Cradle for the newborn Monochamus saltuarius: Microbial associates to ward off entomopathogens and disarm plant defense

The Japanese pine sawyer, Monochamus saltuarius, as a beetle vector of Bursaphelenchus xylophilus (pine wood nematode), is an economically important forest pest in Eurasia. To feed on the phloem and xylem of conifers, M. saltuarius needs to overcome various stress factors, including coping with entomopathogenic bacteria and also various plant secondary compounds (PSCs). As an important adaptation strategy to colonize host trees, M. saltuarius deposit eggs in oviposition pits to shield their progeny. These pits harbor bacterial communities that are involved in the host adaptation of M. saltuarius to the conifers. However, the composition, origin, and functions of these oviposition pit bacteria are rarely understood. In this study, we investigated the bacterial community associated with M. saltuarius oviposition pits and their ability to degrade PSCs. Results showed that the bacterial community structure of M. saltuarius oviposition pits significantly differed from that of uninfected phloem. Also, the oviposition pit bacteria were predicted to be enriched in PSC degradation pathways. The microbial community also harbored a lethal strain of Serratia, which was significantly inhibited. Meanwhile, metatranscriptome analysis indicated that genes involved in PSCs degradation were expressed complementarily among the microbial communities of oviposition pits and secretions. In vitro degradation showed that bacteria cultured from oviposition pits degraded more monoterpenes and flavonoids than bacteria cultured from uninfected phloem isolates. Disinfection of oviposition pits increased the mortality of newly hatched larvae and resulted in a significant decrease in body weight in the early stages. Overall, our results reveal that M. saltuarius construct oviposition pits that harbor a diverse microbial community, with stronger PSCs degradation abilities and a low abundance of entomopathogenic bacteria, resulting in the increased fitness of newly hatched larvae.


Introduction
Phytophagous insects are a highly successful group of animals with considerable species diversity and multiple lifestyles, spanning various ecological niches worldwide (Farrell, 1998;Hansen & Moran, 2014;Forister et al., 2015). Plants have evolved defensive mechanisms to deter herbivores by producing a wide range of plant secondary compounds (PSCs) (e.g., flavonoids, glucosinolates, and terpenoids) that function as antifeedants, repellents, or direct toxins to phytophagous insects (Berenbaum & Zangerl, 1998;Ceja-Navarro et al., 2015;Vilanova et al., 2016;Berasategui et al., 2017;Cheng et al., 2018;Yang et al., 2022). Beyond the defensive reactions to insect feeding, plants also respond to pest oviposition through various strategies, including the active abscission of egg-carrying leaves or increasing the secretion of ovicidal substances. Some plants can also cope with herbivore eggs by producing secondary volatiles that attract parasitoids (Hilker & Meiners, 2006. Besides plant defense, several studies have demonstrated that some plant-associated entomopathogenic bacteria may produce systemic insect resistance and some may also harbor strain-specific arsenals of antifungal metabolites, which enable them to inhibit pathogen growth through direct antibiosis (Raaijmakers et al., 2010). Some of the best-characterized biocontrol strains in Pseudomonas and Serratia were shown to have potent insecticidal activity in both systemic and oral infections. A strain of Serratia marcescens can even kill Anoplophora glabripennis larvae through contact infection only (Deng et al., 2008;Ruffner et al., 2013). Once infected, these plant-associated bacteria exhibit extremely high insecticidal activity by rapidly multiplying and causing larval death within a few days, and even hours, and thus serve as biofertilizers and biopesticides (Deng et al., 2008;Kupferschmied et al., 2013;Flury et al., 2016). However, as a co-evolutionary system, phytophagous insects have evolved distinct strategies to defeat plant defenses, such as the manipulation of plant defenses to reduce toxicity, the modification of feeding strategies to avoid poisoning, or the reduction of target sensitivity to increase resistance (Despres et al., 2007).
