Conifer-killing bark beetles locate fungal symbionts by detecting volatile fungal metabolites of host tree resin monoterpenes
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Authors
Kandasamy, Dineshkumar
Zaman, Rashaduz
Nakamura, Yoko
Zhao, Tao
Hartmann, Henrik
Andersson, Martin N.
Hammerbacher, Almuth
Gershenzon, Jonathan
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Public Library of Science
Abstract
Outbreaks of the Eurasian spruce bark beetle (Ips typographus) have decimated millions of hectares of conifer forests in Europe in recent years. The ability of these 4.0 to 5.5 mm long insects to kill mature trees over a short period has been sometimes ascribed to two main factors: (1) mass attacks on the host tree to overcome tree defenses and (2) the presence of fungal symbionts that support successful beetle development in the tree. While the role of pheromones in coordinating mass attacks has been well studied, the role of chemical communication in maintaining the fungal symbiosis is poorly understood. Previous evidence indicates that I. typographus can distinguish fungal symbionts of the genera Grosmannia, Endoconidiophora, and Ophiostoma by their de novo synthesized volatile compounds. Here, we hypothesize that the fungal symbionts of this bark beetle species metabolize spruce resin monoterpenes of the beetle's host tree, Norway spruce (Picea abies), and that the volatile products are used as cues by beetles for locating breeding sites with beneficial symbionts. We show that Grosmannia penicillata and other fungal symbionts alter the profile of spruce bark volatiles by converting the major monoterpenes into an attractive blend of oxygenated derivatives. Bornyl acetate was metabolized to camphor, and α- and β-pinene to trans-4-thujanol and other oxygenated products. Electrophysiological measurements showed that I. typographus possesses dedicated olfactory sensory neurons for oxygenated metabolites. Both camphor and trans-4-thujanol attracted beetles at specific doses in walking olfactometer experiments, and the presence of symbiotic fungi enhanced attraction of females to pheromones. Another co-occurring nonbeneficial fungus (Trichoderma sp.) also produced oxygenated monoterpenes, but these were not attractive to I. typographus. Finally, we show that colonization of fungal symbionts on spruce bark diet stimulated beetles to make tunnels into the diet. Collectively, our study suggests that the blends of oxygenated metabolites of conifer monoterpenes produced by fungal symbionts are used by walking bark beetles as attractive or repellent cues to locate breeding or feeding sites containing beneficial microbial symbionts. The oxygenated metabolites may aid beetles in assessing the presence of the fungus, the defense status of the host tree and the density of conspecifics at potential feeding and breeding sites.
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10.6084/m9.figshare.21692156.v1.
SUPPORTING INFORMATION : S1 Fig. Adult beetles prefer spruce bark agar (SBA) inoculated with two species of symbiotic fungi over uninoculated SBA. Adult beetles did not prefer O. bicolor, O. piceae, and Trichoderma sp., the latter two species are saprophytes. Deviation of response indices against zero was tested using Wilcoxon’s test (n = 20 or 25). The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S2 Fig. Volatile emission pattern differed between spruce bark inoculated with different fungi and uninfected bark 4 d after inoculation, as depicted in a sparse partial least squares discriminant analysis (sPLS-DA). Analysis was performed using 59 compounds listed in S2 Table. Principal components (PC1 and PC2) explain 41.4% and 11.2% of the total variation, respectively, and ellipses denote 95% confident intervals around each species. The sPLS-DA plot was generated by using MetaboAnalyst 3.0 software with normalized data (both log transformed and range scaled). The data underlying this Figure can be found at https://doi. org/10.6084/m9.figshare.21692156.v1. (TIF) S3 Fig. Changes in volatile emission profiles of fungal-infested vs. uninfested spruce bark over an 18-d time course for three other I. typographus symbiotic fungi besides G. penicillata. Compounds are classified into six groups according to chemical structures. Complete volatile emission data by compound and time point for each fungal species are given in S3–S6 Tables. (n = 3 or 5). The