mortality, cadaver rate, LT50, spore production, spore germination rate, and growth rate of D. helophoroide adults were calculated based on 0-20 days data after spore suspension and powder contact. Results and discussion: The lethality rate of BbMQ, BbFD, and BbMH-03 strains to D. helophoroides exceeded 50%, and the cadaver rate reached 70.6%, among which the mortality rate (82.22%), cadaver rate (47.78%), spore production (1.32 × 109 spores/ml), spore germination rate (94.71%), colony dimension (49.15 mm2), and LT50 (10.62 d) of the BbMQ strain were significantly higher than those of other strains (P < 0.01), and the mortality of D. helophoroides adults increased significantly with increased spore suspension concentration, with the highest mortality reaching 92.22%. This strain was identified as Beauveria bassiana by morphological and molecular methods, while the BbWYS strain had a minimum lethality of only 5.56%, which was safer compared to other strains of adult D. helophoroide. Consequently, the biological characteristics and pathogenicity of different Beauveria bassiana strains varied significantly in their effects on D. helophoroide adults, and the safety of different strains should be assessed when they are released or sprayed to control multiple pests in the forest. The BbMQ strain should not be simultaneously sprayed with releasing D. helophoroide adults in the same forest, while the BbWYS strain can be used in concert with D. helophoroide to synergize their effect.

Heterologous Expression and Bioactivity Determination of Monochamus alternatus Antibacterial Peptide Gene in Komagataella phaffii (Pichia pastoris).

Insects have evolved to form a variety of complex natural compounds to prevent pathogen infection in the process of a long-term attack and defense game with various pathogens in nature. Antimicrobial Peptides (AMPs) are important effector molecules of the insect immune response to the pathogen invasion involved in bacteria, fungi, viruses and nematodes. The discovery and creation of new nematicides from these natural compounds is a key path to pest control. A total of 11 AMPs from Monochamus alternatus were classified into 3 categories, including Attacin, Cecropin and Defensin. Four AMP genes were successfully expressed by Komagataella phaffii KM71. The bioassay results showed that the exogenous expressed AMPs represented antimicrobial activity against Serratia (G-), Bacillus thuringiensis (G+) and Beauveria bassiana and high nematicide activity against Bursaphelenchus xylophilus. All four purified AMPs' protein against B. xylophilus reached LC50 at 3 h (LC50 = 0.19 mg·mL-1 of MaltAtt-1, LC50 = 0.20 mg·mL-1 of MaltAtt-2 and MaltCec-2, LC50 = 0.25 mg·mL-1 of MaltDef-1). Furthermore, the AMPs could cause significant reduction of the thrashing frequency and egg hatching rate, and the deformation or fracture of the body wall of B. xylophilus. Therefore, this study is a foundation for further study of insect biological control and provides a theoretical basis for the research and development of new insecticidal pesticides.

Spatiotemporal dynamics of fluopyram trunk-injection in Pinus massoniana and its efficacy against pine wilt disease.

BACKGROUND: Pine wilt disease (PWD) is a destructive disease of pine trees caused by the pinewood nematode, Bursaphelenchus xylophilus. Fluopyram, a novel nematicide compound with systemic activity, is a prospective trunk-injection agent against pinewood nematodes. The disadvantage of current trunk-injection agents is that they were not evenly distributed in tree tissues and were poor in the persistence of effect and efficiency. Therefore, we investigated the spatiotemporal transport pattern and residue behavior of fluopyram following its injection into the trunk of Pinus massoniana. RESULTS: Fluopyram transport in the trunk occurred through radial diffusion and vertical uptake within 1 week of the injection, reaching all tissues of P. massoniana, including apical branches and needles. Three years after the field test, the infection of PWD declined substantially with treatment using the fluopyram trunk-injection agent, which demonstrated 100% efficacy in both the mild and moderate occurrence areas, and 71.1% efficacy in the severe occurrence area. Fluopyram as trunk-injection agent exerted substantial control over PWD, with its efficacy being influenced by the infection time of PWD. The half-life of 10% fluopyram in treated pine trees was 346.6 days with 3-year persistence. CONCLUSION: The advantages of overall distribution and long persistence of fluopyram in the tree after injection help explain its evident efficacy against PWN. Overall, fluopyram trunk-injection has potential to prevent PWD. © 2023 Society of Chemical Industry.