Wood-boring pests are an important group of herbivores with unique ecological niches, as their beneathbark feeding behavior may protect them from most predators. These wood-feeding insects encounter an extremely challenging environment, with scarce nitrogen content (Hölttä et al., 2013), an indigestible carbohydrate source in the form of lignocellulose (Scully et al., 2013;Tokuda et al., 2014), the threat of infection from potentially pathogenic microorganisms, and a variety of toxic PSCs (Ericsson et al., 1988;Keeling & Bohlmann, 2006;Faccoli & Schlyter, 2007). However, symbiotic microorganisms play an important role in the interactions between borers and their host trees. Symbionts such as gut endophytic bacteria have been reported to help wood-feeding beetles degrade a variety of toxic PSCs, including monoterpenes, diterpenes, flavonoids, and isothiocyanates (Xu et al., 2015;Berasategui et al., 2017;Cheng et al., 2018). Furthermore, some symbiotic bacteria supply the host with nutrition by decomposing PSCs (Berasategui et al., 2017). Several studies have reported the role of symbiotic microorganisms to assist hosts in eradicating microbial infections (Wong et al., 2011;Zhou et al., 2020). However, few studies have focused on the potential role of microbial symbioses in the oviposition pits of borers. Their relationship with PSCs and entomopathogenic bacteria in insect host adaptation remains unknown.
The Japanese pine sawyer Monochamus saltuarius (Coleoptera: Cerambycidae) feeds on the phloem and xylem of a wide range of conifer species. It is considered a serious forest pest in Eurasia because of its catastrophic impacts on conifers. This pest acts as a vector for an invasive pathogenic nematode, Bursaphelenchus xylophilus, causing pine wilt disease (Takizawa & Shoji, 1982;Kwon et al., 2006;Yu & Wu, 2018;Hou et al., 2021). The oviposition behavior of M. saltuarius has been studied in detail as the initial stage in colonizing host trees (Fig. 1). The female gnaws at the surface of the bark to produce an entrance to the vascular tissue after a considerable period of positioning (Fig. 1C). Subsequently, the female turns around and excavates an oviposition pit with the ovipositor, and then deposits a single egg beneath the bark (Fig. 1D). The female adult usually leaves after sealing the wounds with secretions and rubbing the wound surface with her abdomen (Anbutsu & Togashi, 2001). Similar multi-step and laborious oviposition behavior also occurs in other longicorn beetles in the subfamily Lamiinae (Mason et al., 2018). It has previously been observed that the bacterial communities of oviposition pits were similar to that of larvae and eggs in A. glabripennis (Scully et al., 2014;Mason et al., 2018). The frass in oviposition pits can serve as a vehicle for the transmission of a subset of beetle gut microbiota (Mason et al., 2018). Several studies have shown that lethal insect pathogenic bacteria exist widely in both the uninfected phloem and the oviposition pits of longicorn beetles (Deng et al., 2008). The discovery of entomopathogenic bacteria and heterogeneous microbe communities in oviposition pits has raised diverse ecological questions. Does the abundance of pathogenic bacteria differ between oviposition pits and uninfected phloem. What ecological advantage may be gained by this transfer from phloem-associated bacteria to an oviposition pit microbial community? Do the bacteria associated with ovipositor secretions participate in the degradation of PSCs? Based on previous research, we hypothesized that M. saltuarius may shape the microbial community associated with oviposition pits, which can be significantly different from that in uninfected phloem. This specific community may possess a low abundance of entomopathogens and may participate in the degradation of some toxic PSCs in vitro, thus protecting newly hatched larvae. Microbes in ovipositor secretions may play an important role in the degradation of PSCs.
In this study, we investigated differences in the quantity and community structure of bacteria associated with oviposition pits and uninfected phloem via Illumina sequencing of the 16S rRNA gene and quantitative realtime polymerase chain reaction (qPCR). After discovering that the bacterial community is changed by oviposition, we isolated and verified the pathogenicity of an insect pathogenic bacteria that had been inhibited in the oviposition pits. To evaluate the PSC degradation ability of the bacterial community, we compared the level of residual PSCs after degradation by bacterial communities cultured from oviposition pits and from uninfected phloem through in vitro experiments. We further analyzed the metatranscriptomes of the cultured oviposition pit symbionts and ovipositor secretion-associated microbes to determine the potential source of PSC-degrading bacteria. Finally, to evaluate the ecological advantages brought by these symbiotic bacteria, we performed bioassays to assess their effect on the survival rate and weight gain of newly-hatched larvae in the early stages.