data underlying this Figure can be found at https://doi.org/10.6084/ m9.figshare.21692156.v1. (TIF) S4 Fig. Volatile metabolites of (−)-β-pinene produced by two fungal symbionts (L. europhioides and O. bicolor) of I. typographus growing on potato dextrose agar. Isopinocamphone was the major biotransformation product (n = 4 or 5). E. polonica produced no detectable products. ND, not detected. The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S5 Fig. Metabolites of (−)-β-pinene produced by various I. typographus symbiotic fungi growing on potato dextrose agar after this monoterpene was administered to cultures of each species. Amounts of metabolites were determined after hexane extraction of the agar. Error bars represent SEM (n = 5 or 11). ND, not detected. Different lowercase letters denote significant differences between treatments (ANOVA, Sidak’s test; P < 0.05). Fungal abbreviations: E. polonica (Ep), L. europhioides (Le), G. penicillata (Gp), O. bicolor (Ob). The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S6 Fig. Metabolites of (−)-α-pinene produced by various I. typographus symbiotic fungi growing on potato dextrose agar after this monoterpene was administered to cultures of each species. Amounts of metabolites were determined after hexane extraction of the agar. Error bars represent SEM (n = 5 to 12). ND, not detected. Different lowercase letters denote significant differences between treatments (ANOVA, Sidak’s test; P < 0.05). Fungal abbreviations: E. polonica (Ep), L. europhioides (Le), G. penicillata (Gp), O. bicolor (Ob). The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S7 Fig. Metabolites of (+)-α-pinene produced by various I. typographus symbiotic fungi growing on potato dextrose agar after this monoterpene was administered to cultures of each species. Amounts of metabolites were determined after hexane extraction of the agar. Error bars represent SEM (n = 5 or 13). ND, not detected. Different lowercase letters denote significant differences between treatments (ANOVA, Sidak’s test; P < 0.05). Fungal abbreviations: E. polonica (Ep), L. europhioides (Le), G. penicillata (Gp), O. bicolor (Ob). The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S8 Fig. Metabolites of (−)-bornyl acetate produced by various I. typographus symbiotic fungi growing on potato dextrose agar after this monoterpene was administered to cultures of each species. Amounts of metabolites were determined after hexane extraction of the agar. Error bars represent SEM (n = 5 or 13). ND, not detected. Different lowercase letters denote significant differences between treatments (ANOVA, Sidak’s test; P < 0.05). Fungal abbreviations: E. polonica (Ep), L. europhioides (Le), G. penicillata (Gp), O. bicolor (Ob). The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S9 Fig. Relative proportion of oxygenated monoterpenes produced by the bark beetle symbiont G. penicillata, a saprophyte Trichoderma sp. and a fungus-free control potato dextrose agar medium amended with a mix of spruce monoterpenes (see S8 Table). The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S10 Fig. Response spectra of olfactory sensory neuron (OSN) classes (originally characterized in [60]) with primary responses to (A) (+)-α-pinene (n = 14), (B) p-cymene (n = 9), and (C) Δ3-carene (n = 4) to their respective most active ligands at the 10-μg screening dose (ligands eliciting average responses <20 Hz are not shown). In addition to responses to the primary ligands, which are monoterpene hydrocarbons, these OSN classes show comparatively strong secondary responses to oxygenated monoterpenes produced by symbiotic fungi from host tree monoterpenes. Error bars represent SEM. The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S11 Fig. Adult beetles did not discriminate between spruce bark agar (SBA) enriched with monoterpenes and unenriched SBA. Error bars represent SEM (n = 25 for each trial). The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S12 Fig. Concentration of the oxygenated monoterpenes (a) trans-verbenol, (b) cis-verbenol, (c) borneol, (d) myrtanal, (e) myrtenol, (f) verbenone, (g) trans-myrtanol, (h) perillaldehyde, (i) nopinone, and (j) pinocarvone produced by live and dead male I. typographus fumigated with the mix of spruce monoterpenes listed in S8 Table. The method for chemical analysis of beetles is in the supplementary methods (S3 