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. 2022 Dec 23;8(51):eadd5051. doi: 10.1126/sciadv.add5051

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

Fanghua Liu 1,2,†, Fangyuan Ye 2,3,†, Chihang Cheng 4,†, Zhiwei Kang 1, Hongru Kou 2,3, Jianghua Sun 1,2,3,*
PMCID: PMC9788770  PMID: 36563163

Abstract

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.

Symbiotic microbes help RTB degrade a deterrent host pine carbohydrate D-pinitol to aid its host adaptation.

INTRODUCTION

During the long coevolutionary interaction with host plants, herbivores have evolved various adaptive strategies to cope with inherent limitations in obtaining nutrition from plants and countering their defensive allelochemicals, including digestive enzymes, detoxification systems, and forming beneficial relationships with associated microbes (1–4). For example, cytochrome P450 genes are essential for the detoxification of xanthotoxin, a linear furanocoumarin with high toxicity in Rutaceae plants, in Papilio polyxenes and Helicoverpa armigera (5, 6). Research on Plutella xylostella has revealed a glucosinolate sulfatase involved in the detoxification of glucosinolates (7). Recently, growing evidence demonstrated that insect-associated microbes positively affect plant-insect interactions by detoxifying and metabolizing plant secondary compounds (8). In bees, gut bacteria can digest plant polysaccharide and metabolize toxic sugars (9, 10). Following this example, gut microbiota in Curculio chinensis, Atta cephalotes, Hylobius abietis, Psylliodes chrysocephala, and Hypothenemus hampei contributes to the degradation of plant secondary compounds (11–15). Elimination of gut microbiota from H. hampei impairs its reproductive fitness and caffeine detoxification (11). However, the underlying mechanism of how these associated microbes use and degrade these plant secondary compounds remains largely elusive.

The red turpentine beetle (RTB), Dendroctonus valens LeConte, is one of the most destructive invasive pests that have killed more than 10 million pine trees in China since its introduction from North America (16, 17). The invasive success and outbreaks of RTB in China are attributed to the mutualistic relationship with Leptographium procerum, the fungus it vectors (18–20). L. procerum is the most frequently isolated fungus from both the exoskeleton of adult RTB and its galleries in China (21). Novel genotypes of L. procerum in China are more pathogenic to Pinus tabuliformis, its primary host pine in China, and also induce a higher release of the RTB volatile attractant 3-carene (18, 20). Earlier lines of evidence have shown that associated bacteria aid the maintenance of this invasive mutualism through nutritional compensation and degradation of the pine defensive chemical naringenin (22). However, the pine phloem that RTB solely consumes and both L. procerum and associated bacteria colonize contains high amounts of d-pinitol (23). d-Pinitol has been found to have antagonistic effects on both insects and powdery mildew fungus (24–28). Pinitol from soybeans inhibited Heliothis zea growth by reducing food ingestion (26, 27). Notably, the pharmacological activity of d-pinitol in animals has been recognized in recent years (29, 30). Oral administration of d-pinitol can be easily absorbed and cleared, and it exerts an insulin-like effect in diabetic animal models (30, 31). This is currently being developed as a natural beneficial dietary supplement for diabetic treatment (31–33). Unexpectedly, given its medicinal value, the degradation mechanism of d-pinitol is still unknown. Our prior studies found that L. procerum can use d-pinitol as a carbon source for growth (34, 35), and RTB-associated bacteria (Serratia liquefaciens B310, Rahnella aquatilis B301, and Pseudomonas sp. 7 B321) produced the bacterial volatile ammonia that accelerates d-pinitol utilization of L. procerum (34, 35). Thus, questions arise as to what extent does d-pinitol influence the maintenance of this invasive mutualism and how exactly is it metabolized?

To answer these questions, the present study first assessed the effects of d-pinitol on the growth of RTB, L. procerum, and associated bacteria. Then, we investigated the degradation capacity of d-pinitol by RTB, L. procerum, and associated bacteria. We show that d-pinitol had a deterrent effect on RTB larvae and reduced the growth of RTB, while it favors both L. procerum and associated bacteria. d-Pinitol is one of the most common carbohydrates in pine trees, and the content of d-pinitol in P. tabuliformis is much higher compared to three species of pine trees (Pinus pinea L., Pinus taeda, and Pinus monticola Dougl.) in North America (36–39). Therefore, the metabolization of d-pinitol by associated microbes is important for RTB to propagate on its host pine and cause outbreaks in China. Degradation experiments indicate that RTB mainly relies on mutualistic fungi L. procerum and two genera of associated bacteria, Erwinia and Serratia, from galleries to degrade d-pinitol. Genomic and transcriptomic analyses combined with functional bioassays reveal that L. procerum and Erwinia sp.2 B304 have the strongest degradation capacity of d-pinitol, and they metabolize d-pinitol via different branches of the inositol pathway. These findings provide new insights into how the mutualistic fungus L. procerum and symbiotic bacteria may serve as an external degradation system to facilitate the RTB invasion and cause outbreaks by degrading the deterrent pine carbohydrate d-pinitol. The mechanism described here may be more widespread among many other destructive pests, especially bark beetle/fungal mutualisms.

RESULTS

d-Pinitol is a deterrent to RTB, which relies on its gut microbes to degrade d-pinitol

d-Pinitol is one of the dominant carbohydrates in P. tabuliformis phloem and shows antagonistic activity in several insects (26–28). Previously, we determined that d-pinitol, compared to d-glucose, was not the preferred carbon source for RTB larvae, L. procerum, and associated bacteria (23). Consistent with our previous result, we found that the RTB larval weight gain in phloem media with d-pinitol was significantly lower than those in desugared phloem media with d-glucose (Fig. 1A). RTB larvae were significantly repelled from diet plugs with elevated d-pinitol concentrations after 6 hours (Fig. 1B). Larval feeding area (Fig. 1C) and larval weight (Fig. 1D) decreased significantly on the media with d-glucose + d-pinitol compared to media with only d-glucose. These findings imply that d-pinitol has an antifeedant activity to RTB larvae as described in similar studies with other insects (26–28).

Fig. 1. d-Pinitol is a deterrent to RTB, which relies on its gut microbes to degrade d-pinitol.

Fig. 1.

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(A) Weight change of larval RTB reared with d-glucose or d-pinitol (n = 39, Student’s t test). (B) Choice of RTB larvae (n = 60) for diet with d-glucose or d-glucose + d-pinitol (chi-square test). (C and D) Mean feeding area (C) and body weight change (D) of RTB larvae reared with d-glucose or d-glucose + d-pinitol (Student’s t test). (E) Elimination of gut microbiota on the metabolic ability of RTB larvae to d-pinitol (Student’s t test). Control means medium without RTB and any microbes. (F) Degradation of d-pinitol and myo-inositol after hemocoel injection in RTB larvae (n = 5 individuals, Student’s t test). (G) Degradation of d-pinitol by gut microbiota (Student’s t test). Control means medium without any microbes. (H) Effects of d-pinitol supplementation on the growth of gut microbes (n = 30, Student’s t test). (I) Histogram shows the difference of gut microbial communities between d-pinitol– and d-glucose–reared RTB larvae at genera level (n = 5). Mean ± SE. Asterisk indicates significant difference (***P < 0.001). The number of biological replicates and P values of (A) to (E) and (G) are shown in the figures.

Having confirmed the deterrent effect of d-pinitol on RTB, we therefore investigated whether d-pinitol can be metabolized by RTB and whether the gut microbiome contributes to the degradation of d-pinitol. First, we treated wild-collected third instar RTB larvae with antibiotics (antibiotic treatment with streptomycin, ampicillin, tetracyline, and nystatin) for 6 days. Then, these treated (antibiotic) and nontreated (control) RTB larvae were fed on the diet with d-glucose + d-pinitol. In addition, we also used axenic RTB larvae that were generated via surface-sterilized eggs and conventional (unmanipulated) RTB larvae to determine the level of involvement of gut microbiota in d-pinitol degradation (fig. S1). The content of d-pinitol in the frass of axenic larvae, which were generated by egg surface sterilization, remained constant as in the control medium, while d-pinitol in frass of conventional larvae was used up (Fig. 1E). Obviously, unlike the conventional beetle, axenic beetle larvae lacked the ability to use d-pinitol. In addition, hemolymph injection with d-pinitol further confirmed that RTB larvae lack the ability to metabolize d-pinitol (Fig. 1F and fig. S2A). d-Pinitol is the 3-O-methyl ether of d-chiro-inositol, and d-chiro-inositol, scyllo-inositol, and myo-inositol are stereoisomers of inositol. Because myo-inositol is the most common inositol stereoisomer, we injected myo-inositol as control. Results showed that it can be degraded by RTB larvae (Fig. 1F). Furthermore, hemolymph injection showed that higher d-pinitol concentrations caused higher RTB larval mortality, whereas higher d-glucose injection led to higher survival rate (fig. S2B).

As gut microbiota is involved in the degradation of d-pinitol, we then conducted degradation bioassays of gut microbiota in vitro. Results indicated that the gut microbiota have the ability to use d-pinitol, which is beneficial to them (Fig. 1, G and H). Next, we assessed the in vivo impact of d-pinitol on the gut microbiota community of RTB larvae. d-Pinitol supplementation in the desugared phloem resulted in an increase in relative abundance of the genera Erwinia, Serratia, and Taibaiella in RTB larval guts (Fig. 1I and fig. S3).