Sample collection
Adults of M. saltuarius were collected with clean pitfall traps at Dahuofang Forest Farm, Fushun City, Liaoning Province, China. After transferring beetles to the laboratory, adults were paired and stored in boxes with moist paper and fresh Korean pine twigs (Pinus koraiensis) at 22-28°C. Adults were brought to the laboratory 3 d before laying eggs for acclimatization. Females with high vitality were then manually selected and moved to rearing chambers containing Korean pine logs to lay eggs for 12 h. The above-mentioned process was carried out 3 times. Then, we dissected the oviposition pits with sterilized forceps and scalpel, retrieved tissues damaged by oviposition, and collected secretions inside the oviposition pits with sterilized 10-μL pipette tips individually. Uninfected phloem tissues from the same batch of logs were also collected for comparison. All samples were stored at 20−80°C until use.

16S rRNA gene sample preparation and sequencing, quantitative PCR, and analysis
We employed 16S rRNA gene sequencing (Illumina, San Diego, CA, USA) and qPCR to investigate the difference in content and diversity of the bacteria associated with oviposition pits and with uninfected phloem. In the experiment, 6 samples from a single oviposition pit and 5 samples of uninfected phloem were used for profiling. Bacterial community genomic DNA of oviposition pit samples was extracted using the DNeasy ® 96 PowerSoil ® Pro QIAcube ® HT kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. DNA extractions were checked on 1% agarose gel. The concentration and purity were then determined with a NanoDrop 2000 UV-Vis spectrophotometer (ThermoFisher Scientific, Wilmington, NC, USA). The hypervariable region V5-V7 of the bacterial 16S rRNA gene was amplified with primer pairs 779F (5 -AACMGGATTAGATACCCKG-3 ) and 1193R (5 -ACGTCATCCCCACCTTCC-3 ) using an ABI GeneAmp ® 9700 PCR thermocycler (Applied Biosystems, Waltham, MA, USA).
Absolute quantification of bacteria was carried out using qPCR targeting the 16S rRNA gene. A mix of 10 μL of ChamQ SYBR Color qPCR Master Mix (2×) (Vazyme Biotech, Shanghai, China), 0.25 μL of 5 μmol/L forward primer, 0.25 μL of 5 μmol/L reverse primer, 7.5 μL of nuclease-free water, and 2 μL of DNA template was added in each reaction. qPCR was performed on a Li-neGene 9600 Plus fluorescent qPCR detection system (Bioer Technology, Hangzhou, China), as follows: initial denaturation at 95°C for 5 min, then 40 cycles of denaturation at 95°C (30 s), annealing at 56°C (30 s), and elongation at 72°C (40 s), followed by a melting curve analysis from 60°C to 95°C in increments of 0.5°C for 5 s each. Each constructed plasmid was diluted by 10-fold serial dilution (90 μL of diluent + 10 μL of plasmid). Between 4 and 6 points (with standard sample concentrations ranging from 10 −2 to 10 −7 ) were selected through a preliminary study to construct a standard curve. We ensured that all qPCR standard curves had efficiency values between 90% and 110% and R 2 values above 0.9.
Subsequently, purified amplicons were pooled at equimolar concentrations and paired-end sequenced (2 × 300) on the Illumina MiSeq PE300 platform, according to the standard protocols of Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Operational taxonomic units (OTUs) were clustered with a similarity cut-off of 97% (Edgar, 2013) by UPARSE 7.1 (Stackebrandt & Goebel, 1994;Edgar, 2013), and chimeric sequences were identified and removed. The taxonomy of each OTU representative sequence was analyzed using RDP Classifier 2.2 (Wang et al., 2007) against the 16S rRNA database SILVA 138 using a confidence threshold of 70%. The alpha-diversity indexes were calculated using mothur 1.30.2 (Schloss et al., 2009). The rarefaction curve and bar plots were generated using the "vegan" package in R (Oksanen et al., 2019). β-Diversity was estimated by QIIME 1.9.1 (Caporaso et al., 2010) and visualized using a principal coordinates analysis (PCoA) based on Bray-Curtis distances performed using the R package vegan 2.5.

Degradation of PSCs by cultured oviposition pit microbes and uninfected phloem microbes
To verify whether the oviposition pit flora has a stronger PSCs degradation ability, we cultivated communities from oviposition pits and from uninfected phloem separately and observed the in vitro degradation of 6 PSCs candidates: five oviposition pit samples and 5 uninfected phloem samples were dissected under sterile conditions and suspended in a centrifugal tube with 1 mL of PBS buffer. Tissues were macerated with a pestle and vortexed at medium speed for 25 s to separate microbial material from the samples. After that, tubes were centrifuged at 3000 × g to pellet sample tissues. A 10 μL quantity of supernatant bacterial suspension was inoculated in 10 mL of 10% tryptic soy broth (TSB) medium and cultured overnight shaking at 180 r/min at 28°C. Overnight cultures were then diluted to an optical density of 0.1 at 600 nm (OD 600 ) with an additional 10% TSB medium. To test whether the oviposition pit symbioses and uninfected phloem microorganisms can degrade PSCs, we inoculated 10 μL of the diluted bacterial culture in tubes with 10 mL of 10% TSB medium containing 6 different toxic PSCs, individually: (i) (+)-catechin, (ii) taxifolin, and (iii) resveratrol were added to 1% of dry media weight; and (iv) naringenin, (v) α-pinene, and (vi) 3-carene were added to 2% of dry media weight. The proportions of the PSCs added follow the methods used previously in studies on the content of corresponding substances in the phloem of healthy conifers (Faccoli & Schlyter, 2007;Cheng et al., 2018). Likewise, 10 μL of 10% TSB medium was inoculated in control tubes of medium amended with the aforementioned PSCs. The experiment was replicated 5 times in each group. All cultures were allowed to grow for 18 h and were taken for analysis at the 9-and 18-h time points.

Monoterpene and flavone content measurement
We measured the residual monoterpene and flavone content after degradation by the 2 microbial groups, using GCMS and HPLC, respectively: for monoterpene (αpinene and 3-carene) degradation by oviposition pits and uninfected phloem microbes the metabolites were salted out from the aqueous phase using 9 mL of ultrapure water, followed by the addition of 10 μL of 10 mg/L toluene-D8. The suspension was homogenized for 20 min at 60 r/min and then held vertically for 10 min for phase separation. Changes in the metabolites of the samples were analyzed via headspace injection. A 1-μL volume of the upper phase was analyzed in a gas chromatograph mass spectrometer (GCMS QP2010-Ultra; Shimadzu, Kyoto, Japan) equipped with an Optima-5 column (30 m × 0.25 mm × 0.25 μm, Agilent DB-WAX capillary column; Agilent, Santa Clara, CA, USA). The temperature program ran as follows: injection port, 25°C; column held for 3 min at 35°C, then temperature increased at a rate of 10°C/min until 120°C, held constant for 0.1 min, then temperature increased at a rate of 20°C/min up to 230°C, and held constant at 230°C for 9 min; detection temperature, 230°C. We integrated and analyzed the peaks using the chromatography data system software Chromeleon TM .
After microbial degradation, the residual contents of the flavones (+)-catechin, naringenin, resveratrol, and taxifolin were analyzed by high-performance liquid chromatography (HPLC). The analysis was carried out on a Waters ACQUITY UPLC instrument equipped with an AB 5000 Triple Quadrupole Mass Spectrometer (Sciex, Framingham, MA, USA). The samples were separated through an ACQUITY UPLC® BEH C18 column (2.1 × 100 mm, 1.7 μm; Waters, Milford, MA, USA), and the column temperature was 40°C. The mobile phases included solvent A (1%-0.1% formic acid) and solvent B (100% methanol). The flow rate was 0.25 mL/min, and the injection volume was 5 μL. The mass spectrometry conditions included an electrospray ionization (ESI) source and negative ion ionization mode. The ion source temperature was 500°C, the ion source voltage was −4500 V, the collision gas was 6 psi, the curtain gas was 30 psi, and both the atomization gas and the auxiliary gas were applied at 50 psi. Multiple reaction monitoring (MRM) was used for scanning.
The samples were quantified based on the standard curves generated from the relative monoterpene or flavonoid components (Table S1).