Method). The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S13 Fig. Confirmation of the structure of synthesized (+)-isopinocamphone by NMR. (A) 1H and 13C signal assignments in deuterated chloroform (CDCl3), (B) 1H NMR spectrum in PLOS BIOLOGY Conifer bark beetles detect volatiles of fungal symbionts produced from host tree resin monoterpenes CDCl3, (C) phase-sensitive heteronuclear single quantum coherence (HSQC) in CDCl3, (D) heteronuclear multiple bond correlation (HMBC) in CDCl3, (E) correlated spectroscopy (COSY) in CDCl3, and (F) 13C attached proton test (APT) in CDCl3. (TIF) S14 Fig. Confirmation of the structure of synthesized β-isophorone by NMR. (A) 1H and 13C signal assignments in acetone-d6, (B) 1H NMR spectrum in acetone-d6, purity- 91%, (C) phase-sensitive heteronuclear single quantum coherence (HSQC) in acetone-d6, (D) heteronuclear multiple bond correlation (HMBC) in acetone-d6, (E) correlated spectroscopy (COSY) in acetone-d6, and (F) 13C spectrum in acetone-d6. (TIF) S15 Fig. Scanning electron micrographs of (A) an elytron of an untreated bark beetle showing (B) spores of an ophiostomatoid fungus in the elytral pit, (C) an empty elytral pit of a fungusfree beetle, and (D) spore mass of G. penicillata in the elytral pit of a fungus-free beetle reinoculated with this fungal species. (TIF) S1 Table. Purity, source, and biological origins of chemicals used in the experiments. (DOCX) S2 Table. Emission of volatile organic compounds identified from the headspace collection of fresh spruce bark 4 d after inoculation with different fungi. Analyses were conducted using GC-FID. Compounds were identified by GC–MS analyses run in parallel. Compounds with significant P values are highlighted in bold. The data underlying this Table can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (DOCX) S3 Table. Relative amounts (mean ± SE, n = 3) of volatiles from uninoculated bark detected after various time periods (4, 8, 12, and 18 d) from the beginning of an experiment with fungal inoculation. Data from the control uninfected treatment are presented here. Data for fungal treatments are given in S3–S6 Tables. Volatiles were collected on polydimethylsiloxane tubes for 2 h and were subjected to GC–MS analysis (see Materials and methods section for details). ND, not detected, NA, not analyzed, TR, trace amounts (<500 TIC counts). The data underlying this Table can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (DOCX) S4 Table. Relative amounts (mean ± SE, N = 4–5) of volatiles detected at various time periods after inoculation of fresh spruce bark with E. polonica (4, 8, and 12 d). Volatiles were collected on polydimethylsiloxane tubes for 2 h and were subjected to GC–MS analysis (see Materials and methods section for details). ND, not detected, NA, not analyzed, TR, trace amounts (<500 TIC counts). The data underlying this Table can be found at https://doi.org/ 10.6084/m9.figshare.21692156.v1. (DOCX) S5 Table. Relative amounts (mean ± SE, N = 5) of volatiles detected at various time periods after inoculation of fresh spruce bark with G. penicillata (4, 8, 12, and 18 d). Volatiles were collected on polydimethylsiloxane tubes for 2 h and were subjected to GC–MS analysis (see Materials and methods section for details). ND, not detected, NA, not analyzed, TR, trace amounts (<500 TIC counts). The data underlying this Table can be found at https://doi.org/ 10.6084/m9.figshare.21692156.v1. (DOCX) S6 Table. Relative amounts (mean ± SE, N = 5) of volatiles detected at various time periods after inoculation of fresh spruce bark with L. europhioides (4, 8, 12, and 18 d). Volatiles were collected on polydimethylsiloxane tubes for 2 h and were subjected to GC–MS analysis (see Materials and methods section for details). ND, not detected, NA, not analyzed, TR, trace amounts (<500 TIC counts). The data underlying this Table can be found at https://doi.org/ 10.6084/m9.figshare.21692156.v1. (DOCX) S7 Table. Relative amounts (mean ± SE, N = 5) of volatiles detected at various time periods after inoculation of fresh spruce bark with O. bicolor (4, 8, 12, and 18 d). Volatiles were collected on polydimethylsiloxane tubes for 2 h and were subjected to GC–MS analysis (see Materials and methods section for details). ND, not detected, NA, not analyzed, TR, trace amounts (<500 TIC counts). The data underlying this Table can be found at https://doi. org/10.6084/m9.figshare.21692156.v1. (DOCX) S8 