Monoterpenyl esters in juvenile mountain pine beetle and sex-specific release of the aggregation pheromone trans-verbenol

Gas chromatography/mass spectrometry (GC/MS) metabolite profiling of freshly emerged male and female MPB revealed sex-specific differences in a set of compounds that were present in extracts of females, but not of males (Fig. 1 and SI Appendix, Figs. S1 and S2 and Table S1). Silica chromatography and ester hydrolysis, followed by GC/MS analysis, revealed these compounds as fatty acid esters of mostly trans-verbenol along with minor amounts of fatty acids of cis-verbenol, myrtenol, myrtanol, and an unknown terpene alcohol (SI Appendix, Figs. S3 and S4). The female-specific esters were identified by comparison with authentic standards as verbenyl palmitate, verbenyl oleate, myrtanyl oleate, and myrtenyl oleate (Fig. 1 and SI Appendix, Fig. S5). cis-Verbenyl oleate and trans-verbenyl oleate did not separate under the GC conditions and could not be distinguished as these two compounds had very similar mass spectra (SI Appendix, Fig. S5). These two compounds were quantified together as “verbenyl oleate” in subsequent analyses of monoterpenyl esters over the life cycle of the beetle and in response to treatments. Monoterpenyl esters were not detected in the phloem collected from the brood trees (SI Appendix, Fig. S6).

Verbenyl esters, which represent the total of cis- and trans-verbenyl oleates and palmitates, as well as myrtenyl oleate and myrtanyl oleate, were present with similar amounts in females and males in early life stages of first instar larvae until pupation (Fig. 2 A–D). In contrast, significant sex-specific differences appeared in the freshly emerged adult beetles that were no longer in contact with tissues of the brood tree. In the emerged adults, only females contained substantial amounts of verbenyl, myrtenyl, and myrtanyl esters (Fig. 2 A–D). During the early life stages until pupation, verbenyl oleate was the most abundant of the monoterpenyl esters in both sexes with 250–1,500 ng/mg of beetle body weight (Fig. 2A), which was at least one order of magnitude higher than any of the other monoterpenyl esters. Levels of verbenyl esters significantly increased in male pupae compared with the larvae stages (Fig. 2 A and B) and then declined and were absent in freshly emerged adult males. In contrast, while levels of verbenyl esters in females were similar to those of males during the four larvae stages, they significantly increased in teneral adult females and remained high in emerged females. In contrast to the verbenyl and myrtanyl esters, the levels of other monoterpenyl esters, specifically perillyl and carvyl oleate, were independent of sex and decreased over the life cycle from early instar larvae to emerged adults (Fig. 2 E and F).

Verbenyl oleate was present in freshly emerged females in the three major body parts—head, thorax and abdomen—where the alimentary canal with the fat body was removed from the abdomen (Fig. 3). Dissection of the alimentary canal showed that verbenyl oleate was most abundant in the layer of perivisceral fat body that surrounds the alimentary canal (Fig. 3), with much lower amounts in the midgut, Malpighian tubules, and hindgut.

Following the identification of verbenyl esters in preadult males and females and their sex-specific abundance in freshly emerged females, we tested to see if their levels were affected by JHIII, which regulates pheromone release in MPB. We also tested if JHIII treatment affected levels of free verbenol and other monoterpene alcohols in female and male emerged adults. Levels of trans-verbenol increased in JHIII-treated females compared with acetone-treated controls (Fig. 4A), while levels of verbenyl esters decreased in JHIII-treated females compared with controls (Fig. 5A). Levels of cis-verbenol and myrtenol also significantly increased in females in response to JHIII (Fig. 4 B and C), while the corresponding esters decreased (Fig. 5 B and C). JHIII treatment did not induce elevated levels of monoterpenols in males, which lacked substantial amounts of the corresponding fatty acid esters (Figs. 4D and 5D).

We tested the effect of α-pinene vapor, which is the presumed host-derived monoterpene precursor of verbenol, on levels of monoterpenols and monoterpenyl esters in freshly emerged males and females. Exposure to a nearly racemic blend of α-pinene caused increased levels of trans-verbenol, cis-verbenol, and myrtenol, as well as increased accumulation of the corresponding esters, in both sexes (Figs. 4 A–C and 5 A–C). Exposure to α-pinene did not induce a significant increase in the levels of myrtanol, a β-pinene–derived monoterpenol, or accumulation of myrtanyl oleate in either sex (Figs. 4D and 5D).