Temperature and metal ions regulate larval diapause termination via the 20-hydroxyecdysone and juvenile hormone pathways in Monochamus alternatus.

BACKGROUND: Diapause allows insects to survive harsh environments, and its termination is crucial for their normal development after diapause. However, little is known about the regulatory pathways and signals involved in insect diapause termination. RESULTS: We discovered that high temperature (25 °C) influenced larval diapause termination in Monochamus alternatus. Likewise, metal ions (Ca2+ ) promoted diapause termination by reducing diapause duration. We combined transcriptomic and metabolomic analyses to investigate changes in gene expression and metabolism in diapause-terminated larvae treated with high temperature (MaHt) and metal ions (MaCa). Hormone biosynthesis and metabolism contained the highest proportion of significant differentially expressed genes (DEGs) in the two groups. 20-hydroxyecdysone (20E) and juvenile hormone (JH) were closely related to diapause termination in M. alternatus. RNA interference (RNAi) experiments verified that 20E biosynthesis (CYP314a1) and degradation (CYP18a1), JH biosynthesis (FOHSDR-1) and degradation (JHEH) genes affected the larval diapause duration significantly. In addition, dysfunction of CYP314a1 resulted in increased larval mortality (P < 0.01), reduced pupation rate and emergence rate (P < 0.05). Enzyme-linked immunosorbent assay (ELISA) analysis showed that the ecdysone content decreased after dsCYP314a1 injection and JH content increased after dsJHEH injection. CONCLUSION: The results indicate that genes CYP314a1, CYP18a1, FOHSDR-1 and JHEH mediated 20E and JH biosynthesis and degradation to regulate diapause termination in M. alternatus. We elucidated the molecular mechanism underlying the regulation of diapause termination and provided a basis for the prevention and control of M. alternatus infestation. © 2022 Society of Chemical Industry.

Expression of PmACRE1 in Arabidopsis thaliana enables host defence against Bursaphelenchus xylophilus infection.

BACKGROUND: Pine wilt disease (PWD) is a destructive disease that endangers pine trees, resulting in the wilting, with yellowing and browning of the needles, and eventually the death of the trees. Previous studies showed that the Avr9/Cf-9 rapidly elicited (PmACRE1) gene was downregulated by Bursaphelenchus xylophilus infection, suggesting a correlation between PmACRE1 expression and pine tolerance. Here, we used the expression of PmACRE1 in Arabidopsis thaliana to evaluate the role of PmACRE1 in the regulation of host defence against B. xylophilus infection. RESULTS: Our results showed that the transformation of PmACRE1 into A. thaliana enhanced plant resistance to the pine wood nematode (PWN); that is, the leaves of the transgenic line remained healthy for a longer period than those of the blank vector group. Ascorbate peroxidase (APX) activity and total phenolic acid and total flavonoid contents were higher in the transgenic line than in the control line. Widely targeted metabolomics analysis of the global secondary metabolites in the transgenic line and the vector control line showed that the contents of 30 compounds were significantly different between these two lines; specifically, the levels of crotaline, neohesperidin, nobiletin, vestitol, and 11 other compounds were significantly increased in the transgenic line. The studies also showed that the ACRE1 protein interacted with serine hydroxymethyltransferase, catalase domain-containing protein, myrosinase, dihydrolipoyl dehydrogenase, ketol-acid reductoisomerase, geranylgeranyl diphosphate reductase, S-adenosylmethionine synthase, glutamine synthetase, and others to comprehensively regulate plant resistance. CONCLUSIONS: Taken together, these results indicate that PmACRE1 has a potential role in the regulation of plant defence against PWNs.

The complete mitochondrial genome of Agriotes fuscicollis Miwa, 1928 (Coleoptera: Elateridae).

In this study, we sequenced the complete mitochondrial genome of Agriotes fuscicollis Miwa, 1928 (Coleoptera: Elateridae). The results showed that the length of complete mitochondrial genome was 15,866 bp with 26.8% GC content, containing 39.6% A, 33.5% T, 16.8% C, 10% G. There were 13 protein-coding genes (PCGs), 22 transfer RNA genes, and 2 ribosomal RNA genes. Phylogenetic analysis showed that A. fuscicollis was closely related to Cryptalaus larvatus, Cryptalaus yamato, Pyrophorus divergens and Ignelater luminosus. The complete mitogenome of A. fuscicollis would contribute to the study of the phylogeny and evolution of Elateridae.