Gallery microbiota can degrade d-pinitol and use it for growth

In our previous studies, we show that RTB and gallery microbiota form a mutualistic relationship and gallery microbiota also serve as an external detoxification system to help RTB eliminate harmful metabolites (18, 20, 22, 40). Thus, we investigated whether d-pinitol can be metabolized by gallery microbiota. d-Pinitol content in the healthy phloem and RTB gallery of P. tabuliformis showed that d-pinitol content in the RTB gallery was significantly lower than in healthy phloem (Fig. 2, A and B). Degradation bioassay results also suggested that gallery microbiota have the ability to use d-pinitol (Fig. 2C).

Fig. 2. Utilization of d-pinitol by RTB gallery microbiota.

Fig. 2.

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(A) Typical healthy pine phloem and RTB gallery of pine stakes. (B) d-Pinitol content in healthy pine phloem and RTB gallery (Student’s t test). DW means dry weight. (C) Degradation of d-pinitol by gallery microbiota (Student’s t test). (D) Effects of d-pinitol supplementation on the growth of gallery microbiota (n = 30, Student’s t test). Asterisks indicate a significant difference (**P < 0.01). (E) Representative growth of L. procerum on desugared phloem medium infused with d-glucose (top) and d-glucose + d-pinitol (bottom). (F and G) Effects of d-pinitol supplementation on the growth (F) and dry weight (G) of L. procerum (n = 8, Student’s t test). Error bars represent the SEM. The number of biological replicates and P values of (B), (C), (F), and (G) are shown in the figures.

Because d-pinitol supplementation favors gut microbiota growth, we characterized the effect of d-pinitol on the biomass of RTB gallery microbiota and L. procerum. No significant differences in optical density (OD) values between control (only d-glucose) and treatment (d-glucose and d-pinitol) were detected for gallery microbiota before 48 hours (Fig. 2D). Compared to the control, gallery microbiota grew better on minimal salt medium after d-pinitol supplementation after 48 hours (Fig. 2D), which suggested that d-pinitol is also beneficial to gallery microbiota. Spore and mycelium densities of L. procerum revealed that d-glucose + d-pinitol significantly increased the growth of L. procerum compared to d-glucose alone (Fig. 2E). The fungal growth rate and biomass yield with d-glucose + d-pinitol were significantly greater than those of control (Fig. 2, F and G). Together, these results strongly supported the notion that associated microbes could play key roles in using d-pinitol within the context of the overall beetle-microbes invasive complex and enable RTB to adapt to the host pine tree. Furthermore, our previous study has shown that nutritional resource competition is ultimately inevitable (34). Utilization of d-pinitol by L. procerum and associated bacteria may alleviate the nutrient competition between RTB and associated microbes.

Mutualistic fungus L. procerum and bacteria from the genera Erwinia and Serratia are primary degraders of d-pinitol for RTB

To determine which members in the RTB gut and gallery are responsible for the metabolism of d-pinitol, we screened mutualistic L. procerum and 44 bacterial species that were isolated from RTB in our previous works to assess their ability to metabolize d-pinitol (table S1) (23, 41). The fungus L. procerum showed strong degradation ability of d-pinitol (Fig. 3A). Consistent with prior 16S ribosomal RNA (rRNA) gene-sequencing results, only eight species of the genera Serratia and Erwinia could degrade d-pinitol, and d-pinitol supplementation favors the growth of all of these bacteria from both genera Serratia and Erwinia (Fig. 3B and fig. S4). Both genera Serratia and Erwinia are common RTB gallery and gut isolates (23, 41, 42). In addition, exposure of associated bacteria released volatile ammonia, which, in turn, enhanced the degradation efficiency of L. procerum (Fig. 3A). This finding indicates an interaction of associated microbes in the utilization of d-pinitol, and this interaction results in higher utilization efficiency benefiting the whole invasive complex.

Fig. 3. Mutualistic fungus L. procerum and members from the genera Erwinia and Serratia were responsible for d-pinitol metabolism.

Fig. 3.

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(A and B) Degradation of d-pinitol by L. procerum (A) (Student’s t test) and 44 different bacterial strains (B) (Student’s t test). Differences of the degradation ability of associated bacteria were compared between control and each bacterial strain. Error bars represent the SEM. Asterisks indicate significant difference (***P < 0.001 and ns, not significant). The number of biological replicates and P value of (A) are shown in the figure.

The metabolic pathway of d-pinitol by mutualistic fungus L. procerum

d-Pinitol, the 3-O-methyl ether of d-chiro-inositol, is a natural compound related to the inositol sugar group. Thus, we first focused on the genes involved with the inositol pathway (43–45). The inositol pathway has two main catabolism branches: myo-inositol oxygenase (MIOX) and iol operon, iolABCDEFGHIJ (46–48). The L. procerum genome shows that the inositol transporter (iolT), which regulates intracellular distribution and uptake of inositol, MIOX, which is the key enzyme degrading myo-inositol to d-glucuronate, and myo-inositol dehydrogenase (iolG), which is responsible for the conversion of myo-inositol to scyllo-inosose, were present (Fig. 4A) (47, 48). However, the main genes of the operon iolABCDEFHIJ were absent (Fig. 4A) (48). Supplementing d-pinitol to desugared phloem medium significantly induced the expression of iolT1, MIOX, and iolG (Fig. 4B and fig. S5). Our previous work showed that ammonia released by RTB-associated bacteria accelerated the consumption of d-glucose in L. procerum (34). In agreement with that, here, we found that exposure of ammonia (ammonium) also enhanced the utilization of d-pinitol (Fig. 3A). Thus, we analyzed the expression of iolT1, MIOX, and iolG following exposure to ammonia. All three of these genes were up-regulated by exposure to ammonia (Fig. 4C and fig. S6). On the basis of these findings, we subsequently generated ΔioT1 and ΔMIOX mutants. The ΔioT1 mutant lost the ability to metabolize d-pinitol (Fig. 4, D and F). However, deletion of MIOX showed no influence on d-pinitol metabolism but instead it led to an accumulation of d-chiro-inositol, scyllo-inositol, and myo-inositol (Fig. 4, E and F). For these three stereoisomers of inositol, wild-type (WT) L. procerum can metabolize d-chiro-inositol and myo-inositol, while ΔMIOX mutant could not (fig. S7). Scyllo-inositol could not be used by L. procerum (fig. S7). When RTB was provided with the intermediate metabolite myo-inositol, RTB larval weight gain was significantly higher than that with d-pinitol in desugared phloem media (fig. S8). RNA sequencing (RNA-seq) showed that deletion of MIOX results in higher expression of iolG2 and scyllo-inositol 2-dehydrogenase (iolW) and lower expression of iolT2 (Fig. 4G). Our findings of the essential role of MIOX in the metabolism of d-chiro-inositol and myo-inositol corroborate an earlier study showing that d-chiro-inositol and myo-inositol can be oxidized by recombinant MIOX (47).

Fig. 4. Genomic, transcriptomic, and functional investigation of genes involved in metabolization of d-pinitol in L. procerum.

Fig. 4.

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(A) Presence and absence of genes in the inositol pathway in the L. procerum genome. (B) Gene expression profiles of genes in the inositol pathway in response to d-pinitol supplementation based on transcriptome [n = 3, asterisks (***) indicate an absolute value of log2ratio ≥ 1 and FDR < 0.001]. (C) Impacts of ammonium exposure on the gene expression in the inositol pathway of L. procerum [asterisk (*) indicates an absolute value of log2ratio ≥ 1 and FDR < 0.05, n = 3]. (D) Efficiency of d-pinitol degradation by different L. procerum mutants on d-pinitol. Differences were compared between control with each mutant and WT L. procerum (Student’s t test). Error bars represent the SEM. Asterisks indicate significant difference (***P < 0.001). (E) Carbohydrates left in ΔMIOX of L. procerum colonized minimal medium. Error bars represent the SEM. (F) GC-MS traces of control, WT, and different mutants of L. procerum on minimal medium with d-pinitol. (G) Effects of deletion of MIOX on the gene expression of the inositol pathway in L. procerum by transcriptomic analyses [asterisk (*) indicates an absolute value of log2ratio ≥ 1 and FDR < 0.05, n = 3].

d-Pinitol is the 3-O-methyl ether of d-chiro-inositol, and d-chiro-inositol, scyllo-inositol, and myo-inositol are stereoisomers of inositol (Fig. 4E). The accumulation of d-chiro-inositol, scyllo-inositol, and myo-inositol in ΔMIOX mutant suggested that demethylation should be required for d-pinitol metabolism. Therefore, we analyzed the expression of demethylase genes in response to d-pinitol supplementation and exposure of ammonia. Three of four pisatin demethylases (PDAs), which are responsible for detoxifying pisatin by demethylation in Nectria haematococca (49), were significantly elicited by d-pinitol and ammonia (Fig. 4, B and C). However, ΔPDA6, ΔPDA9-1, and ΔPDA9-3 single mutants showed no influence on the metabolism of d-pinitol (Fig. 4D). On the basis of the quantitative polymerase chain reaction (qPCR results), we found that deletion of either PDA resulted in higher expression of other PDAs that compensated the deletion (fig. S9). Unfortunately, resistance screening results showed that L. procerum is resistant to chlorazosulfuron, benomyl, and glufosinate. Thus, we could not generate PDA triple mutants using resistance screening, but we could reasonably speculate that PDAs should be essential in the metabolism of d-pinitol.

