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

Abstract

A recent outbreak of mountain pine beetle (MPB) has spread over more than 25 million hectares of pine forests in western North America, affecting pine species of sensitive boreal and mountain ecosystems. During initial host colonization, female MPB produce and release the aggregation pheromone trans-verbenol to coordinate a mass attack of individual trees. trans-Verbenol is formed by hydroxylation of α-pinene, a monoterpene of the pine oleoresin defense. It is thought that adult females produce and immediately release trans-verbenol when encountering α-pinene on a new host tree. Here, we show that both sexes of MPB accumulate the monoterpenyl esters verbenyl oleate and verbenyl palmitate during their development in the brood tree. Verbenyl oleate and verbenyl palmitate were retained in adult female MPB until the time of emergence from brood trees, but were depleted in males. Adult females released trans-verbenol in response to treatment with juvenile hormone III (JHIII). While both sexes produced verbenyl esters when exposed to α-pinene, only females responded to JHIII with release of trans-verbenol. Accumulation of verbenyl esters at earlier life stages may allow adult females to release the aggregation pheromone trans-verbenol upon landing on a new host tree, independent of access to α-pinene. Formation of verbenyl esters may be part of a general detoxification system to overcome host monoterpene defenses in both sexes, from which a specialized and female-specific system of pheromone biosynthesis and release may have evolved.

Keywords: bark beetle; forest health; pheromone; pinene; verbenol.

Fig. 1.

GC/MS chromatogram of the ester fraction of freshly emerged adult beetles (A) and monoterpenyl ester standards (B–F). The standards and ester fraction were injected onto an HP-5 column at 30 °C using a cool on-column injector. Verbenyl palmitate, trans- and cis-verbenyl oleate, myrtenyl oleate, and myrtanyl oleate were all present in the emerging female ester fraction (red) but were not present in the emerging male ester fraction (blue). The mass spectra of peaks labeled 1–5 are shown in SI Appendix, Fig. S5.

Fig. 2.

The abundance of monoterpenyl esters in females (pink) and males (blue) over the life cycle of MPB. Columns in the same graph with the same letter were not significantly different by Conover’s test (α ≥ 0.05). During the larval instar to the teneral adult stage, developing beetles remain in the brood tree. Tenerals are adult beetles that do not have fully sclerotized cuticle, a stage that lasts 7–14 d. Emerged beetles are fully mature adults, which leave their brood tree in search of a new host. The amounts of (A) verbenyl oleate, (B) verbenyl palmitate, and (D) myrtanyl oleate were significantly different between female and male emerged beetles as indicated by the arrows, but not for (C) myrtenyl oleate or (F) perillyl oleate. No significant differences were found for (E) carvyl oleate (n = 4).

Fig. 3.

The abundance of verbenyl oleate in dissected tissues of emerging female MPB. The head, thorax, and abdomen were separated, and then the alimentary canal with Malpighian tubules and perivisceral fat body were removed from the abdomen for further dissection into the fat body, midgut, Malpighian tubules, and hindgut. The asterisk denotes the abdomen with the alimentary canal removed (n = 4).

Fig. 4.

The presence of monoterpene alcohol pheromone components in MPB after treatment with acetone, JHIII, or 44(+):56(−) α-pinene. Columns in the same graph with the same number [(+) enantiomer] or same letter [(−) enantiomer] were not significantly different by Conover’s test (α ≥ 0.05). Compared with the acetone control, JHIII treatment significantly increased (A) trans-verbenol, (B) cis-verbenol, and (C) myrtenol production in female, but not male beetles. α-Pinene treatment increased (A) trans-verbenol, (B) cis-verbenol, and (C) myrtenol production in both sexes. (D) α-Pinene did not increase trans-myrtanol, a product of β-pinene metabolism. Enantiomeric ratios are shown where relevant (n = 6).

Fig. 5.

The presence of monoterpenyl esters in MPB after treatment with acetone, JHIII, or α-pinene. Columns in the same graph with the same letter were not significantly different by Conover’s test (α ≥ 0.05). (A) Verbenyl oleate, (B) verbenyl palmitate, and (D) myrtanyl oleate declined significantly with JHIII treatment in females. (A) Verbenyl oleate, (B) verbenyl palmitate, and (C) myrtenyl oleate increased significantly with α-pinene treatment in both sexes (n = 6).

Fig. 6.

GC/MS chromatograms of extracts of female and male beetles treated with acetone, (−)-limonene, (−)-β-pinene, and (−)-β-phellandrene. The monoterpenyl esters, (A) carvyl oleate and perillyl oleate, were identified in both sexes of limonene-treated MPB; (B) pinocarvyl oleate and myrtanyl oleate were identified in both sexes of β-pinene–treated MPB; and (C) a monoterpenyl ester was present in β-phellandrene–treated MPB, but could not be identified. The mass spectra of peaks labeled 1–6 are shown in SI Appendix, Fig. S8.