The stereochemistry of the monoterpenols detected in MPB is expected to be dependent upon the stereochemistry of the proposed precursor monoterpenes (SI Appendix, Fig. S7). We compared the enantiomeric ratio of the monoterpenols induced by treatment with JHIII or α-pinene to the enantiomeric ratio of the presumed monoterpene precursors.

The enantiomeric ratio of trans-verbenol detected in beetles differed between the JHIII and α-pinene treatments. The average enantiomeric ratio of trans-verbenol in JHIII-treated females was 3(+):97(−) for trans-verbenol (Fig. 4A). This ratio was similar to the ratio 4(+):96(−) of the trans-verbenol that comprised the verbenyl esters in freshly emergent females (SI Appendix, Fig. S3). The enantiomeric ratio of trans-verbenol found in freshly emerged beetles exposed to a nearly racemic α-pinene vapor was 34(+):66(−) in females (Fig. 4A) and 35(+):65(−) in males (Fig. 4B), which was closer to the 44(+):56(−) ratio of α-pinene used for treatment.

Following the observation that exposure to α-pinene vapor resulted in increased levels of verbenyl and myrtenyl esters in both females and males, we also exposed freshly emerged adult beetles to vapors of other monoterpenes that are present in host trees to test if the occurrence of monoterpenyl esters was specific to α-pinene or a general effect of monoterpene exposure. Females and males were individually exposed to (−)-β-pinene, (−)-limonene and (−)-β-phellandrene. Exposure to all of these monoterpenes resulted in the production of monoterpene alcohols and the corresponding monoterpenyl esters in the beetles (Fig. 6). Often, multiple products were produced from the treatment with a single monoterpene. However, the identity of only a few of the esters from (−)-β-pinene and (−)-limonene could be verified by our synthetic standards (Fig. 6 and SI Appendix, Fig. S8).

The following chemicals were obtained from Sigma-Aldrich: racemic JHIII [catalog no. J2000, Chemical Abstracts Service (CAS) no. 24198–95-6], N,N′-dicyclohexylcarbodiimide (DCC, catalog no. D8002), 4-(dimethylamino)pyridine (DMAP, catalog no. 39405), N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA, catalog no. 15209), (+)-α-pinene (CAS no. 7785–70-8), (−)-α-pinene (CAS no. 7785–26-4), (−)-carveol (catalog no. 192384), (−)-myrtenol (catalog no. W343900), oleic acid (catalog no. O1008), palmitic acid (catalog no. P0500), (S)-(−)-perillyl alcohol (catalog no. 218391), stearic acid (catalog no. 85679), (−)-transpinocarveol (catalog no. 80613), (−)-β-pinene (catalog no. 112089), (+)-3-carene (catalog no. 441619), (−)-limonene (catalog no. 218367), and myrcene (catalog no. M100005). trans-Verbenol [∼20(+):80(−) optical purity, lot no. W06-00141], and cis-Verbenol [∼20(+):80(−) optical purity, lot no. CV001129] were obtained from PheroTech. The (−)-trans-myrtanol (catalog no. 5134 S) was obtained from Extrasynthese, and (−)-β-phellandrene was obtained by purification from lodgepole pine (Pinus contorta) turpentine by Synergy Semiochemicals.

MPB-infested lodgepole pine (P. contorta) stems were collected near Whistler, British Columbia, Canada (50°12′46.6″ N, 122°53′20.8″ W). In June 2016, MPB pheromone baits (Contech) were attached to trees and removed after attack 4 wk later. In September 2016, infested trees were felled and the logs placed in screened cages at room temperature. Phloem samples were collected from the infested logs. Larvae, pupae, and teneral adults were dissected from infested logs and stored at −80 °C. Larval instar was estimated by head capsule width according to Bleiker and Régnière (45). In addition, emerging adults were collected every 3–4 d from inside the screened cages. Adult MPB were sexed using the shape of the seventh abdominal tergite according to Lyon (46), and larvae and pupae were sexed by PCR.