Table. Composition of synthetic monoterpene mixture used in bioassays. $The purity of each compound was calculated from GC–MS analysis. (DOCX) S9 Table. Average colony forming units (CFUs/mL) from untreated, fungus-free, and fungus- free G. penicillata-reinoculated I. typographus bark beetles (n = 5 or 6 beetles). Wash, supernatant from beetles immersed in 0.05% Triton X in 500 μL PBS buffer (pH 7.4); lysate, crushed beetles in 500 μL PBS buffer (pH 7.4); NP, not present. The data underlying this Table can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (DOCX) S10 Table. Colony forming units (CFUs/mL) obtained from bark beetle gallery samples infested by fungus-free beetles, and fungus-free beetles reinoculated with G. penicillata, and untreated control beetles. Approximately 300 mg of bark samples were dissolved in 1 mL PBS buffer solution, and dilutions were plated on PDA. NP, not present. $Only one gallery sample was tested due to low sample availability. (DOCX) S1 Method. Preparation of bark beetle diet for eliminating fungal symbionts. (DOCX) S2 Method. Analysis of G. penicillata and Trichoderma sp. headspace volatiles. (DOCX) S3 Method. Beetle pheromone analysis. (DOCX)
SUPPORTING INFORMATION : S1 Fig. Adult beetles prefer spruce bark agar (SBA) inoculated with two species of symbiotic fungi over uninoculated SBA. Adult beetles did not prefer O. bicolor, O. piceae, and Trichoderma sp., the latter two species are saprophytes. Deviation of response indices against zero was tested using Wilcoxon’s test (n = 20 or 25). The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S2 Fig. Volatile emission pattern differed between spruce bark inoculated with different fungi and uninfected bark 4 d after inoculation, as depicted in a sparse partial least squares discriminant analysis (sPLS-DA). Analysis was performed using 59 compounds listed in S2 Table. Principal components (PC1 and PC2) explain 41.4% and 11.2% of the total variation, respectively, and ellipses denote 95% confident intervals around each species. The sPLS-DA plot was generated by using MetaboAnalyst 3.0 software with normalized data (both log transformed and range scaled). The data underlying this Figure can be found at https://doi. org/10.6084/m9.figshare.21692156.v1. (TIF) S3 Fig. Changes in volatile emission profiles of fungal-infested vs. uninfested spruce bark over an 18-d time course for three other I. typographus symbiotic fungi besides G. penicillata. Compounds are classified into six groups according to chemical structures. Complete volatile emission data by compound and time point for each fungal species are given in S3–S6 Tables. (n = 3 or 5). The data underlying this Figure can be found at https://doi.org/10.6084/ m9.figshare.21692156.v1. (TIF) S4 Fig. Volatile metabolites of (−)-β-pinene produced by two fungal symbionts (L. europhioides and O. bicolor) of I. typographus growing on potato dextrose agar. Isopinocamphone was the major biotransformation product (n = 4 or 5). E. polonica produced no detectable products. ND, not detected. The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S5 Fig. Metabolites of (−)-β-pinene produced by various I. typographus symbiotic fungi growing on potato dextrose agar after this monoterpene was administered to cultures of each species. Amounts of metabolites were determined after hexane extraction of the agar. Error bars represent SEM (n = 5 or 11). ND, not detected. Different lowercase letters denote significant differences between treatments (ANOVA, Sidak’s test; P < 0.05). Fungal abbreviations: E. polonica (Ep), L. europhioides (Le), G. penicillata (Gp), O. bicolor (Ob). The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S6 Fig. Metabolites of (−)-α-pinene produced by various I. typographus symbiotic fungi growing on potato dextrose agar after this monoterpene was administered to cultures of each species. Amounts of metabolites were determined after hexane extraction of the agar. Error bars represent SEM (n = 5 to 12). ND, not detected. Different lowercase letters denote significant differences between treatments (ANOVA, Sidak’s test; P < 0.05). Fungal abbreviations: E. polonica (Ep), L. europhioides (Le), G. penicillata (Gp), O. bicolor (Ob). The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S7 Fig. Metabolites of (+)-α-pinene produced by various I. typographus symbiotic fungi growing on potato dextrose agar after