Proteolytic Activation of Bacillus thuringiensis Cry3Aa Toxin in the Red Palm Weevil (Coleoptera: Curculionidae).

The red palm weevil (RPW), Rhynchophorus ferrugineus (Oliver) is an important pest of palms that causes significant damage by boring into and feeding within palm stem tissues. Here, we studied the proteolytic process of Cry3Aa in the RPW to understand the mechanism of Cry toxicity. The bioassays showed that Cry3Aa toxin is weakly toxic to the RPW. Proteolytic activation assays indicated the Cry3Aa protein is digested into smaller fragments than the 55-kDa activated fragments under different conditions. In particular, at higher mass ratios of gut protease and Cry3Aa protein (5:1, 2:1, and 1:1, respectively), and at 36.9°C for 16 h in a solution of pH 8.6, the Cry3Aa protoxin is over-digested by the gut proteases of weevil larvae. Moreover, the zymogram analysis of the gut proteases revealed the RPW larvae harbors intestinal digestive enzymes mainly composed of serine proteases. This study describes the proteolytic activation process of Cry3Aa in the midgut of RPW larvae.

Gut Bacterial Communities of Lymantria xylina and Their Associations with Host Development and Diet.

The gut microbiota of insects has a wide range of effects on host nutrition, physiology, and behavior. The structure of gut microbiota may also be shaped by their environment, causing them to adjust to their hosts; thus, the objective of this study was to examine variations in the morphological traits and gut microbiota of Lymantria xylina in response to natural and artificial diets using high-throughput sequencing. Regarding morphology, the head widths for larvae fed on a sterilized artificial diet were smaller than for larvae fed on a non-sterilized host-plant diet in the early instars. The gut microbiota diversity of L. xylina fed on different diets varied significantly, but did not change during different development periods. This seemed to indicate that vertical inheritance occurred in L. xylina mutualistic symbionts. Acinetobacter and Enterococcus were dominant in/on eggs. In the first instar larvae, Acinetobacter accounted for 33.52% of the sterilized artificial diet treatment, while Enterococcus (67.88%) was the predominant bacteria for the non-sterilized host-plant diet treatment. Gut microbe structures were adapted to both diets through vertical inheritance and self-regulation. This study clarified the impacts of microbial symbiosis on L. xylina and might provide new possibilities for improving the control of these bacteria.

A new species of the genus Colopalpus Pritchard and Baker (Trombidiformes: Tenuipalpidae) from China, with ontogenetic patterns in chaetotaxy.

A new species, Colopalpus hibiscus sp. nov. (Acari: Tenuipalpidae) is described from Hibiscus in Hainan province, China. The ontogenetic changes in the idiosoma and leg chaetotaxy on adults (female and male) and immature stages of this new species are presented. A key to species of Colopalpus is also provided.

The complete mitochondrial genome of Plagiodera versicolora (Laicharting)(Coleoptera: Chrysomelidae).

Plagiodera versicolora (Laicharting) is a leaf-eating pest widely distributed in the world. In this study, the first complete mitochondrial genome of P. Versicolora (Laicharting) was assembled and analyzed. The complete mitochondrial genome of P. Versicolora (Laicharting) is 16,857 bp with 22.39% GC containing, 13 protein-coding genes, 22 transfer RNA (tRNA), 2 ribosomal RNA (rRNA), as well as an AT-rich region. Phylogenomic analysis indicated that P. Versicolora (Laicharting) is sister to Chrysomela populiThis study provides useful information for the identification of this species and the study of genetic evolution with other species of Chrysomelidae.

The complete mitochondrial genome of Chrysolina aeruginosa Fald (Coleoptera: Chrysomelidae).

In this study, the first complete mitochondrial genome of Chrysolina aeruginosa Fald was assembled and analyzed. The total length of this mitochondrial genome is 16,335 base pairs. It consists of 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and an AT-rich region. Phylogenomic analysis indicated that C. aeruginosa Fald is sister to Chrysodinopsis sp. This study provides new molecular data for the further taxonomic and phylogenetic studies of the Chrysomelidae of Coleoptera.

Characterization of Bacterial Communities Associated with Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) and its Host Phoenix sylvestris.

