DATA AVAILABILITY STATEMENT : All relevant data are
either within the manuscript and its supporting
information files, or available online https://DOI.org/
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)