Isolation, preliminary identification, and injection assays of a potential pathogen, S. marcescens (OTU231)
To investigate the entomopathogenicity and taxonomic status of OTU231, the following experiments were conducted. The microbe cultures described above were diluted to 1 × 10 −6 and spread onto potato dextrose agar (PDA) plates. After shaking at 180 r/min at 28°C for 48 h, single colonies were selected and inoculated in 10% TSB and cultured as described above. Genomic DNA of pure cultures was extracted using the Bacterial gDNA Isolation Kit (BIOMIGA, Hangzhou, China). The bacterial 16S rRNA gene was amplified with primer pairs 27F (5 -AGAGTTTGATCCTGGCTCA-3 ) and 1492R (5 -GGTTACCTTGTTACGACTT-3 ). Sequences for the 16S rRNA gene in Serratia species were retrieved from the National Center for Biotechnology Information (NCBI) Assembly database (Table S2). Sequences with a high similarity of OTU231 were included in the phylogeny. In Serratia species other than S. marcescens, only the type strain sequences were considered. Sequences were aligned with MUSCLE, and the phylogenetic tree was obtained with the MEGAX maximum-likelihood algorithm using a bootstrap of n = 1000. Serratia marcescens was clustered with OTU231 and was thus regarded as a preliminary identification result.
Injection assays were performed as described by Flury et al. (2016). The washed bacterial cells (10 μL) suspended in 0.9% sterile NaCl solution and adjusted to the desired concentration were injected into the hemolymph of ultimate-instar M. saltuarius larvae (Fushun, Liaoning, China). Sterile 0.9% NaCl solution served as the control. Each treatment had 10 larvae, and the experiment was replicated 3 times. The injected larvae were kept in a sterilized 2-mL centrifuge tube at 25°C in the dark for 48 h. The larvae were considered dead if they did not show any reaction after being probed repeatedly.

Metatranscriptomic sequencing of cultured microflora from oviposition pits and ovipositor secretions
To determine the potential source of PSCs-degrading bacteria in communities cultured from oviposition pits and ovipositor secretions, we compared the gene expressions involved in the related pathways among them. Five oviposition pit samples and five ovipositor secretion samples were, respectively, collected under sterile conditions and suspended in a centrifugal tube with 1 ml of PBS buffer. The culture and dilution steps of the microbial samples were the same as described previously. Samples were collected from overnight cultures of diluted solutions. Three replicates were collected in each group. Total RNAs were extracted using the E.Z.N.A. ® Soil RNA Kit (Omega Bio-tek, Norcross, GA, USA), according to manufacturer's protocols. The RNA concentration and purity were quantified with NanoDrop2000 (ThermoFisher Scientific). RNA quality was determined with 1% agarose gel electrophoresis. RNA quality was assessed using an RNA6000 Nano chip (total RNA) in an Agilent 2100 Bioanalyzer. Total RNA of samples was subjected to an rRNA removal procedure using the Ribo-zero Magnetic kit, according to the manufacturer's instructions (Epicentre Biotechnologies, an Il-lumina® company, Madison, WI, USA). cDNA libraries were constructed using the TruSeq TM RNA sample prep kit (Illumina). The barcoded libraries were paired-end sequenced on the Illumina Hiseq 2500 platform at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China), using the HiSeq 4000 PE Cluster Kit and the HiSeq 4000 SBS Kit, according to the manufacturer's instructions. The 3 and 5 ends were stripped using SeqPrep (https: //github.com/jstjohn/SeqPrep). Low-quality reads (with a length of <50 bp, with a quality value of <20, or with N bases) were removed using Sickle (https://github. com/najoshi/sickle). rRNA reads were removed using SortMeRNA (http://bioinfo.lifl.fr/RNA/sortmerna/) and aligning to the SILVA 128 database (https://www.arbsilva.de/). Open reading frames (ORFs) from each sample were predicted using TransGeneScan (http://sourceforge. net/projects/transgenescan/). All sequences with a 95% sequence identity (90% coverage) were clustered as the nonredundant gene catalog using CD-HIT (http://www. bioinformatics.org/cd-hit/). Reads after quality control were mapped to the representative genes with 95% identity, and TPM values were evaluated using RSEM (http: //deweylab.biostat.wisc.edu/rsem/). BLASTP 2.2.28+ (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was employed for taxonomic annotations by aligning nonredundant gene catalogs against the NCBI NR database with evalue cut-offs of 1e −5 . GO annotation was performed using Blast2go (http://www.blast2go.com/b2ghome), and aligning sequences to the GO database (Gene Ontology, http://www.geneontology.org). Clusters of orthologous genes (COG) annotation was performed using BLASTP against the eggNOG database (evolutionary genealogy of genes: Non-supervised Orthologous Groups, v4.5; http: //eggnog.embl.de/). KEGG annotation was performed using BLASTP against the KEGG database (Kyoto Encyclopedia of Genes and Genomes, http://www.genome. jp/kegg/). All the blast e-value cut-offs were 1e −5 . The heat map of secondary metabolite degradation gene expression was generated with the "pheatmap" library in R using the scale = "row" parameter.