The metabolic pathway of d-pinitol by associated bacteria

To better understand how these bacteria from the genera Erwinia and Serratia metabolize d-pinitol, we conducted genomic and transcriptomic sequencing of Erwinia sp.2 B304 and S. liquefaciens B314 that efficiently degrade d-pinitol (fig. S4). Unlike L. procerum, MIOX was absent from the genome of E. sp.2 B304 and S. liquefaciens B314, while mainly operons iolABCDEFGHIJ and iolT genes were found (Fig. 5A and fig. S10). d-Pinitol supplementation significantly increased the expressions of iolT1, iolG, inosose isomerase (iolI), inosose dehydratase (iolE), 3d-(3,5/4)-trihydroxycyclohexane-1,2-dione acylhydrolase (iolD), 5-deoxy-glucuronate isomerase (iolB), and 5-dehydro-2-deoxygluconokinase (iolC) in both E. sp.2 B304 and S. liquefaciens B314 (Fig. 5, B and C, and fig. S10). ΔioT1, ΔiolG, and ΔiolE mutants of E. sp.2 B304 lost the ability to metabolize d-pinitol (Fig. 5, D and E). Similarly, iolG has been found to be involved in d-pinitol depletion in Bacillus subtilis (46). Consistent with L. procerum, E. sp.2 B304 also degraded d-chiro-inositol and myo-inositol but lacked the ability to degrade scyllo-inositol (fig. S11). Deletion of ioT1, iolG, and iolE impaired the degradation of d-chiro-inositol and myo-inositol in E. sp.2 B304 (fig. S11).

Fig. 5. Genomic, transcriptomic, and functional investigation of genes involved in metabolizing of d-pinitol in E. sp.2 304.

Fig. 5.

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(A) Presence and absence of genes in the inositol pathway in the E. sp.2 304 genome. (B) Gene expression profiles of genes in the inositol pathway in response to d-pinitol supplementation based on transcriptome [n = 3, asterisks indicate an absolute value of log2ratio ≥ 1with FDR < 0.001 (**) and 0.001 (***)]. (C) Verification of transcriptome results by qPCR (Student’s t test). (D) Metabolic ability of different E. sp.2 304 mutants on d-pinitol. Differences were compared between control, each mutant, and WT E. sp.2 304 (Student’s t test). (E) GC-MS traces of control, WT, and different mutants of E. sp.2 304 on minimal medium with d-pinitol. Mean ± SE. Asterisks in (C) and (D) indicate significant difference (**P < 0.01, ***P < 0.001, and ns, not significant).

Our prior results found that demethylation of d-pinitol are required in the degradation of d-pinitol by L. procerum. Thus, we also tested whether demethylation is required in the degradation process of E. sp.2 B304. We only identified two demethylase genes (vanB1 and vanB2) in the genome of E. sp.2 B304, and both of them were down-regulated by the supplementation of d-pinitol (Fig. 5, B and C). Single mutants ΔvanB1 and ΔvanB2, and the double mutant ΔvanB1/ΔvanB2 can degrade d-pinitol normally, which strongly indicates that demethylation is not required in the d-pinitol degradation of E. sp.2 B304 (Fig. 5, D and E).

Given these findings, we subsequently used the ΔioT1 mutants of L. procerum and E. sp.2 B304 to investigate the impact of the abolishment of d-pinitol metabolism on the performance of RTB larvae. We found that loss of the d-pinitol degradation ability led to weight loss of RTB larvae but no difference in survival (Fig. 6). When RTB larvae were reared on the desugared medium without d-pinitol, there were no differences of the weight gain and survival between the reintroduction of WT and ΔioT1 mutants of L. procerum and E. sp.2 B304 (fig. S12). As our prior result showed that there was no weight gain of conventional larvae reared on d-pinitol–containing diet (Fig. 1D and fig. S8), these results suggested that associated microbes benefit RTB larvae via external d-pinitol degradation more than those in the gut.

Fig. 6. Effects of d-pinitol on the performance of antibiotic-treated RTB larvae with the reintroduction of WT or ΔiolT1 mutants of L. procerum and E. sp.2 304.

Fig. 6.

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(A) d-Pinitol concentration in the frass of antibiotic-treated RTB larvae with the reintroduction of WT or ΔiolT1 mutants. Control means desugared medium without RTB and any microbes (n = 10, Student’s t test). (B and C) Effects of d-pinitol on the larval weight changes (B) and survival (C) of antibiotic-treated RTB larvae with the reintroduction of WT or ΔiolT1 mutants (Student’s t test). Mean ± SE.

DISCUSSION

The present study provides new important advances in the understanding of the RTB invasive complex and functional roles of key associated microbes in insect-plant interactions. First, we found that pine phloem enriched with d-pinitol depressed the larval growth of RTB, while it benefits the growth of RTB mutualistic fungal partner L. procerum and associated bacteria. Degradation experiments showed that the RTB larva relies on L. procerum and associated bacteria to degrade d-pinitol, which is partially consistent with our previous findings. Last, we conducted genomic and transcriptomic studies combined with functional verification to disentangle the metabolic mechanism of D-pinitol by RTB mutualistic partner L. procerum and symbiotic bacteria. Results showed that RTB mutualistic L. procerum and symbiotic bacteria metabolized d-pinitol via two different branches of the inositol pathway (Fig. 7 and fig. S13). Reinoculation experiments together with the feeding behavior and larval weight gain in desugared phloem medium with d-pinitol and d-glucose revealed that microbes benefit RTB larvae mainly by degrading pinitol in the medium (pine phloem or in the artificial medium) rather than in the gut of the larvae. All of these results demonstrate that larvae are deterred by d-pinitol but not intoxicated. The association with microbes may benefit the beetle via the external degradation of d-pinitol rather than in the gut of the beetle larvae to produce d-pinitol–deficient diet, leading to higher feeding activity of the RTB larvae.

Fig. 7. Model of the d-pinitol metabolic mechanism in RTB mutualistic L. procerum and associated bacteria, Erwinia and Serratia.

Fig. 7.

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RTB mutualistic L. procerum relies on the demethylation of PDAs to convert d-pinitol into three stereoisomers of inositol, and then, these three stereoisomers of inositol were oxidized by MIOX. Last, its metabolites enter the pentose pathway. As to associated bacteria, d-pinitol can be directly used by the iol divergon, comprising the operons iolGEDBC, and its metabolites enter the glycolysis/gluconeogenesis pathway and trichloroacetic acid (TCA) cycle. The transportation of d-pinitol by iolT is required in both L. procerum and associated bacteria, Erwinia and Serratia. Associated bacteria, including Serratia, released volatile ammonia and significantly enhanced the utilization of d-pinitol in L. procerum.

In its native North America, RTB is a secondary pest and mainly attacks weakened pine trees or fresh stumps, rarely killing healthy pines (16). However, when it was inadvertently introduced into China, RTB devastated healthy pine forests (16, 17). Normally, healthy pines have stronger plant defense response and contain higher secondary metabolites, which are usually involved in resistance to insects (16, 23, 50). RTB solely consumes pine phloem, which contains a high amount of d-pinitol. Previous studies reported that d-pinitol exhibits antagonistic effects on several insects (26–28). Notably, d-pinitol content in P. tabuliformis in China is much higher than that in three species of pine trees in North America, and its content in fresh phloem of healthy P. tabuliformis from our previous studies were also significantly higher than that in the phloem of felled P. tabuliformis trees used in this study (23, 36–38, 40). Furthermore, d-pinitol negatively affected the growth of RTB larvae. Thus, how to deal with the higher content of d-pinitol in healthy pines becomes a problem that must be overcome for invasive RTB to successfully colonize hosts. Elimination of gut microbiota and injection of RTB larvae with d-pinitol showed that RTB lacks the ability to degrade d-pinitol. However, RTB mutualistic fungus L. procerum and two genera of symbiotic bacteria, Erwinia and Serratia, were empirically shown to have high efficiency in the utilization of this deterrent pine carbohydrate. In H. hampei, gut microbiota Pseudomonas fulva contributes to the detoxification of caffeine, and elimination of gut microbiota impairs its reproductive fitness (11). Moreover, the bee gut symbiont Gilliamella apicola can degrade toxic carbohydrates to improve the dietary tolerance of its host bees (9). In contrast, our results indicated that the external degradation of the gallery symbiotic microbes plays a bigger role than gut microbes in d-pinitol degradation. In nature, female RTBs initiate attack and construct galleries in pine phloem for mating and egg hatching (16). Thus, when female RTBs attack and feed on the healthy pine phloem, gut microbiota can help its host to degrade d-pinitol, and gut microbiota also serves as the main source of gallery microbiota. During the gallery construction, L. procerum and bacteria (especially from the genera Erwinia and Serratia) from RTB’s exoskeleton and gut as well as in frass were inoculated into the RTB gallery. Thus, before the offspring hatch from the egg, L. procerum and these associated bacteria have grown in the RTB gallery for a while. Thereby, the offspring can feed on d-pinitol–deficient pine phloem. Thus, in this way, the process is beneficial to the RTB’s offspring, thereby contributing to the outbreaks of RTB. Together, these results indicate that RTB mutualistic L. procerum and symbiotic bacteria, Erwinia and Serratia, from galleries may serve as an external degradation system to increase the fitness of RTB by degrading the deterrent pine carbohydrate d-pinitol, thus contributing to its invasiveness and facilitating outbreaks in China. Notably, in its native range (United States), RTB was occasionally associated with ophiostomatoid fungi with high variability of prevalence, and L. procerum only presented in the eastern U.S. RTB population (42, 51). While in invasive range (China), L. procerum is closely related with RTB at high prevalence (21). The bacterial community associated with RTB in its native range was also different from invasive populations in China (23, 42). However, bacteria from the genera Erwinia and Serratia can be isolated in both native and invasive ranges (23, 42). Furthermore, Serratia has been found as a dominant member of bacteria community in many Dendroctonus bark beetles, and Erwinia is a relaxed core bacterium in Dendroctonus bark beetles (42, 52). Thus, we speculated that d-pinitol degradation of Serratia and Erwinia may contribute to the adaptation of Dendroctonus bark beetles to living on pine. Future work can aim at a more in-depth comparison of the microbiome differences between invasive and noninvasive RTB populations.