this monoterpene was administered to cultures of each species. Amounts of metabolites were determined after hexane extraction of the agar. Error bars represent SEM (n = 5 or 13). ND, not detected. Different lowercase letters denote significant differences between treatments (ANOVA, Sidak’s test; P < 0.05). Fungal abbreviations: E. polonica (Ep), L. europhioides (Le), G. penicillata (Gp), O. bicolor (Ob). The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S8 Fig. Metabolites of (−)-bornyl acetate produced by various I. typographus symbiotic fungi growing on potato dextrose agar after this monoterpene was administered to cultures of each species. Amounts of metabolites were determined after hexane extraction of the agar. Error bars represent SEM (n = 5 or 13). ND, not detected. Different lowercase letters denote significant differences between treatments (ANOVA, Sidak’s test; P < 0.05). Fungal abbreviations: E. polonica (Ep), L. europhioides (Le), G. penicillata (Gp), O. bicolor (Ob). The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S9 Fig. Relative proportion of oxygenated monoterpenes produced by the bark beetle symbiont G. penicillata, a saprophyte Trichoderma sp. and a fungus-free control potato dextrose agar medium amended with a mix of spruce monoterpenes (see S8 Table). The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S10 Fig. Response spectra of olfactory sensory neuron (OSN) classes (originally characterized in [60]) with primary responses to (A) (+)-α-pinene (n = 14), (B) p-cymene (n = 9), and (C) Δ3-carene (n = 4) to their respective most active ligands at the 10-μg screening dose (ligands eliciting average responses <20 Hz are not shown). In addition to responses to the primary ligands, which are monoterpene hydrocarbons, these OSN classes show comparatively strong secondary responses to oxygenated monoterpenes produced by symbiotic fungi from host tree monoterpenes. Error bars represent SEM. The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S11 Fig. Adult beetles did not discriminate between spruce bark agar (SBA) enriched with monoterpenes and unenriched SBA. Error bars represent SEM (n = 25 for each trial). The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S12 Fig. Concentration of the oxygenated monoterpenes (a) trans-verbenol, (b) cis-verbenol, (c) borneol, (d) myrtanal, (e) myrtenol, (f) verbenone, (g) trans-myrtanol, (h) perillaldehyde, (i) nopinone, and (j) pinocarvone produced by live and dead male I. typographus fumigated with the mix of spruce monoterpenes listed in S8 Table. The method for chemical analysis of beetles is in the supplementary methods (S3 Method). The data underlying this Figure can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (TIF) S13 Fig. Confirmation of the structure of synthesized (+)-isopinocamphone by NMR. (A) 1H and 13C signal assignments in deuterated chloroform (CDCl3), (B) 1H NMR spectrum in PLOS BIOLOGY Conifer bark beetles detect volatiles of fungal symbionts produced from host tree resin monoterpenes CDCl3, (C) phase-sensitive heteronuclear single quantum coherence (HSQC) in CDCl3, (D) heteronuclear multiple bond correlation (HMBC) in CDCl3, (E) correlated spectroscopy (COSY) in CDCl3, and (F) 13C attached proton test (APT) in CDCl3. (TIF) S14 Fig. Confirmation of the structure of synthesized β-isophorone by NMR. (A) 1H and 13C signal assignments in acetone-d6, (B) 1H NMR spectrum in acetone-d6, purity- 91%, (C) phase-sensitive heteronuclear single quantum coherence (HSQC) in acetone-d6, (D) heteronuclear multiple bond correlation (HMBC) in acetone-d6, (E) correlated spectroscopy (COSY) in acetone-d6, and (F) 13C spectrum in acetone-d6. (TIF) S15 Fig. Scanning electron micrographs of (A) an elytron of an untreated bark beetle showing (B) spores of an ophiostomatoid fungus in the elytral pit, (C) an empty elytral pit of a fungusfree beetle, and (D) spore mass of G. penicillata in the elytral pit of a fungus-free beetle reinoculated with this fungal species. (TIF) S1 Table. Purity, source, and biological origins of chemicals used in the experiments. (DOCX) S2 Table. Emission of volatile organic compounds identified from the headspace collection of fresh spruce bark 4 d after inoculation with different fungi. Analyses were conducted using GC-FID. Compounds were identified by GC–MS analyses run in parallel. Compounds with significant P values are highlighted in bold. The