Effects of oviposition pit disinfection on the survival of newly-hatched larvae and their initial weight gain
We manipulated the bacterial community structure of oviposition pits to evaluate the influence on the fitness of newly hatched larvae. Single newly hatched larvae from the oviposition pits were randomly assigned to a feeding tube. Each tube contained the same volume of Korean pine fresh phloem tissue as the diet prepared for larvae. Each tube was randomly assigned to 1 of 3 treatments: (i) control (n = 10); (ii) sterilized (n = 10); and (iii) reinfected (n = 10). The larvae from the control group, associated with the original oviposition pits, received no treatment. By contrast, the larvae of the sterilized group were immediately moved to a sterile environment after hatching. Their oviposition pits were sterilized twice (with 15-min intervals) with 10 μL of 75% alcohol before the larvae were returned to their original pits. The treatment given to the reinfected group was similar to that of the sterilized group, but their oviposition pits were treated with 10 μL of microbial suspension after disinfection. The suspension was generated by crushing the tissues of 5 untreated oviposition pits in 1 mL of PBS with vortexing. Larvae were subjected to the experiment for 1 week, and each larva was provided with fresh phloem every 48 h. The survival of the larvae was observed every 12 h. All surviving larvae were weighed on days 1, 3, and 7. During the experiments, the general growth conditions for the larvae were 25°C and 60% humidity in a dark environment. The survival rate of M. saltuarius larvae was calculated using the Kaplan-Meier survival analysis. The comparison between survival curves was further tested using the logrank (Mantel-Cox) method, implemented in GraphPad Prism 8 (GraphPad, San Diego, CA, USA).

Data availability
All sequencing data have been uploaded to NCBI under the following BioProject numbers: PRJNA816634 and PRJNA820901.

General profile of the 16S rRNA gene sequencing data from oviposition pits and host phloem
A total of 1 313 361 high-quality and filtered sequences were obtained from 6 M. saltuarius oviposition pits and 5 uninfected Korean pine phloem samples, with an average read length of 377 bp per sample. After subsampling the reads to an equal sequencing depth (105 780 sequences), a total of 619 OTUs were clustered with 97% sequence identity. The rarefaction curves suggested that all samples showed convergence in their sampling-effort curves (Fig. S1), indicating a sufficient sequencing depth capturing most of the bacterial diversity.

Bacterial richness and within-habitat diversity differences between oviposition pits and uninfected phloem
We performed qPCR on 2 groups of samples to determine whether there was any difference in the total quantity of bacteria found in the oviposition pits and in the phloem. The results showed that there was no significant difference (Mann-Whitney U-test, P > 0.05) in the mean number of copies of bacterial 16S rRNA gene between the groups (Fig. 2B), thus indicating that the total quantity of bacteria in the oviposition pits should be roughly the same as that found in the phloem.
Among the 619 OTUs observed in all samples, 259 OTUs were shared between the groups and 243 OTUs were considered to be unique to the oviposition pits. 117 unique OTUs were detected in samples from uninfected phloem (Fig. S2). The bacterial communities in both groups were mainly composed of 3 phyla: Proteobacteria (oviposition pits, 93.47% ± 6.25% SD; uninfected phloem, 96.78% ± 2.15% SD), Actinobacteria (oviposition pits, 4.77% ± 4.7% SD; uninfected phloem, 1.24% ± 0.61% SD), and Firmicutes (oviposition pits, 0.67% ± 0.57% SD; uninfected phloem, 1.4% ± 1.35% SD). There was no significant difference observed in dominant phyla between groups (P > 0.05). To evaluate the variation in bacteria richness and diversity on the genus level, 3 indices (Shannon, Simpson, and Chao1 indices) were employed to estimate the alpha diversity. The results showed no significant difference in all alpha diversity indices (Welch's t-test, Chao1, Shannon, and Simpson, P > 0.05; Table S3). At the genus level ( Fig. 2A), we found that the top-4 genera with an average abundance of >2% in uninfected phloem samples were Serratia, unclassified_f__Enterobacteriaceae, Ralstonia, and Burkholderia-Caballeronia-Paraburkholderia, and that 7 dominant genera were observed in the oviposition pits: Serratia, unclassified_f__Enterobacteriaceae, Ralstonia, Pseudoxanthomonas, Sphingomonas, Burkholderia-Caballeronia-Paraburkholderia, and Methylobacterium-Methylorubrum. Serratia was the only dominant genus with significantly uneven distribution (oviposition pits, 26.66% ± 24.27% SD; uninfected phloem, 74.97% ± 15.14% SD) (P < 0.01). To further understand the differences in abundance of Serratia between groups, we compared the abundances of OTUs belonging to the genus (Fig. 2C). Only 2 OTUs were identified as Serratia. However, both of them were observed in all samples: OTU231 was enriched in healthy phloem, whereas its abundance in oviposition pits showed a steep decrease (Mann-Whitney test, P < 0.005). Conversely, OTU417 showed no significant difference in abundance between groups (Mann-Whitney test, P > 0.05). We have noticed that some new synonyms have been pointed out in the taxonomy of bacteria (Oren & Garrity, 2021), which may still be used in this study, e.g. Proteobacteria=Pseudomonadota, Actinobacteria=Actinomycetota, Firmicutes=Bacillota. However, due to the wide use of the above names (especially in databases), we still retain their usage in this article.