Few studies explore the metabolic mechanism of how associated microbes degrade plant secondary compounds. The science of symbiont-mediated secondary compound detoxification in herbivores is largely at a nascent stage, and these studies focus mainly on genome sequencing to figure out the presence and absence of genes, transcriptomic sequencing to explore the potential key genes by gene expression, and metabolomic analyses to identify the potential products (9, 10, 12, 22). Reverse genetics research approaches on the verification of potential molecular mechanism are lacking. In this study, to gain comprehensive insights into how RTB mutualistic L. procerum and symbiotic bacteria metabolize d-pinitol, genomic and transcriptomic analyses combined with functional investigations were performed. Our genome results revealed that genes iolI, iolE, iolD, iolC, and iolJ are absent from L. procerum genomes, while iolT, MIOX, iolG, and iolU are present. Functional investigation of iolT1 and MIOX showed that iolT1 serves as the transporter of d-pinitol, and MIOX plays an essential role in metabolizing d-pinitol (Fig. 3). Unexpectedly, unlike the fungus L. procerum, the bacteria Erwinia and Serratia both lost MIOX but retained iolI, iolE, iolD, iolC, and iolJ, which were lost in L. procerum. Mutations of iolT1, iolG, and iolE resulted in a loss of the ability to metabolize d-pinitol (Fig. 5). Our findings are consistent with earlier studies, showing that bee gut bacteria show extensive species-level diversity in the ability to metabolize toxic carbohydrates and digest plant polysaccharides (9, 10). Likewise, metagenomes of leaf-cutter ant fungus garden bacteria isolates indicated that different bacterial isolates carry different genes involved in plant secondary compound degradation (12). Moreover, our results also found that demethylation is required for the metabolization of d-pinitol in L. procerum but not in associated bacteria Erwinia and Serratia (Fig. 6). It important to note that RTB has an intact inositol pathway and myo-inositol can be degraded, whereas it lacks the ability to degrade d-pinitol, which suggests that gene presence does not mean biological ability (fig. S14). Together, all of these results demonstrated that RTB relies on mutualistic L. procerum and symbiotic bacteria, Erwinia and Serratia, metabolizing d-pinitol via different branches of the inositol pathway (Fig. 6).

Multiple interactions of insect and associated microbes have recently been implicated in host plant adaptation and insect invasion (10, 12, 16, 19, 20). For example, bacteria from the fungus garden of leaf-cutter ants can degrade plant secondary compounds, potentially enabling leaf-cutter ants to feed on a wide variety of plants (12), whereas L. procerum has been demonstrated to contribute to RTB invasion success and outbreaks in China by its pathogenicity and induction of 3-carene in P. tabuliformis (18, 20). Recent studies have shown that associated bacteria can help RTB degrade naringenin and α-pinene, both of which are pine defensive compounds (22, 40). Furthermore, bacteria of the genera Pseudomonas, Serratia, Rahnella, and Lactococcus have the ability to convert cis-verbenol to verbenone (41), and ammonia released from RTB-associated bacteria activates the conversion of starch into glucose in L. procerum supplementing RTB larval growth (34). Subsequently, the d-glucose consumption of L. procerum was accelerated by ammonia (34). In this study, we also found that ammonia exposure enhanced the degradation of d-pinitol in L. procerum. Transcriptomic and functional results indicated that ammonia exposure significantly induced the expression of iolT1 and MIOX, resulting in the acceleration of d-pinitol consumption and metabolism in L. procerum. Our finding of the d-pinitol degradation ability of RTB mutualistic partners L. procerum and their symbiotic bacteria, Erwinia and Serratia, provides a novel insight into the adaptive strategy of how pine pests can live on host pines in a nutrient-limited environment. These findings collectively suggest that associated microbes may contribute to RTB invasive success and cause outbreaks in China to a certain extent. Supplementation of d-pinitol promotes the growth of RTB’s mutualistic partner L. procerum and symbiotic bacteria. Because of the poor nutritional condition in pine phloem, utilization of d-pinitol by L. procerum and symbiotic bacteria also alleviates the nutrient competition between RTB and associated microbes by leaving higher amounts of other nutrients, such as d-glucose, for RTB (34). In addition, various species of the Erwinia genus have been known as phytopathogens causing soft rots, bacterial wilt, and fire blight disease (53–55). Thus, the pathogenicity of RTB-associated Erwinia warrants further investigation.

In recent years, d-pinitol has been used as a bioactive compound for many diseases, especially diabetes (29, 30, 56). Oral ingestion of d-pinitol significantly improves the insulin-glucose metabolic homeostasis of diabetic patients, streptozotocin-diabetic rats, and glucose-loaded mice (32, 33, 57, 58). Further studies showed that d-pinitol acts through the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway (57, 59). However, the mechanisms of action of d-pinitol on diabetes remain inconclusive. In this study, we found that in L. procerum, d-pinitol was metabolized by MIOX, which is a kidney-specific enzyme degrading myo-inositol to d-glucuronate in rats, mice, and humans (47, 60). Furthermore, MIOX was also found to be involved in diabetes (39, 60–62). Thus, we speculate that MIOX might be the modulator of d-pinitol in diabetic animals. As myo-inositol and glucose pathway are interdependent, myo-inositol depletion has been observed in diabetic animal models (57, 62, 63). Supplementation of d-pinitol might alleviate the depletion of myo-inositol in diabetes and thereby improves the clinical treatment of diabetes. In addition, in Wistar rats, oral ingested d-pinitol is rapidly absorbed and cleared in plasma and liver (31). Our findings of the roles of iolT and MIOX in the transportation and metabolism on d-pinitol also provided an example for potential d-pinitol transport and metabolic mechanism investigations in mammals. Furthermore, gut microbiota might also participate in the pharmacological activity of d-pinitol.

In summary, our study reveals how RTB, a destructive pine pest, overcomes the deterrent pine carbohydrate d-pinitol and is a successful invader. We found that RTB’s mutualistic partner L. procerum and symbiotic bacteria, Erwinia and Serratia, are responsible for d-pinitol degradation, while RTB cannot metabolize it directly. Genomic and transcriptomic analyses combined with functional studies illustrate the metabolic mechanism governing d-pinitol utilization in L. procerum and symbiotic bacteria, Erwinia and Serratia. This finding uncovers how symbiotic microbes enable RTB to live exclusively on pine phloem. On the basis of these results and results from previous studies, RTB-associated microbes seem to perform a range of different tasks to accelerate its adaptation in China, thereby contributing to its invasive success and facilitate outbreaks in China (18, 20, 22, 34, 41). Generally, there was an abundance of microbes in the galleries of inner-bark borers (16, 19, 42). Thus, we hypothesized that these associated microbes may serve as an external detoxification system to benefit these insects, especially bark beetles, which forms tight relationship with associated microbes. Our findings further enrich and consummate the RTB-microbes invasion complex theory and provide new insights into the understanding of plant-insect interactions. Because of the hypoglycemic activity of d-pinitol on diabetes, our results also provide valuable information on the effects and mechanism of actions of d-pinitol, which has important medicinal value for humans.

MATERIALS AND METHODS

RTB and microbial strains

Adults of RTB were captured in newly attacked pine stumps in the Tunlanchuan Forestry Station (N 37°48′, E 111°44′; average elevation, 1400 m, Shanxi, China). Axenic RTB larvae were generated via oral antibiotic treatment with egg surface sterilization as previously described with little modification (64). First, RTB eggs were washed by sterile water and then placed into sterile petri dishes containing washing solution (10% bleach and 10% ethanol) for 1 min. Last, these eggs were rinsed 5× in sterile water. The efficacy of elimination of symbiotic microbes was confirmed by plating beetle larvae and phloem powder in its habitat onto Luria-Bertani (LB) agar plates and performing PCR analysis using bacterial 16S rRNA gene universal primers (table S2). Surface sterilization of larvae was conducted by washing RTB larvae 3× with sterile water for 5 s and then immersing them into the aforementioned washing solution for 5 s, followed by immersion in sterile water for 5 s. The fungal strain L. procerum CMW25614 used in this study, commonly associated with RTB and reported as a mutualistic fungal associate for RTB larvae, was previously isolated from exoskeleton of RTB adults and its galleries in infested P. tabuliformis forests in China (21). The 44 bacterial strains used in this study were previously isolated from the RTB gut and its galleries in infested P. tabuliformis forests in China (table S1) (23, 41).