Sex-specific PCR primers were designed to identify the sex of MPB larvae and pupae (SI Appendix, Figs. S9 and S10 and Table S2). Similar to a method developed to identify the sex in Tribolium casteneum (47), these primers amplify regions of the neo-X and neo-Y chromosomes in a multiplex PCR. PCR of males (neo-X and neo-Y) gives two major DNA products, and PCR of females (neo-X and neo-X) gives one major DNA product as detected on an agarose gel. Primers were validated with 60 individual male and female adults that were sexed using morphological characteristics (46). DNA was extracted from larval and pupal samples that had previously been extracted with pentane for trans-verbenyl ester analysis. A volume of about 1 mm³ MPB sample was used to produce a 20-µL DNA extract using the prepGEM insect kit (ZyGEM) according to the manufacturer’s instructions.

Emerging adult female and male MPB were separately treated with acetone, JHIII, α-pinene, and other monoterpenes to test for the production of trans-verbenol. For treatment with JHIII, 0.5 μL of JHIII (20 mg/mL in acetone) was topically applied to the abdomen. Topical application of 0.5 μL acetone served as a control. Treated beetles were placed individually into sealed 20-mL glass vials. Both enantiomers of α-pinene were used to treat beetles, as both enantiomers are present in the pine hosts of MPB. A nearly racemic mixture [44(+):56(−)] of α-pinene was used in treatments, of which 2 μL was applied to a 1-cm² Whatman filter paper and placed into a sealed 20-mL glass vial with an individual beetle. Beetles were allowed to come in contact with the α-pinene carrying filter paper. Other monoterpene treatments were done with 2 μL of (−)-β-phellandrene, 2 μL of (−)-β-pinene, or 1 μL of (−)-limonene applied to a 1-cm² Whatman filter. Beetles were treated for 24 h and then removed from the vial, frozen in liquid N₂, and stored at −80 °C until extraction.

Emerging females were dissected into head, thorax, and abdomen. The alimentary canal was removed from the dissected abdomen and separated into the fat body (perivisceral layer), midgut, Malpighian tubules, and hindgut. The perivisceral layer of the fat body surrounds the alimentary canal and was removed cleanly without contamination from other tissues. The rest of the fat body (parietal layer), located between the muscles in the abdomen, thorax, and head, could not be removed cleanly or completely and was left in place.

Frozen beetles were crushed in a 2-mL Safe-Lock tube (Eppendorf) on dry ice using a cold glass stir rod and extracted with 0.5 mL pentane containing 1 ng/µL of tridecane as internal standard. Smaller specimens (first and second instar larvae and dissected alimentary canal) were extracted with 0.1–0.25 mL pentane. A single larva, pupa, or adult was extracted per tube. Samples were removed from dry ice, allowed to thaw for a few minutes, and centrifuged for 20 s at 2,000 × g. Samples were frozen again on dry ice, and the pentane supernatant was transferred into an amber 2-mL glass vial (Agilent). Beetles were extracted a second time with 0.5 mL pentane, and the two extracts were combined. To remove excess amounts of fatty acids from the sample, 400 μL of 1 mM ammonium carbonate (pH 8) was added to the combined pentane extract and vortexed. The sample was centrifuged for 10 min at 3,000 × g, and the pentane layer was removed for analysis by GC/MS. Phloem samples were extracted with 1.5 mL of methyl tertiary butyl ether (MTBE) (Sigma-Aldrich).

To identify female-specific compounds found as esters in emerged beetles, male and female beetle extracts were separately processed by silica chromatography and ester hydrolysis. Beetle extracts were evaporated to dryness, redissolved in 0.2 mL hexane, loaded onto a silica column (300 mg) (catalog no. S2509; Sigma), and washed with 4 mL hexane to remove alkanes. Esters were eluted with 6 mL of 1% (vol/vol) MTBE in hexane. To hydrolyze the esters, 3 mL of the ester fraction was evaporated to dryness and hydrolyzed by redissolving in 0.5 mL of 0.3 M methanolic potassium hydroxide and incubation at 75 °C for 1 h in a sealed amber glass 2-mL vial (catalog no. 5182–0716; Agilent). The released alcohols were then extracted twice with 0.5 mL pentane. The ester fraction and the alcohols obtained by hydrolysis were analyzed using GC/MS.