An entomopathogenic bacteria (OTU231), S. marcescens, exhibiting insecticidal activity towards M. saltuarius
To further identify OTU231, we sequenced the 16S rRNA gene on its pure culture and compared the sequence on NCBI databases. Phylogeny analyses indicated that OTU231 clustered with S. marcescens and Serratia nematodiphila (Fig. 3A). Based on the environment in which we isolated the strain, we preliminarily identified OTU231 as S. marcescens (because S. nematodiphila was usually reported as a nematode-specific symbiont; Zhang et al., 2009;Xu et al., 2017). To assess whether the strain was pathogenic to M. saltuarius, we mimicked a systemic infection by injecting bacteria into the hemolymph of beetle larvae. After 48 h of injection, the corrected mortality rate of longicorn beetle larvae reached 95.83% ± 4.3% SE, and the strain was successfully re-isolated from the dead larvae. Dead larvae injected with the strain showed little difference in morphology from living larvae but had a matte cuticle and relatively soft habitus (Fig. 3C). Within control groups, although some melanization symptoms were observed, most individuals were alive 48 h after injection and had a glossy body surface (Fig. 3D).

Oviposition pit bacteria community differs from that of uninfected phloem, with stronger plant secondary metabolite degradation potential
To investigate differences in community structure between groups, we conducted principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS) using Bray-Curtis dissimilarity distances. We checked for the presence of statistically supported groups using Palmanova clustering algorithms at both genus and OTU levels (Fig. 4). Almost all samples were divided into 2 groups, with clusters represented by oviposition pits and uninfected phloem at the genus level (permutational multivariate analysis of variance, PERMANOVA: P = 0.022; R 2 = 0.2327; F = 2.7298). This indicated that the egg-laying behavior shaped a different bacterial community in the oviposition pits compared with that found in uninfected phloem. Further, bacterial communities at the OTU level showed a more significant separation among groups (PERMANOVA: P = 0.003; R 2 = 0.3520; F = 4.8894), thus indicating that oviposition pit bacteria showed more distinct specificity at a lower taxonomic level. To reveal the latent functional differences of the bacterial communities, we compared the potential abundance differences in related PSC pathways (flavone degradation, phenol degradation, and terpene degradation) (Fig. 4E): among the 9 selected pathways, the bacterial community from oviposition pits was predicted to have significantly stronger PSC-degradation functions than the phloem community in 7 pathways (Mann-Whitney test, P < 0.05). Whereas there was no significant difference between the groups in another 2 pathways (Mann-Whitney test, P > 0.05). The above results suggest that the bacterial community in the oviposition pits is likely to degrade diverse toxic PSCs.

Metatranscriptomes show that bacteria cultured from oviposition pits and secretions express genes involved in the degradation of toxic secondary metabolites
We investigated the expression of genes related to the degradation of toxic PSCs between microbes found in oviposition pits and in secretions. The results showed that the 4 genes involved in limonene and pinene degradation (ko00903) ranged from 0 to 4436.332 TPM (Fig. 5C). The 25 genes annotated in benzoate degradation (ko00362) ranged from 0 to 5134.434 TPM in all samples. In addition, genes involved in aminobenzoate degradation (ko00627) (6 genes), styrene degradation (ko00643) (5 genes), geraniol degradation (ko00281) (4 genes), naphthalene degradation (ko00626) (4 genes), polycyclic aromatic hydrocarbon degradation (ko00624) (2 genes), and ethylbenzene degradation (ko00642) (1 gene) were also expressed ( Fig. 5C; Table S4). These benzoate degradation-related pathways are important intermediate pathways for the degradation of phenolic compounds, which could be further degraded into fatty acids. The terminal product acetyl-CoA could be utilized in the citrate cycle metabolic pathway. Intriguingly, although a few genes were not stably expressed within the group, the expression of most genes from the oviposition pit and the secretion groups showed strong exclusivity and complementarity (Fig. 5C).

Oviposition pit microbes reduce more plant secondary metabolites than uninfected phloem microbes in vitro
We isolated bacteria from the 2 groups to determine whether the communities from oviposition pits and uninfected phloem degrade monoterpenoids and flavonoids in vitro. Diluted bacterial cultures were inoculated into the medium treated with 6 monoterpenes or flavonoids. Compared with phloem microorganisms, the oviposition pit microorganisms significantly degraded α-pinene and 3-carene at both the 9-and 18-h time points (analysis of variance, ANOVA: P < 0.0027) ( Fig. 6A; Supplementary Table S5). However, for the degradation of Fig. 4 The β-diversity of the microbial community significantly differed between the oviposition pits and the uninfected phloem; oviposition pit bacteria were predicted to have stronger plant secondary compound (PSC) degradation ability. (A, B) Principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS) plots of 16S rRNA gene weighted Bray-Curtis distances on the genus level for bacteria from oviposition pits and uninfected phloem. (C, D) PCoA and NMDS plots of 16S rRNA gene weighted Bray-Curtis distances on the operational taxonomic unit (OTU) level for bacteria from oviposition pits and uninfected phloem. (E) Predicted differences in abundance between bacterial communities from oviposition pits and uninfected phloem in pathways related to the degradation of monoterpenes and flavonoids (Mann-Whitney U-test, *P < 0.05, **P < 0.01). the flavonoids, (+)-catechin and resveratrol few significant differences were observed between groups except the remaining resveratrol in uninfected phloem was significantly higher than that in oviposition pit groups (ANOVA: P < 0.0129) in both 9 and 18 h experiments (ANOVA: P > 0.05) (Fig. 6B,C; Table S5). By contrast, oviposition pit bacteria degraded significantly more taxifolin and naringenin content than the uninfected phloem bacteria (ANOVA: P < 0.0348) (Fig. 6B,C; Table S5). Our results showed that oviposition pit bacteria could harbor stronger degradation ability for various secondary metabolites than phloem microorganisms.