Effect of d-pinitol on the performance of RTB

To determine the effect of dominant carbohydrate d-pinitol on the growth of beetle larvae and symbiotic microbes, two treatments were set. Briefly, medium supplemented with 0.3% d-pinitol was set as P and medium supplemented only with 0.3% d-glucose or myo-inositol was set as G or MI, respectively. Second, medium supplemented with 0.3% d-glucose and 0.3% d-pinitol was set as G&P and medium supplemented only with 0.3% d-glucose was set as G. We use the desugared phloem medium to study the effect of d-pinitol on larval growth (34). To test the diet choice of RTB larvae between G&P and G, one second to third surface-sterilized RTB larva was placed equidistant from the plugs (diameter: 1.5 cm) of G&P and G in a petri dish (diameter: 3.5 cm) (65). Petri dishes (n = 60) were kept in 25°C in darkness. Diet choice was recorded after 6 hours. To test feeding areas (n ≥ 20 per treatment), second to third instar RTB larvae were randomly selected and pre-starved for 30 min and then allowed to feed on phloem diet of G&P and G, respectively, for 6 hours. The feeding areas were photographed and measured using the software ImageJ (65). To test body weight change of larvae, the second to third instar RTB larvae were surface-sterilized and transferred into phloem medium for 7 days. Active larvae (n = 39) were selected and pre-starved for 30 min and then randomly separated into two groups, and each individual larva was weighed and transferred to each petri dish containing medium. Petri dishes were incubated at 25°C for 6 days, after which RTB larvae within each dish were weighed.

Effect of d-pinitol on the growth of symbiotic microbes

For gallery microbiota, we collected RTB gallery tissues from naturally infested P. tabuliformis trees at Tunlanchuan Forest Station, which were washed using 10% PBS buffer through the method previously described (22). For the gut microbiota, individual guts of third instar RTB larvae were obtained by dissection of surface-sterilized beetle larvae and then crushed in LB broth. M9 minimal salt medium (pH 7.4; 6 g Na2HPO4, 3 g KH2PO4, 0.5 g NaCl, and 1 g NH4Cl per liter) was used for growth of gallery and gut microbiota. Tissue samples from 60 galleries were separately washed using 10% PBS buffer to obtain gallery microbiota, which were transferred into 4 ml of LB broth and incubated about 12 hours when cultures were adjusted to an OD600 of 0.5. Then, a dilution of 1:100 of each gallery microbiota was made in 4 ml of M9 minimal salt medium. Absorbance at 600 nm was measured at 0, 4, 8, 12, 16, 24, 36, 48, 72, 96, and 120 hours after the gallery microbiota were inoculated and regarded as the sole parameter to compare bacterial growth rate. M9 minimal salt medium containing d-pinitol (3 g/liter) plus d-glucose (3 g/liter) was set as treatment, while only d-glucose (3 g/liter) was set as control. The same approach was used to examine the effect of d-pinitol on gut microbiota by setting individual gut as a sample (n = 60 guts were used in the experiment). Effect of d-pinitol supplementation on the growth of bacteria from the genera Erwinia and Serratia were determined as described above. To assess the effect of d-pinitol on the growth of L. procerum, standard minimal medium (1 g of KH2PO4, 1 g of NH4Cl, 0.5 g of MgSO4·7H2O, 0.02 g of FeSO4·7H2O, 0.02 g of ZnCl2, 0.01 g of MnCl2, 0.01 g of pyridoxine, and 25 g of agar were dissolved into 1 liter of distilled water) with d-pinitol (3 g/liter) plus d-glucose (3 g/liter) or only d-glucose (3 g/liter) was used. Growth rate, dry weight, and density of L. procerum were measured as previously described.

Utilization of d-pinitol by RTB and symbiotic microbes

The preceding results showed that d-pinitol, the dominant carbohydrate in P. tabuliformis phloem, was a deterrent to RTB larvae and, at the same time, was beneficial to symbiotic microbial growth. Therefore, we aimed to determine the utilization of d-pinitol by RTB larvae, mutualistic fungal strain L. procerum, and symbiotic bacterial strains.

To clarify the utilization of d-pinitol by the beetles, 0.3% (w/v) of d-pinitol was added into desugared phloem medium. To eliminate the effects of microbes, we used the axenic larvae, which were hatched from surface-sterilized eggs, to further clarify the utilization of d-pinitol by the beetles. At day 8 after emergence, the larvae were maintained on desugared phloem medium for 6 days. The frass of beetles was sampled on day 6 after the introduction of beetles, and carbohydrate composition was measured as before (35). For each treatment, nine biological replicates were used, and the frass were also analyzed using the same methods. Particularly, the axenic larvae were set as treatment, and the conventional larvae were set as control. To further confirm the ability of beetle to use d-pinitol, d-pinitol solution and myo-inositol were injected into surface-sterilized RTB larvae. Briefly, d-pinitol/myo-inositol was dissolved in water with a final concentration of 0.1, 0.5, and 1 M, and 1 μl of d-pinitol was injected into surface-sterilized larvae. After injection, surface-sterilized larvae were kept at 25°C for 0, 3, 6, and 12 hours, respectively, snap-frozen in liquid nitrogen, and kept at −80°C until carbohydrate analysis. The method of carbohydrate extraction and analysis of beetles was described as below. In addition, we also injected different concentrations (0.1, 0.5, and 1 M) of d-pinitol and d-glucose into surface-sterilized beetle larvae to analyze the effect of d-pinitol on the survival rate of RTB larvae. In addition, the larvae were collected for RNA-seq and qRT-PCR analysis at 12 hours after injection. Surface-sterilized larvae that were not injected with d-pinitol were parallel-cultured as controls. Three biological repeats were established for each treatment.

To investigate the utilization of d-pinitol by the symbiotic fungus L. procerum, 0.1% (w/v) of d-pinitol was added into standard minimal medium. A mycelial plug (3 mm in diameter) of L. procerum growing on malt extract agar (MEA) was transferred to the center of each plate (d = 90 mm). The medium without fungus was set as control. Our previous work showed that ammonia released by RTB-associated bacteria accelerated the consumption of d-glucose in L. procerum (34). We therefore analyzed the effect of ammonia exposure on the degradation of d-pinitol by adding ammonium or H2O (Control) into standard minimal medium with d-pinitol. All of the cultures were grown under 25°C and 70% relative humidity in darkness for 13 days until the mycelia covered all of the medium. Carbohydrate composition was analyzed as previously described (23). We collected one sample per plate, and eight biological replicates were used for each treatment. In addition, the mycelial mat was scraped for RNA-seq and qRT-PCR analysis at 4 days after the fungus was inoculated on the standard minimal medium. Particularly, the standard minimal medium with d-pinitol was set as treatment and d-glucose was set as control. Three biological repeats were established for each treatment.

To investigate the utilization of d-pinitol by the associated bacteria, 0.1% (w/v) of d-pinitol was added into M9 minimal salt medium. First, gallery microbiota or gut microbiota was added into the above M9 minimal salt medium, and the medium carbohydrate composition was measured after 12 hours with shaking at 180 rpm. In addition, to further characterize the gut microbial communities associated with beetle larvae feeding on d-pinitol, we constructed and sequenced 16S rDNA libraries of RTB larvae gut microbiotas (described below). Furthermore, to explore which associated bacterial strains were involved in degrading d-pinitol, 44 bacterial strains (table S1) related to RTB (23, 41) were individually made in the above M9 minimal salt medium to incubate for 12 hours and then we analyzed the content of d-pinitol left in the medium.

On the basis of the above results, two bacterial strains, E. sp.2 B304 and S. liquefaciens B314, with high d-pinitol degradation efficiency were used to explore the mechanism of d-pinitol utilization by bacteria. To measure the two bacterial strains’ d-pinitol degradation efficiency, 0.3% (w/v) of d-pinitol was added into 20 ml of LB medium. Bacteria were added into the above LB medium, and the medium carbohydrate composition was measured at 0, 6, 9, 12, 15, and 18 hours with shaking at 180 rpm, which was replicated six times per time point. In addition, the bacteria were collected for RNA-seq and qRT-PCR analysis at 12 hours after inoculation. In parallel, bacteria were cultured in LB medium without d-pinitol as controls. Three biological replicates were established for each treatment.