This study measured the changes of microorganisms in the midgut and habitat niche of Rhynchophorus ferrugineus Olivier, an invasive quarantine pest, by Illumina sequencing. The bacterial diversity in the R. ferrugineus larvae midgut and their habitat niche was compared to the uninfected P. sylvestris. The Proteobacteria and Firmicutes occupied a dominant position in the R. ferrugineus midgut and infected P. sylvestris, while in the uninfected P. sylvestris the predominant bacterial phylum was the Cyanobacteria. Enterobacter, Dysgonomonas, and Entomoplasma were the dominant bacterial genera in R. ferrugineus midgut and also within the infected trees and uninfected trees with low relative abundance. These bacteria could be exploited as the biopesticide vector to control R. ferrugineus population. Besides, Sphingobacterium, Shinella, and Rhodobacter genera had the same distribution pattern in the infected and uninfected P. sylvestris, and these bacteria were not found in the midgut of R. ferrugineus. Interestingly, Paludibacter and Parabacteroides were only distributed in the wood fiber of the infected P. sylvestris, which could be used as potential microbial markers to detect if the palm plants are damaged by the R. ferrugineus. The results of this study will be beneficial to the development of control strategies for R. ferrugineus.

Identification and Characterization of Aminopeptidase-N as a Binding Protein for Cry3Aa in the Midgut of Monochamus alternatus (Coleoptera: Cerambycidae).

Bacillus thuringiensis Cry proteins have been widely used over the past decades for many different insect pests, which are safe for users and the environment. The coleopteran-specific Cry3Aa toxin from B. thuringiensis exhibits toxicity to the larvae of Monochamus alternatus. Receptors play a key role in the mechanisms underlying the toxic action of Cry. However, the binding receptor for Cry3Aa has yet to be identified in the midgut of M. alternatus larvae. Therefore, the aim of this study was to identify the receptor for Cry3Aa toxin in the brush border membrane vesicles (BBMVs) of M. alternatus larvae. Our results indicate that the Cry3Aa toxin binds to the BBMVs (Kd = 247 nM) of M. alternatus via a 107 kDa aminopeptidase N (APN) (Kd = 57 nM). In silico analysis of the APN protein predicted that an 18 amino acid sequence in the N-terminal acted as a signal peptide, and that the Asn residue, located at position 918 in the C-terminus is an anchored site for glycosyl phosphatidyl inositol. Further analysis showed that M. alternatus APN exhibits 75% homology to the APN from Anoplophora glabripenis. Our work, therefore, confirmed that APN, which is localized in the BBMVs in the midgut of M. alternatus larvae, acts as a binding protein for Cry3Aa toxins.

Characterization of bacterial communities associated with the pinewood nematode insect vector Monochamus alternatus Hope and the host tree Pinus massoniana.

BACKGROUND: Monochamus alternatus Hope is one of the insect vectors of pinewood nematode (Bursaphelenchus xylophilus), which causes the destructive pine wilt disease. The microorganisms within the ecosystem, comprising plants, their environment, and insect vectors, form complex networks. This study presents a systematic analysis of the bacterial microbiota in the M. alternatus midgut and its habitat niche. METHODS: Total DNA was extracted from 20 types of samples (with three replicates each) from M. alternatus and various tissues of healthy and infected P. massoniana (pines). 16S rDNA amplicon sequencing was conducted to determine the composition and diversity of the bacterial microbiota in each sample. Moreover, the relative abundances of bacteria in the midgut of M. alternatus larvae were verified by counting the colony-forming units. RESULTS: Pinewood nematode infection increased the microbial diversity in pines. Bradyrhizobium, Burkholderia, Dyella, Mycobacterium, and Mucilaginibacter were the dominant bacterial genera in the soil and infected pines. These results indicate that the bacterial community in infected pines may be associated with the soil microbiota. Interestingly, the abundance of the genus Gryllotalpicola was highest in the bark of infected pines. The genus Cellulomonas was not found in the midgut of M. alternatus, but it peaked in the phloem of infected pines, followed by the phloem of heathy pines. Moreover, the genus Serratia was not only present in the habitat niche, but it was also enriched in the M. alternatus midgut. The colony-forming unit assays showed that the relative abundance of Serratia sp. peaked in the midgut of instar II larvae (81%). CONCLUSIONS: Overall, the results indicate that the bacterial microbiota in the soil and in infected pines are correlated. The Gryllotalpicola sp. and Cellulomonas sp. are potential microbial markers of pine wilt disease. Additionally, Serratia sp. could be an ideal agent for expressing insecticidal protein in the insect midgut by genetic engineering, which represents a new use of microbes to control M. alternatus.