Beneficial effects of oviposition pit bacteria on larval development and survival
To assess the benefaction and protection of oviposition pit microorganisms on newly hatched larvae, we manipulated the microbial abundance and subsequently measured the survival rate and weight gain of larvae with different types of treatment (Fig. 7). No significant difference among survival curves were observed in treatment between groups (Mantel-Cox test, P = 0.52; Breslow test, P = 0.46) after 7 d (Fig. 7A). At the beginning of the experiment, the weight of the larvae in each group was evenly distributed. However, 3 d after the treatment, the average weight of larvae from a sterilized oviposition pit was significantly lower (ANOVA: P < 0.05) than in any of the other groups (Fig. 7B). No significant differences were observed among individuals in all 3 groups on day 7, indicating that larvae of the sterilized oviposition pit group obtained weight supplementation after feeding for some time.

Discussion
The ecological significance of the longicorn beetle laying eggs into oviposition pits is to reduce the resistance of host trees to the eggs and newly hatched larvae (Ji et al., 2002). The existence of oviposition pits enhances the ability of longicorn beetles to infest healthy trees. Culture-independent analyses have previously suggested that the microbial community of plant tissues infected by xylophagous beetles (e.g., gallery, frass, oviposition pit, etc.) significantly differed from that of healthy plants. The PSC degradation ability was highly correlated with the β-diversity of the microbial symbioses communities (Cheng et al., 2018;Mason et al., 2018). Similarly, our results combining culture-independent analyses, metatranscriptomes, and in vitro degradation have provided clear evidence that the oviposition pits of M. saltuarius hold a heterogeneous associated bacterial community that possess the ability to degrade PSCs, with a lower abundance of entomopathogenic bacteria.
Our findings showed that the oviposition pits had a specific bacterial community that considerably differed from the phloem bacteria in terms of β-diversity. However, there was less difference in terms of α-diversity and the total quantity of bacteria. We speculated that this difference was conducive to the survival and development of longicorn eggs and newly hatched larvae. A lower quantity of microorganisms could be conducive to the formation of plant callus, and the rapid formation of these tissues may kill the developing eggs. Conversely, the excessive proliferation of microorganisms in ovipo-sition pits may accelerate the decay of the surrounding tissues, thus resulting in a water imbalance of the microenvironment, which may increase the risk of pathogen infection of the eggs. Notably, although the abundance of Serratia in oviposition pits was significantly inhibited, it still served as the dominant bacterial genus. Further analysis showed that the distribution of Serratia was uneven, mostly contributed by OTU231 (but significantly lower in oviposition pits). This OTU was identified as S. marcescens, which has repeatedly been reported as an entomopathogenic bacteria in the preliminary molecular identification and is recognized as an opportunistic pathogenic bacterium for Monochamus alternatus (Zhang et al., 2021). Serralysin from S. marcescens was shown to promote hemolymph bleeding in the silkworm (Bombyx mori) (Ishii et al., 2014). Previous studies have noted that the abundant and diverse chitinase genes from S. marcescens strongly promote their infection process in insects (Lysenko, 1959(Lysenko, , 1976Okay et al., 2010). We used this bacterium with high insecticidal activity in the following larval infection tests. Our finding was consistent with that of Deng et al. (2008), who isolated a strain of S. marcescens from both the oviposition pits of the Asian longicorn beetle (A. glabripennis) and the healthy phloem. The strain was reported to show insecticidal activity from both oral intake and contact injection. On the other hand, Serratia has been identified from large microenvironments of the interaction between Monochamus and conifer species (Guo et al., 2020;Ge et al., 2021;Tian et al., 2022). Species of this genus have strong stability, with rapid adaptation to the environment (Grimont & Grimont, 1978;Anand et al., 2010). Some were reported as cellulose-and terpene-degrading bacteria, and thus symbioses beneficial for the colonization of wood tissues (Boone et al., 2013;Vicente et al., 2013). We hypothesized that the oviposition behavior of M. saltuarius may shape a selective environment for Serratia, which is conductive to the growth of strains that benefit beetles and reduce the abundance of potential entomopathogenic strains.
Metatranscriptomes of oviposition pits and secretions indicated that genes involved in monoterpene and flavonoid degradation were present and expressed jointly. Intriguingly, the expression of most genes in oviposition pits and secretion groups showed exclusivity and complementarity. Our results suggested that both oviposition pit bacteria and secretion bacteria metatranscriptomes contained the genes necessary to degrade or transform the PSCs that could harm the longicorn beetles. This was also confirmed by the degradation of PSCs in vitro. We specifically tested the PSC degradation ability of oviposition pit bacteria and uninfected phloem bacteria for monoterpenes and flavonoids that have been identified from host tree tissues in our previous investigations . Compared with the uninfected phloem, oviposition pit cultures maintained significantly lower levels of α-pinene and 3-carene, whereas the control group showed the lowest monoterpene level. This could be because of the rapid growth of microbes, which made the culture dense in a short time and inhibited the volatilization of the 2 monoterpenes to a great extent. In the degradation of flavonoids, the residual levels of the 4 substances used in the experiment, (+)-catechin, naringenin, resveratrol, and taxifolin, in oviposition pit cultures were lower than those in the uninfected phloem group and in the control group. However, significant differences compared with the other 2 groups were only observed for naringenin and taxifolin. With the incomplete pathways found in the metatranscriptomes of both oviposition pit bacteria and secretion bacteria (Fig. 5B), we believe that many of them may lack an entire pathway for the degradation of a specific compound, evident, for example, in the insignificant degradation of (+)-catechin and resveratrol. However, their joint activation as part of the microenvironment of oviposition pits may increase the degradation range and ability of PSCs.