Quantification of d-pinitol and inositols by GC-MS/FID

For the quantification of d-pinitol and inositols, these target metabolites were extracted from solid medium (200 mg), liquid medium (200 μl), and RTB larva (one larva) samples. Solid medium and RTB larvae were crushed in 400 μl of precooled methanol:ethanol:chloroform (8:1:1, v/v/v) solution individually. Liquid medium can be directed mixed with this precooled extraction solution. Then, the mixture added into 2 μl of adonitol (30 mg/ml) as an internal quantitative standard was sealed with a cap and incubated at −20°C for 1 hour. After centrifugation at 8000g for 15 min, the supernatant was transferred to a new 1.5-ml centrifuge tube. After the extracts were dried in a vacuum container, 40 μl of methoxyamination reagent was added into the samples, and the mixtures were shaken at 37°C for 1 hour. Then, 50 μl of MSTFA reagent was added and shaken at 37°C for 0.5 hour. After the addition of 0.4 ml of hexane, the carbohydrates were extracted into the hexane layer by centrifuging at 14,000g for 10 min. The supernatants were transferred into a gas chromatography–mass spectrometry (GC-MS) or GC-falme ionization detector (GC-FID) target vial and analyzed as previously described (34).

Constructing and sequencing 16S rDNA libraries of RTB larvae gut microbiotas

Gut DNA from surface-sterilized RTB larvae feeding on desugared phloem medium containing d-glucose versus d-pinitol for 6 days were separately used as templates (100 ng) for PCR using bacterial universal primers (table S2). For each population, total gut DNA was extracted from a pool of 15 RTB larval guts. We amplified the V3-V4 hypervariable region of the bacterial 16S rRNA gene and sequenced amplicons on an Illumina HiSeq2500 platform [read length of 250 base pairs (bp)]. Sequencing and data processing of 16S rDNA libraries of RTB larvae gut microbiotas were performed at Novogene Bioinformatic Technology Co. Ltd. (Tianjin, China) and were completed as previously described (40). Microbial community genomic DNA was extracted from gut samples of RTB larvae using the cetyltrimethylammonium bromide (CTAB)-SDS method. The DNA extract was checked on 1% agarose gel, and DNA concentration and purity were determined with a NanoDrop 2000 ultraviolet-visible spectrophotometer (Thermo Fisher Scientific, Wilmington, USA). According to the concentration, DNA was diluted to l μg/μl using sterile water.

Whole-genome sequence and analysis

Fungus L. procerum and bacterial strains of E. sp.2 B304 and S. liquefaciens B314 were used for genome sequencing. Both of the L. procerum and associated bacterial genomic DNA were extracted with the SDS method, and then, high-quality and high-quantity genomic DNA were used for library construction using the PacBio Sequel platform (with the insert size of fungus and bacteria being 20 and 10 kb, respectively) and the Illumina NovaSeq platform (66). All sequencing, assembly, and annotation of L. procerum and bacteria were performed at the Beijing Novogene Bioinformatics Technology Co. Ltd. The whole genome was sequenced using the PacBio Sequel platform and Illumina NovaSeq PE150. Genome preliminary assembly from PacBio reads was done with SMRT Link v5.0.1, followed by patching using the variant Caller module of the SMRT Link software. For fungi, by default, we used the Augustus 2.7 program to retrieve the related coding gene. For bacteria, we used the GeneMarkS program to retrieve the related coding gene. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, KOG (Clusters of Orthologous Groups), NR (Non-Redundant Protein Database databases), Transporter Classification Database, P450, and Swiss-Prot were used to predict gene functions. A whole-genome Blast search (E-value less than 1 × 10−5, minimal alignment length percentage larger than 40%) was performed against the above seven databases.

RNA-seq analyses and qRT-PCR analysis

RNA extractions, RNA-seq analyses, complementary DNA (cDNA) synthesis, and qRT-PCR of fungus L. procerum, bacteria E. sp.2 B304, and beetle larvae were performed as previously described (34). RNA-seq analyses of bacteria E. sp.2 B304 were carried out with minor modification. A total amount of 3 μg of RNA per sample was used as input material for the RNA sample preparations. mRNA was purified from total RNA using probes to remove rRNA. Fragmentation was carried out using divalent cations under elevated temperature in first-strand synthesis reaction buffer (5×). First-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase [ribonuclease (RNase) H−]. Second-strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3′ ends of DNA fragments, adaptors with a hairpin loop structure were ligated to prepare for hybridization. Then, USER Enzyme was used to degrade the second strand of cDNA containing U. To select cDNA fragments of preferentially 370 to 420 bp in length, the library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, USA). Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. Last, PCR products were purified (AMPure XP system), and library quality was assessed on the Agilent Bioanalyzer 2100 system.

The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina NovaSeq platform, and 150-bp paired-end reads were generated. Both building index of reference genome and aligning clean reads to reference genome (from above) used Bowtie2-2.2.3. HTSeq v0.6.1 was used to count the read numbers mapped to each gene. Then, the FPKM of each gene was calculated on the basis of the length of the gene and read count mapped to this gene. FPKM, the expected number of fragments per kilobase of transcript sequence per millions base pairs sequenced, considers the effect of sequencing depth and gene length for the read count at the same time and is currently the most commonly used method for estimating gene expression levels. Differential expression analysis was performed using the DESeq R package (1.18.0). DESeq provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate (FDR). Genes with an adjusted P value <0.05 found by DESeq were assigned as differentially expressed. Three biological replicates were used for both RNA-seq and qRT-PCR. The primers used here are included in table S2.

Construction of fungal and bacterial deletion strains

Gene deletions of fungus and bacteria were performed by homologous recombination (34). Gene deletions of fungus iolT11, PDA6, PDA9-1, PDA9-3, and MIOX were performed as previously described (34). To construct the E. sp.2 B304 (ΔiolT1) deletion strains, the homologous arms upstream and downstream of the gene were amplified using different paired primer pairs (table S2), which respectively contained Nde I and Xba I restriction enzyme sites. Then, amplified fragments were inserted into pRE112, a mobilizable plasmid to form pRE112-ΔiolT1. The assembled construct was transformed into Escherichia coli S17-1 λpir (Biomedal) to form pRE112-ΔiolT1. To conjugate cells, recipient (E. sp.2 B304) and donor cells (E. coli) were inoculated from overnight cultures. The conjugation puddle was scraped in LB broth and incubated at 28°C on LB agar plates. All the constructed strains were validated by PCR and sequenced. The iolG, iolE, vanB1, vanB2, and vanB1/vanB2 mutants of E. sp.2 B304 were generated using the same procedure. The primers used here are included in table S2.

Effects of reintroduction of ΔiolT1 mutants on the performance of RTB larvae

The third surface-sterilized instar RTB larvae were maintained on phloem medium with antibiotics [streptomycin sulfate (4.1 μg/μl), ampicillin sodium salt (4.1 μg/μl), tetracyline HCl 4.1 (μg/μl), and nystatin (4.1 μg/μl)] for 6 days. After that, larvae (n = 30 per treatment) were transferred into a medium with WT or ΔiolT1 mutants of L. procerum and E. sp. 2 B304. Medium used for the reintroduction of these symbionts and RTB bioassays was antibiotic free and inoculated at 25°C for 6 days. Survival and body weight changes of RTB larvae in the desugared phloem medium with d-glucose and d-pinitol or only d-glucose were recorded. Elimination of gut microbiota by antibiotic was determined and confirmed by plating beetle larvae onto LB agar plates and performing PCR analysis using bacterial 16S rRNA gene universal primers (table S2). The frass of beetles was sampled on day 6 after the reintroduction of these microbes, and d-pinitol concentration was measured as before (35).

Statistical analysis

The statistical significance of the difference in RTB larval and microbial performance between G&P and G, and d-pinitol degradation between control and different treatments were analyzed using Student’s t test. All of the qPCR results and bioassays of different mutants were also determined using Student’s t test. For diet choice, statistical significance was calculated using a chi-square test. Asterisk indicates significant difference at *P < 0.05, **P < 0.01, ***P < 0.001, and ns, not significant. Biological replicates are described in Materials and Methods and Results.

Acknowledgments

We are grateful to J. D. Wickham (Chinese Academy of Science, China) for discussion on experimental design and C. Zhang (Ministry of Agriculture and Rural Affairs of the People’s Republic of China) for assistance in experimental methods. We thank Y. Xu for help during the experiment. Thanks are also extended to R. Blanchette (University of Minnesota) for comments and critical revision of the earlier version of the manuscript. We appreciate the constructive comments from three anonymous reviewers that greatly improved the quality of our paper.

Funding: This work was supported by the National Natural Science Foundation of China (grant nos. 32088102, 32101537, and 32061123002), the National Key Research and Development Program of China (2021YFC2600100), and the Hebei Natural Science Foundation (C2022201042).

Author contributions: F.L., and J.S. conceived and designed the experiment. F.L., F.Y., C.C., Z.K., and H.K. performed the experiments. F.L., F.Y., and Z.K. analyzed the data. F.L. and Z.K. wrote the manuscript. F.L., C.C., Z.K., and J.S. revised the manuscript.

Competing interests: The authors declare that they have no competing interests.

Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Genome and transcriptome sequences obtained here have been deposited in the Sequence Read Archive Database of NCBI (L. procerum genome and transcriptome: PRJNA808223; E. sp.2 B304 genome and transcriptome: PRJNA808186; S. liquefaciens B314 genome and transcriptome: PRJNA808204; 16S rRNA sequences of RTB gut microbiota: PRJNA808487).