Monoterpenyl esters were synthesized by Steglich esterification (48) for the identification of the female-specific esters. Amounts of 35 mmol of fatty acid and 105 mmol of monoterpenol were combined in 1 mL of CH₂Cl₂ and placed on ice, and 0.2 mL of an ice-cold CH₂Cl₂ solution containing 39 mmol of DCC and 3.5 mmol of DMAP was added. Amber GC vials (Agilent) containing the reaction mixture were briefly shaken and then held on ice for 10 min before being placed on a shaker at room temperature for 2 h. The subsequent work-up involved washing with 1 M sodium acetate (pH 5.2), washing with saturated NaCl, and then drying over anhydrous MgSO₄. The CH₂Cl₂ solution was evaporated under a flow of nitrogen gas, and the product was redissolved in 0.5 mL hexane. The product was purified on a 300-mg silica column by first passing 4 mL hexane through the column and then eluting the ester product with 6 mL of 1% (vol/vol) MTBE in hexane. Monoterpenyl esters were analyzed using GC/MS. However, verbenyl oleate was thermally unstable at the high-temperature conditions of the GC inlet (250 °C), resulting in several breakdown products detected with multiple peaks in the chromatogram of standards. In addition, the menthatrienes, cymenes, and verbenes (decomposition products of verbenol) were detected in the GC/MS of beetle extracts whenever the esters were present, even when hydrocarbons had been removed by silica chromatography (SI Appendix, Fig. S1). Cool (30 °C) on-column injection of ester standards and beetle ester fractions prevented the decomposition of the esters (Fig. 1).

Monoterpenyl esters were separated and analyzed using an Agilent VF-5 column (5% phenyl methyl siloxane, 27.4 m length, 250 μm i.d., 0.25-μm film thickness) at 0.9 mL⋅min⁻¹ He on an Agilent 7890A system GC, Agilent 7683B series GC Sampler, and a 7000A GC/MS triple quad MS detector at 70 eV. The GC temperature program was as follows: 40 °C for 2 min, increase at 18 °C min⁻¹ to 300 °C, hold for 7 min, using a pulsed splitless injector held at 250 °C. Fatty acids were derivatized with BSTFA before analysis.

Due to their thermal instability, the monoterpenyl ester standards were also analyzed using a cool-on column injector on an Agilent HP-5 column (5% phenyl methyl siloxane, 30 m length, 250 μm i.d., 0.25-μm film thickness) at 0.9 mL⋅min⁻¹ He on an Agilent 6890A system GC, Agilent 7683 series GC Sampler, and an Agilent 5973 Mass Selective Detector at 70 eV. The GC temperature program was as follows: 30 °C for 1 min, increase at 20 °C min⁻¹ to 300 °C, and hold for 7 min. The temperature of the cool on-column injector increased at 20 °C min⁻¹, tracking the temperature of the column.

Enantiomeric purity of trans- and cis-verbenol extracted from beetles was analyzed on an Agilent CyclodexB column (10.5% β-cyclodextrin, 25.7 m length, 250 μm i.d., 0.25-μm film thickness) at 0.9 mL⋅min⁻¹ He on an Agilent 7890A system GC, Agilent GC Sampler 80, and a 7000A GC/MS triple quad M5975C inert XL MSD with triple axis detector at 70 eV. The GC temperature program was as follows: 40 °C for 2 min, increase at 10 °C min⁻¹ to 100 °C, 20 °C min⁻¹ to 230 °C, hold for 7 min with pulsed splitless injector held at 250 °C.

Compound quantities were analyzed using nonparametric tests because they failed tests for normality and homogeneous variances (Shapiro–Wilk normality test and Barlett test of homogeneity of variances). We used the Kruskal–Wallis rank sum test followed by the Conover’s test for pairwise comparisons with the P values adjusted by the Benjamini, Hochberg, and Yekutieli correction method for multiple comparisons.