To assess the impacts of oviposition pit bacteria on M. saltuarius performance, we measured the survival rate and weight of newly hatched larvae with and without the disinfection of oviposition pit bacteria. However, although larvae in the disinfection group showed the highest mortality and earlier death events, no significant difference was observed in the survival analysis. Compared with the other 2 groups, the larval weight in the disinfection group was significantly lower on day 3 but recovered to the same level on day 7. Various hypotheses might explain the above survival experiment findings. For example, removing oviposition pit microorganisms may reduce newly hatched larval fitness to some extent. However, we did not disinfect microorganisms on the surface or endophytes of eggs, which may collectively play a role in protecting the development of newly hatched larvae. Another explanation for the findings is that the sterilization effectively killed larvae-beneficial oviposition pit microbes and depressed potential pathogenic microbes to an extremely low level, potentially reducing the risk of pathogenic infection of the disinfected larvae in the early stages of the experiment. We suggest that oviposition pit bacteria were significantly beneficial to weight gain in the early stages of larval development, which may be attributed to the in vitro degradation of toxic PSCs by oviposition pit bacteria. These bacteria may also colonize the larval gut as the initial intestinal bacteria through feeding and improve the stress resistance of newly hatched larvae. For the weight recovery of the sterilized group on day 7, we supposed that the sterilization of oviposition pit bacteria served as a selection pressure that screened out individuals with poor physique among the group. The survivors actively recruited their symbiotic bacteria to help them resist PSCs, which helped them regain weight.
Although a comprehensive understanding of the ecological significance of oviposition pits to reproduction still needs extensive research, our study revealed an enhancement of host adaptation for the longicorn beetle (Fig. 8). Some Lamiinae longicorn beetles used a timeconsuming and complex process to make an oviposition pit for each egg and were sealed with their secretions. These secretions contain pheromones that regulate the spatial distribution of population by inhibiting the oviposition of other individuals (Anbutsu & Togashi, 2001, thus reducing intraspecific offspring competition. The moderate level of bacteria in oviposition pits maintains a suitable environment for the development of newly hatched larvae. The community structure in oviposition pits is manipulated by the females, inhibiting the abundance of entomopathogenic bacteria and shaping a stronger PSCs-degrading community, which is conducive to the development of larvae. The aforementioned uniparous and oviposition pit reproduction habits greatly consume the energy and time of female adults, which may result in their relatively low fecundity (Hwang et al., 2008;Han et al., 2016;Li et al., 2021). This indicates that to some extent these longicorn beetles may be K-strategists. Compared with the oviposition pit reproduction of Lamiinae, some longicorn beetles have amazing reproductive ability (e.g., Arhopalus spp.). Most of them lay clutches of multiple eggs directly in the gaps of bark. Offspring of these r-strategists must bore through the rough bark to enter the phloem and face a relatively high abundance of entomopathogens and a considerable number of PSCs, as well as encountering higher intraspecific competition pressure.

Conclusion
In conclusion, our findings highlighted the ecological significance of oviposition pit reproduction in aspects of microbial symbioses. Our research showed that M. saltuarius could form an oviposition pit bacterial community with low entomopathogenic bacteria abundance and strong PSC degradation abilities through oviposition behavior, to benefit their offspring. This provides a promising model system to investigate how such symbiotic systems play roles in insect-host adaptation. Considering that most species of the subfamily Lamiinae exhibit an oviposition pit reproduction habit, the ecological function we identified here must be only the tip of an iceberg. We expect that subsequent research will reveal the role of symbionts in oviposition pits of longicorn beetles on a broader and much more in-depth scale.

Supporting Information
Additional supporting information may be found online in the Supporting Information section at the end of the article. Fig. S1. Rarefaction curves of bacterial communities in oviposition pits and uninfected phloem samples. Fig. S2. Venn diagrams of OTUs shared between oviposition pits and uninfected phloem. Table S1. Standard curves of monoterpene and flavonoid components involved in this study. Table S2. Information on 16S rRNA gene sequences of Serratia spp. involved in phylogenetic analysis. Table S3. Difference in α-diversity of bacteria between oviposition pits and uninfected phloem samples. Table S4. Gene expression involved in monoterpene and flavonoid degradation pathways in oviposition pit cultures and secretion cultures. Table S5. Detailed information on ANOVA and multiple comparisons.