Supplementary Materials

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Figs. S1 to S14

Tables S1 and S2

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REFERENCES AND NOTES

  • 1.Despres L., David J. P., Gallet C.,The evolutionary ecology of insect resistance to plant chemicals. Trends Ecol. Evol. 22,298–307 (2007). [DOI] [PubMed] [Google Scholar]
  • 2.Erb M., Reymond P.,Molecular interactions between plants and insect herbivores. Annu. Rev. Plant Biol. 70,527–557 (2019). [DOI] [PubMed] [Google Scholar]
  • 3.Jing T. Z., Qi F. H., Wang Z. Y.,Most dominant roles of insect gut bacteria: Digestion, detoxification, or essential nutrient provision? Microbiome 8,38 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Nallu S., Hill J. A., Don K., Sahagun C., Zhang W., Meslin C., Snell-Rood E., Clark N. L., Morehouse N. I., Bergelson J., Wheat C. W., Kronforst M. R.,The molecular genetic basis of herbivory between butterflies and their host plants. Nat. Ecol. Evol. 2,1418–1427 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Wen Z. M., Pan L. P., Berenbaum M. R., Schuler M. A.,Metabolism of linear and angular furanocoumarins by Papilio polyxenes CYP6B1 co-expressed with NADPH cytochrome P450 reductase. Insect Biochem. Mol. Biol. 33,937–947 (2003). [DOI] [PubMed] [Google Scholar]
  • 6.Wang H. D., Shi Y., Wang L., Liu S., Wu S. W., Yang Y. H., Feyereisen R., Wu Y. D.,CYP6AE gene cluster knockout in Helicoverpa armigera reveals role in detoxification of phytochemicals and insecticides. Nat. Commun. 9,4820 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Ratzka A., Vogel H., Kliebenstein D. J., Mitchell-Olds T., Kroymann J.,Disarming the mustard oil bomb. Proc. Natl. Acad. Sci. U.S.A. 99,11223–11228 (2002). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Itoh H., Tago K., Hayatsu M., Kikuchi Y.,Detoxifying symbiosis: Microbe-mediated detoxification of phytotoxins and pesticides in insects. Nat. Prod. Rep. 35,434–454 (2018). [DOI] [PubMed] [Google Scholar]
  • 9.Zheng H., Nishida A., Kwong W. K., Koch H., Engel P., Steele M. I., Moran N. A.,Metabolism of toxic sugars by strains of the bee gut symbiont Gilliamella apicola. MBio 7,e01326-16 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Zheng H., Perreau J., Powell J. E., Han B. F., Zhang Z. J., Kwong W. K., Tringe S. G., Moran N. A.,Division of labor in honey bee gut microbiota for plant polysaccharide digestion. Proc. Natl. Acad. Sci. U.S.A. 116,25909–25916 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ceja-Navarro J. A., Vega F. E., Karaoz U., Hao Z., Jenkins S., Lim H. C., Kosina P., Infante F., Northen T. R., Brodie E. L.,Gut microbiota mediate caffeine detoxification in the primary insect pest of coffee. Nat. Commun. 6,7618 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Francoeur C. B., Khadempor L., Moreira-Soto R. D., Gotting K., Book A. J., Pinto-Tomás A. A., Keefover-Ring K., Currie C. R.,Bacteria contribute to plant secondary compound degradation in a generalist herbivore system. MBio 11,e02146-20 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Zhang S. K., Shu J. P., Xue H. J., Zhang W., Zhang Y. B., Liu Y. N., Fang L. X., Wang Y. D., Wang H. J.,The gut microbiota in camellia weevils are influenced by plant secondary metabolites and contribute to saponin degradation. mSystems 5,e00692-19 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Shukla S. P., Beran F.,Gut microbiota degrades toxic isothiocyanates in a flea beetle pest. Mol. Ecol. 29,4692–4705 (2020). [DOI] [PubMed] [Google Scholar]
  • 15.Berasategui A., Salem H., Paetz C., Santoro M., Gershenzon J., Kaltenpoth M., Schmidt A.,Gut microbiota of the pine weevil degrades conifer diterpenes and increases insect fitness. Mol. Ecol. 26,4099–4110 (2017). [DOI] [PubMed] [Google Scholar]
  • 16.Sun J. H., Lu M., Gillette N. E., Wingfield M. J.,Red turpentine beetle: Innocuous native becomes invasive tree killer in China. Annu. Rev. Entomol. 58,293–311 (2013). [DOI] [PubMed] [Google Scholar]
  • 17.Yan Z. L., Sun J. H., Don O., Zhang Z. N.,The red turpentine beetle, Dendroctonus valens LeConte (Scolytidae): An exotic invasive pest of pine in China. Biodivers. Conserv. 14,1735–1760 (2005). [Google Scholar]
  • 18.Lu M., Wingfield M. J., Gillette N. E., Mori S. R., Sun J. H.,Complex interactions among host pines and fungi vectored by an invasive bark beetle. New Phytol. 187,859–866 (2010). [DOI] [PubMed] [Google Scholar]
  • 19.Lu M., Hulcr J., Sun J. H.,The role of symbiotic microbes in insect invasions. Annu. Rev. Ecol. Evol. Syst. 47,487–505 (2016). [Google Scholar]
  • 20.Lu M., Wingfield M. J., Gillette N. E., Sun J. H.,Do novel genotypes drive the success of an invasive bark beetle-fungus complex? Implications for potential reinvasion. Ecology 92,2013–2019 (2011). [DOI] [PubMed] [Google Scholar]
  • 21.Lu M., Zhou X. D., De Beer Z. W., Wingfield M. J., Sun J. H.,Ophiostomatoid fungi associated with the invasive pine-infesting bark beetle, Dendroctonus valens, in China. Fungal Divers. 38,133–145 (2009). [Google Scholar]
  • 22.Cheng C. H., Wickham J. D., Chen L., Xu D. D., Lu M., Sun J. H.,Bacterial microbiota protect an invasive bark beetle from a pine defensive compound. Microbiome 6,132 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Zhou F. Y., Lou Q. Z., Wang B., Xu L. T., Cheng C. H., Lu M., Sun J. H.,Altered carbohydrates allocation by associated bacteria-fungi interactions in a bark beetle-microbe symbiosis. Sci. Rep. 6,20135 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Chen J., Dai G. H.,Effect of d-pinitol isolated and identified from Robinia pseudoacacia against cucumber powdery mildew. Sci. Horti. 176,38–44 (2014). [Google Scholar]
  • 25.Chen J., Zhang H. Y., Chen Y. J., Wu T. L., Yu W. J., Dai G. H.,Optimization of d-pinitol extraction from vegetable soybean leaves and its potential application in control of cucumber powdery mildew. Crop Prot. 60,20–27 (2014). [Google Scholar]
  • 26.Dreyer D. L., Binder R. G., Chan B. G., Waiss A. C. Jr., Hartwig E. E., Beland G. L.,Pinitol, a larval growth inhibitor for Heliothis zea in soybeans. Experientia 35,1182–1183 (1979). [DOI] [PubMed] [Google Scholar]
  • 27.Reese J. C., Chan B. G., Waiss A. C. Jr.,Effects of cotton condensed tannin, maysin (corn) and pinitol (soybeans) on Heliothis zea growth and development. J. Chem. Ecol. 8,1429–1436 (1982). [DOI] [PubMed] [Google Scholar]
  • 28.Chaubal R., Pawar P. V., Hebbalkar G. D., Tungikar V. B., Puranik V. G., Deshpande V. H., Deshpande N. R.,Larvicidal activity of Acacia nilotica extracts and isolation of d-pinitol—A bioactive carbohydrate. Chem. Biodivers. 2,684–688 (2005). [DOI] [PubMed] [Google Scholar]
  • 29.Sánchez-Hidalgo M., León-González A. J., Gálvez-Peralta M., González-Mauraza N. H., Martin-Cordero C.,d-Pinitol: A cyclitol with versatile biological and pharmacological activities. Phytochem. Rev. 20,211–224 (2021). [Google Scholar]
  • 30.Bates S. H., Jones R. B., Bailey C. J.,Insulin-like effect of pinitol. Brit. J. Pharmacol. 130,1944–1948 (2000). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Navarro J. A., Decara J., Medina-Vera D., Tovar R., Suarez J., Pavón J., Serrano A., Vida M., Gutierrez-Adan A., Sanjuan C., Baixeras E., de Fonseca F. R.,d-Pinitol from Ceratonia siliqua is an orally active natural inositol that reduces pancreas insulin secretion and increases circulating ghrelin levels in Wistar rats. Nutrients 12,2030 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Kim J. I., Kim J. C., Kang M. J., Lee M. S., Kim J. J., Cha I. J.,Effects of pinitol isolated from soybeans on glycaemic control and cardiovascular risk factors in Korean patients with type II diabetes mellitus: A randomized controlled study. Eur. J. Clin. Nutr. 59,456–458 (2005). [DOI] [PubMed] [Google Scholar]
  • 33.Kim H. J., Park K. S., Lee S. K., Min K. W., Han K. A., Kim Y. K., Ku B. J.,Effects of pinitol on glycemic control, insulin resistance and adipocytokine levels in patients with type 2 diabetes mellitus. Ann. Nutr. Metab. 60,1–5 (2012). [DOI] [PubMed] [Google Scholar]