data underlying this Table can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (DOCX) S3 Table. Relative amounts (mean ± SE, n = 3) of volatiles from uninoculated bark detected after various time periods (4, 8, 12, and 18 d) from the beginning of an experiment with fungal inoculation. Data from the control uninfected treatment are presented here. Data for fungal treatments are given in S3–S6 Tables. Volatiles were collected on polydimethylsiloxane tubes for 2 h and were subjected to GC–MS analysis (see Materials and methods section for details). ND, not detected, NA, not analyzed, TR, trace amounts (<500 TIC counts). The data underlying this Table can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (DOCX) S4 Table. Relative amounts (mean ± SE, N = 4–5) of volatiles detected at various time periods after inoculation of fresh spruce bark with E. polonica (4, 8, and 12 d). Volatiles were collected on polydimethylsiloxane tubes for 2 h and were subjected to GC–MS analysis (see Materials and methods section for details). ND, not detected, NA, not analyzed, TR, trace amounts (<500 TIC counts). The data underlying this Table can be found at https://doi.org/ 10.6084/m9.figshare.21692156.v1. (DOCX) S5 Table. Relative amounts (mean ± SE, N = 5) of volatiles detected at various time periods after inoculation of fresh spruce bark with G. penicillata (4, 8, 12, and 18 d). Volatiles were collected on polydimethylsiloxane tubes for 2 h and were subjected to GC–MS analysis (see Materials and methods section for details). ND, not detected, NA, not analyzed, TR, trace amounts (<500 TIC counts). The data underlying this Table can be found at https://doi.org/ 10.6084/m9.figshare.21692156.v1. (DOCX) S6 Table. Relative amounts (mean ± SE, N = 5) of volatiles detected at various time periods after inoculation of fresh spruce bark with L. europhioides (4, 8, 12, and 18 d). Volatiles were collected on polydimethylsiloxane tubes for 2 h and were subjected to GC–MS analysis (see Materials and methods section for details). ND, not detected, NA, not analyzed, TR, trace amounts (<500 TIC counts). The data underlying this Table can be found at https://doi.org/ 10.6084/m9.figshare.21692156.v1. (DOCX) S7 Table. Relative amounts (mean ± SE, N = 5) of volatiles detected at various time periods after inoculation of fresh spruce bark with O. bicolor (4, 8, 12, and 18 d). Volatiles were collected on polydimethylsiloxane tubes for 2 h and were subjected to GC–MS analysis (see Materials and methods section for details). ND, not detected, NA, not analyzed, TR, trace amounts (<500 TIC counts). The data underlying this Table can be found at https://doi. org/10.6084/m9.figshare.21692156.v1. (DOCX) S8 Table. Composition of synthetic monoterpene mixture used in bioassays. $The purity of each compound was calculated from GC–MS analysis. (DOCX) S9 Table. Average colony forming units (CFUs/mL) from untreated, fungus-free, and fungus- free G. penicillata-reinoculated I. typographus bark beetles (n = 5 or 6 beetles). Wash, supernatant from beetles immersed in 0.05% Triton X in 500 μL PBS buffer (pH 7.4); lysate, crushed beetles in 500 μL PBS buffer (pH 7.4); NP, not present. The data underlying this Table can be found at https://doi.org/10.6084/m9.figshare.21692156.v1. (DOCX) S10 Table. Colony forming units (CFUs/mL) obtained from bark beetle gallery samples infested by fungus-free beetles, and fungus-free beetles reinoculated with G. penicillata, and untreated control beetles. Approximately 300 mg of bark samples were dissolved in 1 mL PBS buffer solution, and dilutions were plated on PDA. NP, not present. $Only one gallery sample was tested due to low sample availability. (DOCX) S1 Method. Preparation of bark beetle diet for eliminating fungal symbionts. (DOCX) S2 Method. Analysis of G. penicillata and Trichoderma sp. headspace volatiles. (DOCX) S3 Method. Beetle pheromone analysis. (DOCX)
Keywords
Feeding, Breeding, Bark beetles, Oxygenated metabolites, Eurasian spruce bark beetle (Ips typographus), Ips typographus, SDG-15: Life on land
Sustainable Development Goals
SDG-15:Life on land
Citation
Kandasamy, D., Zaman, R., Nakamura, Y., Zhao, T., Hartmann, H., Andersson, M.N. et al. (2023) Conifer-killing bark beetles locate fungal symbionts by detecting volatile fungal metabolites of host tree resin monoterpenes. PLoS Biology 21(2): e3001887.
https://doi.org/10.1371/journal.pbio.3001887.