  • 34.Liu F. H., Wickham J. D., Cao Q. J., Lu M., Sun J. H.,An invasive beetle–fungus complex is maintained by fungal nutritional-compensation mediated by bacterial volatiles. ISME J. 14,2829–2842 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Zhou F. Y., Xu L. T., Wang S. S., Wang B., Lou Q. Z., Lu M., Sun J. H.,Bacterial volatile ammonia regulates the consumption sequence of d-pinitol and d-glucose in a fungus associated with an invasive bark beetle. ISME J. 11,2809–2820 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Ruiz-Aceituno L., Ramos L., Martinez-Castro I., Sanz M. L.,Low molecular weight carbohydrates in pine nuts from Pinus pinea L. J. Agric. Food Chem. 60,4957–4959 (2012). [DOI] [PubMed] [Google Scholar]
  • 37.Pullman G. S., Buchanan M.,Identification and quantitative analysis of stage-specific carbohydrates in loblolly pine (Pinus taeda) zygotic embryo and female gametophyte tissues. Tree Physiol. 28,985–996 (2008). [DOI] [PubMed] [Google Scholar]
  • 38.Lindstedt G.,Constituents of pine heartwood. XIV. The heartwood of Pinus monticola Douglas. Acta Chem. Scand. 3,1147–1152 (1949). [Google Scholar]
  • 39.Sharma I., Dutta R. K., Singh N. K., Kanwar Y. S.,High glucose-induced hypomethylation promotes binding of Sp-1 to myo-inositol oxygenase: Implication in the pathobiology of diabetic tubulopathy. Am. J. Pathol. 187,724–739 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Xu L. T., Shi Z. H., Wang B., Lu M., Sun J. H.,Pine defensive monoterpene α-pinene influences the feeding behavior of Dendroctonus valens and its gut bacterial community structure. Int. J. Mol. Sci. 17,1734 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Xu L. T., Lou Q. Z., Cheng C. H., Lu M., Sun J. H.,Gut-associated bacteria of Dendroctonus valens and their involvement in verbenone production. Microb. Ecol. 70,1012–1023 (2015). [DOI] [PubMed] [Google Scholar]
  • 42.Dohet L., Gregoire J. C., Berasategui A., Kaltenpoth M., Biedermann P. H.,Bacterial and fungal symbionts of parasitic Dendroctonus bark beetles. FEMS Microbiol. Ecol. 92,fiw129 (2016). [DOI] [PubMed] [Google Scholar]
  • 43.Kanter U., Becker M., Friauf E., Tenhaken R.,Purification, characterization and functional cloning of inositol oxygenase from Cryptococcus. Yeast 20,1317–1329 (2003). [DOI] [PubMed] [Google Scholar]
  • 44.Kohler P. R., Zheng J. Y., Schoffers E., Rossbach S.,Inositol catabolism, a key pathway in sinorhizobium meliloti for competitive host nodulation. Appl. Environ. Microbiol. 76,7972–7980 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Yoshida K., Yamaguchi M., Morinaga T., Kinehara M., Ikeuchi M., Ashida H., Fujita Y.,myo-Inositol catabolism in Bacillus subtilis. J. Biol. Chem. 283,10415–10424 (2008). [DOI] [PubMed] [Google Scholar]
  • 46.Morinaga T., Yamaguchi M., Makino Y., Nanamiya H., Takahashi K., Yoshikawa H., Kawamura F., Ashida H., Yoshida K.,Functional myo-inositol catabolic genes of Bacillus subtilis natto are involved in depletion of pinitol in natto (fermented soybean). Biosci. Biotechnol. Biochem. 70,1913–1920 (2006). [DOI] [PubMed] [Google Scholar]
  • 47.Arner R. J., Prabhu K. S., Thompson J. T., Hildenbrandt G. R., Liken A. D., Reddy C. C.,myo-Inositol oxygenase: Molecular cloning and expression of a unique enzyme that oxidizes myo-inositol and d-chiro-inositol. Biochem. J. 360,313–320 (2001). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Schneider S.,Inositol transport proteins. FEBS Lett. 589,1049–1058 (2015). [DOI] [PubMed] [Google Scholar]
  • 49.VanEtten H. D., Matthews P. S., Tegtmeier K. J., Dietert M. F., Stein J. I.,The association of pisatin tolerance and demethylation with virulence on pea in Nectria haematococca. Physiol. Plant Pathol. 16,257–268 (1980). [Google Scholar]
  • 50.Endara M. J., Coley P. D., Ghabash G., Nicholls J. A., Dexter K. G., Donoso D. A., Stone G. N., Pennington R. T., Kursar T. A.,Coevolutionary arms race versus host defense chase in a tropical herbivore-plant system. Proc. Natl. Acad. Sci. U.S.A. 114,E7499–E7505 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Taerum S. J., Duong T. A., De Beer Z. W., Gillette N., Sun J. H., Owen D. R., Wingfield M. J.,Large shift in symbiont assemblage in the invasive red turpentine beetle. PLOS ONE 8,e78126 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Hernández-García J. A., Briones-Roblero C. I., Rivera-Ordua F. N., Zúiga G.,Revealing the gut bacteriome of Dendroctonus bark beetles (curculionidae: scolytinae): Diversity, core members and co-evolutionary patterns. Sci. Rep. 7,13864 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Pérombelon M. C. M.,Ecology of the soft rot Erwinias. Annu. Rev. Phytopathol. 18,361–387 (1980). [Google Scholar]
  • 54.Cellini A., Giacomuzzi V., Donati I., Farneti B., Rodriguez-Estrada M. T., Savioli S., Angeli S., Spinelli F.,Pathogen-induced changes in floral scent may increase honeybee-mediated dispersal of Erwinia amylovora. ISME J. 13,847–859 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Shapiro L. R., Paulson J. N., Arnold B. J., Scully E. D., Zhaxybayeva O., Pierce N. E., Rocha J., Klepac-Ceraj V., Holton K., Kolter R.,An introduced crop plant is driving diversification of the virulent bacterial pathogen Erwinia tracheiphila. MBio 9,e01307-18 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Lin T. H., Tan T. W., Tsai T. H., Chen C. C., Hsieh T. F., Lee S. S., Liu H. H., Chen W. C., Tang C. H.,d-Pinitol inhibits prostate cancer metastasis through inhibition of αVβ3 integrin by modulating FAK, c-Src and NF-κB pathways. Int. J. Mol. Sci. 14,9790–9802 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Medina-Vera D., Navarro J. A., Tovar R., Rosell-Valle C., Gutiérrez-Adan A., Ledesma J. C., Sanjuan C., Pavón F. J., Baixeras E., de Fonseca F. R., Decara J.,Activation of PI3K/Akt signaling pathway in rat hypothalamus induced by an acute oral administration of d-pinitol. Nutrients 13,2268 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Dang N. T., Mukai R., Yoshida K., Ashida H.,d-Pinitol and myo-inositol stimulate translocation of glucose transporter 4 in skeletal muscle of C57BL/6 mice. Biosci. Biotechnol. Biochem. 74,1062–1067 (2010). [DOI] [PubMed] [Google Scholar]
  • 59.Gao Y. F., Zhang M. N., Wu T. C., Xu M. Y., Cai H. N., Zhang Z. S.,Effects of d-pinitol on insulin resistance through the PI3K/Akt signaling pathway in type 2 diabetes mellitus rats. J. Agric. Food Chem. 63,6019–6026 (2015). [DOI] [PubMed] [Google Scholar]
  • 60.Sharma I., Deng F., Liao Y., Kanwar Y. S.,Myo-inositol oxygenase (MIOX) overexpression drives the progression of renal tubulointerstitial injury in diabetes. Diabetes 69,1248–1263 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Tominaga T., Dutta R. K., Joladarashi D., Doi T., Reddy J. K., Kanwar Y. S.,Transcriptional and translational modulation of myo-inositol oxygenase (Miox) by fatty acids: Implications in renal tubular injury induced in obesity and diabetes. J. Biol. Chem. 291,1348–1367 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Nayak B., Kondeti V. K., Xie P., Lin S., Viswakarma N., Raparia K., Kanwar Y. S.,Transcriptional and post-translational modulation of myo-inositol oxygenase by high glucose and related pathobiological stresses. J. Biol. Chem. 286,27594–27611 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Kawa J. M., Przybylski R., Taylor C. G.,Urinary chiro-inositol and myo-inositol excretion is elevated in the diabetic db/db mouse and streptozotocin diabetic rat. Exp. Biol. Med. 228,907–914 (2003). [DOI] [PubMed] [Google Scholar]
  • 64.Coon K. L., Vogel K. J., Brown M. R., Strand M. R.,Mosquitoes rely on their gut microbiota for development. Mol. Ecol. 23,2727–2739 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Cheng C. H., Xu L. T., Xu D. D., Lou Q. Z., Lu M., Sun J. H.,Does cryptic microbiota mitigate pine resistance to an invasive beetle-fungus complex? Implications for invasion potential. Sci. Rep. 6,33110 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Lim H. J., Lee E. H., Yoon Y., Chua B., Son A.,Portable lysis apparatus for rapid single-step DNA extraction of Bacillus subtilis. J. Appl. Microbiol. 120,379–387 (2016). [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Figs. S1 to S14

Tables S1 and S2

Click here for additional data file. (1.9MB, pdf)

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